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1 DIFFERENTIATION OF CARBOHYDRATE ISOMERS BY TUNABLE INFRARED MULTIPLE PHOTON DISSOCIATION AND FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY By SARAH ELIZABETH STEFAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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1

DIFFERENTIATION OF CARBOHYDRATE ISOMERS BY TUNABLE INFRARED MULTIPLE PHOTON DISSOCIATION AND FOURIER TRANSFORM ION CYCLOTRON

RESONANCE MASS SPECTROMETRY

By

SARAH ELIZABETH STEFAN

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009

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© 2009 Sarah Elizabeth Stefan

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To my Mom and Dad

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ACKNOWLEDGMENTS

Many people have supported and helped me throughout my graduate career. First, I would

like to thank my parents. Their support and unconditional love have made my studies possible. I

want to thank them for their understanding and encouragement when tough times occurred; I

appreciate all their help and love more than they will ever know. I also thank my family for their

support and for always making life interesting.

I am grateful for my friends, old and new, who gave a helping hand and an ear for listening

when I needed them. All the laughs and conversations over these past four years have lifted my

spirits and helped me to keep going. I want to acknowledge my lab mates, past and present, for

their help, knowledge and conversations have been instrumental in my work.

I would also like to thank all my professors at Wheaton College, specifically Drs. Elita

Pastra-Landis and Laura Muller, whose support and investment in me opened my eyes and mind

to the potential of graduate school. Their enthusiasm and support have made all the difference.

I have the deepest gratitude to all the people with whom I collaborated; they have made my

project possible. First, I wish to thank my advisor, Dr. John Eyler, for his guidance, patience and

support during my graduate career. I want to thank Dr. Brad Bendiak for all the samples, advice

and support that he has provided throughout this project. His guidance and suggestions were

well needed and helped tremendously. I would also like to thank Dr. David Powell for use of his

instrument for the negative disaccharide work. Next, I want to thank my other committee

members, Drs. Nicolo Omenetto, Nicolas Polfer and Carrie Haskell-Luevano, whose questions

and conversations have helped me along the way. Finally, I would like to thank Drs. Jos

Oomens and Jeffrey Steill for their help and effort with the work performed at the Free Electron

Laser for Infrared eXperiments (FELIX) facility. Without all of these people, this dissertation

would not be possible.

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Finally I need to thank the one person who has had to listen to me late at night and early in

the morning, whose patience and loving shoulder made it easier to continue when I wanted to

give up, Mr. Brad House. His immense computer knowledge and lack of chemistry knowledge

helped me survive the past four years.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES...........................................................................................................................9

LIST OF FIGURES .......................................................................................................................10

ABSTRACT...................................................................................................................................13

CHAPTER

1 INTRODUCTION ..................................................................................................................15

Carbohydrates .........................................................................................................................15 Monosaccharides .............................................................................................................15 Disaccharides...................................................................................................................18 Oligo- and Polysaccharides .............................................................................................19

Differentiation of Mono- and Disaccharides ..........................................................................21 Separation of Oligosaccharides .......................................................................................22 Analysis Methods ............................................................................................................24 Mass Spectrometry: Ionization Techniques ....................................................................26 Fragmentation Methods...................................................................................................27 Charged Ions....................................................................................................................29

Objective of This Research.....................................................................................................31 Overview.................................................................................................................................32

2 FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY.................................................................................................................39

History ....................................................................................................................................39 Apparatus................................................................................................................................40

Magnet.............................................................................................................................40 Vacuum System...............................................................................................................40 Analyzer Cell...................................................................................................................41 Data System.....................................................................................................................41

Theory.....................................................................................................................................42 Cyclotron Motion ............................................................................................................42 Trapping Motion..............................................................................................................43 Magnetron Motion...........................................................................................................44

Basic FTICR-MS Operation and Data Acquisition ................................................................46 Mass Resolution......................................................................................................................49 Tandem Mass Spectrometry ...................................................................................................51 Dissociation Techniques.........................................................................................................51 Conclusions.............................................................................................................................53

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3 INFRARED MULTIPLE PHOTON DISSOCIATION .........................................................58

Introduction.............................................................................................................................58 Mechanism of Infrared Multiple Photon Dissociation ...........................................................59 Lasers Used for IRMPD .........................................................................................................60

4 DIFFERENTIATION OF MONOSACCHARIDES IN THE POSITIVE ION MODE BY IRMPD WITH A TUNABLE CO2 LASER.....................................................................66

Introduction.............................................................................................................................66 Procedure ................................................................................................................................67 Reproducibility .......................................................................................................................68 Results and Discussion ...........................................................................................................69

Methyl-glucopyranosides ................................................................................................69 Unknown Study of Methyl-glucopyranosides.................................................................71 Methyl-galactopyranosides..............................................................................................71 Unknown Study of both Methyl-gluco- and galactopyranosides ....................................72

Conclusions.............................................................................................................................73

5 DIFFERENTIATION OF DISACCHARIDES IN THE POSITIVE ION MODE WITH A TUNABLE CO2 LASER ....................................................................................................83

Introduction.............................................................................................................................83 Procedure ................................................................................................................................84

Fragmentation Study .......................................................................................................84 Anomeric Configuration Study .......................................................................................85

Results and Discussion ...........................................................................................................85 Differentiation of Disaccharides......................................................................................85 Determination of the Anomeric Configurations..............................................................86 Differentiation of Unknowns...........................................................................................87

Conclusions.............................................................................................................................88

6 IRMPD STUDIES OF NEGATIVELY CHARGED DISACCHARIDES WITH A TUNABLE CO2 LASER ........................................................................................................93

Introduction.............................................................................................................................93 Procedure ................................................................................................................................94

Deprotonated Disaccharides ............................................................................................94 Chlorinated Disaccharides...............................................................................................95 Reproducibility: Deprotonated Disaccharides.................................................................96 Reproducibility: Chlorinated Disaccharides....................................................................96

Results and Discussion ...........................................................................................................97 Deprotonated Disaccharides ............................................................................................97 Chlorinated Disaccharides...............................................................................................98 Identification of Fragment Ions .....................................................................................102

Conclusions...........................................................................................................................102

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7 DIFFERENTIATION OF DISACCHARIDES IN THE NEGATIVE ION MODE WITH FREE ELECTRON LASER INFRARED MULTIPLE PHOTON DISSOCIATION ..................................................................................................................115

Introduction...........................................................................................................................115 Procedure ..............................................................................................................................115 Results and Discussion .........................................................................................................116

Disaccharides.................................................................................................................116 Monosaccharide Anion Produced from Disaccharides .................................................118

Conclusions...........................................................................................................................119

8 CONCLUSIONS AND FUTURE WORK...........................................................................127

LIST OF REFERENCES.............................................................................................................131

BIOGRAPHICAL SKETCH .......................................................................................................139

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LIST OF TABLES

Table page 5-1 Table of ratios used to determine the laser power used for fragmentation........................91

6-1 Major fragment ions observed for the chlorinated disaccharides when the precursor ion (m/z 377) was almost depleted by infrared mulitple photon dissociation (IRMPD) at 9.588 μm ......................................................................................................................108

6-2 Comparison of the fragments produced by collision induced dissociation (CID) and IRMPD for the chlorinated disaccharides........................................................................108

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LIST OF FIGURES

Figure page 1-1 Fischer projection for D- and L-glucose. ...........................................................................34

1-2 Example of the numbering system for the carbons of monosaccharides...........................34

1-3 Fischer projections for the D-hexoses of the aldose family...............................................34

1-4 Anomers of D-glucose. ......................................................................................................35

1-5 Inter-conversion of the ring structures for the 6-membered ring, pyranose, and the 5-membered ring, furanose, of D-glucose .........................................................................35

1-6 Examples of disaccharides composed of two glucose (Glc) monosaccharides. ................36

1-7 Structures of two common oligosaccharide derivatives. ...................................................36

1-8 Typical steps for analysis of glycans. ................................................................................37

1-9 Fragmentation nomenclature for oligosaccharides. ...........................................................38

1-10 Possible fragmentation pathways for fragmentation by infrared multiple photon dissociation (IRMPD). .......................................................................................................38

2-1 Ion cyclotron motion..........................................................................................................54

2-2 Schematic diagram of the components of a Bruker 4.7 T FTICR (Fourier transform ion cyclotron) mass spectrometer. .....................................................................................54

2-3 Figures of merit for FTICR-MS as a function of magnetic field strength .........................55

2-4 Two of the typical analyzer cells used for in FTICR mass spectrometers.........................55

2-5 General schematic of a typical experimental sequence. ....................................................56

2-6 Various domains and spectra obtained from an FTICR-MS experiment. .........................56

2-7 Effect of number of data points acquired and Fourier transform on mass resolution........57

3-1 Energy potential well. ........................................................................................................64

3-2 Depiction of the IRMPD mechanism in polyatomic molecules. .......................................64

3-3 Schematic of an undulator used for free elctrom lasers (FELs).........................................65

3-4 Layout schematic of Free Electrom Laser for Infrared eXperiments (FELIX) .................65

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4-1 Structures of the O-methylated monosaccharides discussed in this chapter......................74

4-2 Experimental set up of the 4.7 T FTICR mass spectrometer.............................................74

4-3 Wavelength-dependent fragmentation patterns for the lithiated O-methyl-glucopyranosides for wavelength from 9.2 to 10.8 μm.....................................75

4-4 Infrared mulitple photon dissociation depletion spectra of the precursor ions (m/z 201) for both α- and β-O-methyl-glucopyranoside – lithium cation complexes. ......76

4-5 Comparison of the fragmentation of β-methyl-glucopyranoside at wavelengths 9.588 and 10.611 μm. ..................................................................................................................77

4-6 Relative percent abundance of fragment ions for both lithiated α- and β-O-methyl-glucopyranosides over the wavelength range from 9.201 to 9.675 μm. ........78

4-7 Spectra of unknowns in single blind study of methyl-glucopyranosides at wavelength 9.588 μm. ...........................................................................................................................79

4-8 Fragmentation patterns over the wavelengths from 9.2 to 10.6 μm. .................................80

4-9 Ratio of m/z 169 to m/z 151 for α- and β-O-methyl-galactopyranoside. ...........................81

4-10 Decision flowchart used to identify the different monosaccharide anomers. ....................81

4-11 Spectra of unknowns identified as galactopyranosides in single blind study obtained at wavelength 9.588 μm.. ...................................................................................................82

5-1 Wavelength-dependent fragmentation for the various linked lithiated disaccharides .......89

5-2 Flow-chart depicting how linkage of the disaccharides was determined. .........................90

5-3 Flow-chart showing ratios of peak heights and values used to determine anomeric configurations. ...................................................................................................................91

5-4 Bar graphs comparing ratios from knowns and unknown lithiated glucose-containing disaccharides at the wavelengths 9.342, 9.472 and 9.588 μm. ..........................................92

6-1 Schematic drawing of the laser/mass spectrometer set-up used for the analysis of deprotonated disaccharides. .............................................................................................104

6-2 Relative percent abundance of the precursor ion (m/z 341) of isomaltose at selected wavelengths......................................................................................................................104

6-3 Wavelength-dependent fragmentation patterns for the various deprotonated disaccharides. ...................................................................................................................105

6-4 Ratio of m/z 161/179 for 1-3 and 1-6 linked disaccharides, showing that this ratio is not optimal for distinguishing the different anomers.......................................................106

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6-5 Comparison of the fragmentation patterns of deprotonated isomaltose on two separate days. ...................................................................................................................107

6-6 Fragmentation spectra for the nearly depleted precursor ion (m/z 377) for the chlorinated disaccharides at 9.588 μm.............................................................................109

6-7 Infrared multiple photon dissociation spectra for chlorinated isomaltose obtained at three wavelengths on two different days.. .......................................................................110

6-8 Average fragmentation spectra for the disaccharides at 9.342, 9.473 and 9.588 μm. .....111

6-9 Decision flow chart used to identify disaccharide samples with unknown identities in a single-blind study. .........................................................................................................112

6-10 Comparison of various ratios used to determine the anomeric configurations of the chlorinated disaccharides. ................................................................................................113

6-11 Identification of some of the fragment ions for the various linked disaccharides. ..........114

7-1 Schematic of the FTICR set-up at FELIX. ......................................................................121

7-2 Infrared multiple photon dissociation fragmentation patterns over the wavelength range of 5.5 to 11 μm for the deprotonated 18O-labeled disaccharides. ..........................122

7-3 Fragmentation pattern of chlorinated unlabeled sophorose. ............................................123

7-4 Comparison of the IRMPD spectra for O18-labeled sophorose and O16-chlorinated sophorose. ........................................................................................................................123

7-5 Comparison of the IRMPD spectra of the monosaccharide anions (m/z 179) produced by deprotonation of glucose and by fragmentation of a disaccharide by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and CO2 laser irradiation. .......................................................................................................124

7-6 Schematic of the possible mechanism leading to the opening of the monosaccharide anion ring. ........................................................................................................................124

7-7 Infrared multiple photon dissociation spectra of various deprotonated monosaccharides. .............................................................................................................125

7-8 Comparison of the IRMPD spectra for anomers of O-methyl-glucopyranoside to the spectrum of deprotonated glucose. ..................................................................................125

7-9 Comparison of the fragmentation patterns of the deprotonated monosaccharides over the wavelength range of 5.5 to 11 μm..............................................................................126

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

DIFFERENTIATION OF CARBOHYDRATE ISOMERS BY TUNABLE INFRARED

MULTIPLE PHOTON DISSOCIATION AND FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY

By

Sarah Elizabeth Stefan

May 2009 Chair: John R. Eyler Major: Chemistry

Carbohydrates and their derivatives play a crucial role in many biological processes

including fertilization, cell growth, inflammation and post-translational protein modification.

The function of carbohydrates in these systems is closely related to their structure, including

monosaccharide sequence, glycosidic linkage and stereochemistry. Unfortunately, the number of

anomeric configurations and possible linkages between monosaccharide units makes analysis of

carbohydrate structures complex. In order to shed light on these larger oligosaccharides, the

fragmentation patterns and infrared multiple photon dissociation (IRMPD) spectra of various

mono- and disaccharides were obtained and compared. For this work, various tunable infrared

sources including a line-tunable continuous-wave carbon dioxide laser and a free electron laser

(FEL) were used in conjunction with Fourier transform ion cyclotron resonance mass

spectrometry (FTICR-MS).

The first three projects used a line-tunable carbon dioxide laser to fragment various mono-

and disaccharides in both the positive and negative ion modes. In the first project, anomers of

lithium-cation attached O-methyl-gluco- and galactopyranosides were fragmented. The identity

and anomeric configuration of each monosaccharide was accurately determined by comparing

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fragmentation patterns and ratios of certain fragments. A second project explored the

fragmentation pattern of lithiated glucose-containing disaccharides having various linkages (1-2,

1-3, 1-4 and 1-6) and anomeric configurations (alpha and beta). Both the linkage and anomeric

configuration of the various disaccharides were successfully identified based on their

fragmentation patterns at several wavelengths. Next, irradiation of deprotonated and chlorinated

glucose-containing disaccharides produced fragmentation patterns in which cleavage of the

glycosidic bond resulted in major abundances of m/z 161 and 179 fragment ions. Along with

differentiating the anomeric configuration for the chlorinated disaccharides, comparison of the

abundances for major fragment ions also resulted in the positive identification of the linkages for

both sets of disaccharides.

Lastly, several deprotonated (negatively charged) mono- and disaccharides were

fragmented with a FEL. The IRMPD spectra of the monosaccharide anions (m/z 179) from both

the deprotonated monosaccharides and those isolated by fragmentation of various disaccharides

were taken. A C-O stretching band characteristic of aldehydes was present in all spectra at

~1720 wavenumbers and gave spectroscopic evidence of the monosaccharide ring opening and

therefore loss of anomericity.

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CHAPTER 1 INTRODUCTION

Carbohydrates and their derivatives are biologically important. They participate in cell-

cell interactions and also act as target structures for microorganisms, toxins and antibodies. 1-3

Carbohydrates also interact with proteins and play a critical role in fertilization, cell growth,

inflammation and post-translational protein modifications.1,3-5 The simplest unit within these

larger carbohydrates is that of the monosaccharide. When two monosaccharides are joined

together, the result is a disaccharide. The disaccharide is the smallest saccharide unit which

contains the glycosidic bond. Depending on the anomeric configurations of the monosaccharides

that react, a disaccharide can either be α- or β-linked. The role of carbohydrates depends not

only on the subunits of sugars which compose them, but also how these units are linked

together.6 Therefore, characterization of the both the anomeric configuration and the linkage of

the different types of mono- and disaccharides is important.

Carbohydrates

Carbohydrates can be categorized based on their degree of polymerization. The smallest

group is that of monosaccharides and their derivatives, all of which are not polymerized. The

next category includes oligosaccharides, that have 2 to 10 degrees of polymerization. The last

category is that of polysaccharides, that have greater than 10 degrees of polymerization. This

chapter will discuss all the possible types of carbohydrates as well as give an overview of the

methods used for carbohydrate analysis.

Monosaccharides

Monosaccharides are the smallest units that compose larger oligosaccharides. There are

several types of monosaccharides and they all have the general formula of (CH2O)n. Typically

the more biologically common isomer of monosaccharides in nature is the D-isomer, but

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L-isomers are also found. The monosaccharide isomer can be determined by drawing the

Fischer projection. In the Fischer projection when the hydroxyl group on the highest numbered

stereocenter is on the right, it is the D-isomer and when the hydroxyl group is on the left it is the

L-isomer, Figure 1-1.7 Since D-isomers of sugars are found much more frequently in nature, this

dissertation will deal only with D-isomers.

The carbons in monosaccharides are numbered sequentially starting with the end of the

chain nearest to the carbonyl group, as seen in Figure 1-2. Carbon number 1, also known as the

anomeric carbon, is where two monosaccharides can be joined together, through a glycosidic

linkage or bond, to form larger oligosaccharides.

The smallest possible monosaccharide has a backbone composed of only 3 carbon atoms,

but 4, 5 and 6 carbons are other possible backbones. The names of these monosaccharides are

trioses, tetroses, pentoses, hexoses, and heptoses, respectively. Monosaccharides that contain a

keto group are called ketose whereas monosaccharides containing an aldehyde are called aldoses.

Typically the names of the family and number of carbons are combined into one systematic

name. For example, a monosaccharide containing both a 4 carbon backbone and an aldehyde

group would be named an aldotetrose (aldo for the aldehyde group and tetrose for the 4 carbon

backbone). For the aldose family, each of the eight D-aldohexoses differs in stereochemistry at

carbon 2, 3 or 4 and has its own unique, common name, such as D-glucose, D-galactose, etc., as

shown in Figure 1-3. When two monosaccharides only differ at one carbon position, they are

epimers. Since they only differ in the position of the hydroxyl group on carbon number 4,

D-glucose and D-galactose are an example of epimers from the aldose family.

Monosaccharides can be found in either the open chain or ring form, but typically the ring

form is more common. In solution, monosaccharides with a 5 or 6 carbon backbone can undergo

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nucleophilic attack of the carbonyl carbon by one of the hydroxyl groups along the chain,

resulting in a ring. Six-membered rings are called pyranoses and 5-membered rings are called

furanoses.8,9 At least four carbons and one oxygen are needed to form a furanose. Therefore,

aldotetroses and higher aldoses and 2-pentuloses and higher ketoses can be found in the furanose

ring. While monosaccharide rings can be either 5- or 6-membered, pyranosides are the most

common form.

When cyclic monosaccharides only differ by the position of the hydroxyl group on the

anomeric carbon, they are anomers. If the hydroxyl group is axial relative to the plane of the

ring then it is said to be in the α-position and if it is equatorial then it is in the β-position,

Figure 1-4. The cyclic monosaccharides can interconvert between α- and β-anomers through a

process known as mutarotation, Figure 1-5. During mutarotation, the ring opens into the chain

form. Once in the chain form, a nucleophilic attack results in the formation of the β-anomer.

Therefore, in solution there is an equilibrium mixture of all possible isomers including the

furanose, pyranose, α-, β- and open chain forms of the monosaccharides. This equilibrium

mixture is different for each monosaccharide, but for D-glucose it is approximately one-third

α-anomer, two-thirds β-anomer and less than 1% of both the open and five-membered ring

forms.7 On the other hand, D-mannose has approximately 69% α-anomer and 31% β-anomer in

solution, thus showing that the equilibrium doesn’t always contain more of the β-anomer than the

α-anomer.

The two cyclic forms of D-glucose are known as hemi-acetals, which are formed by the

reaction of the hydroxyl group on carbon number 5 and the aldehyde group. Typically any

monosaccharide that contains a hemiacetal group is a reducing sugar and can react further. A

reducing sugar is one that reacts with Tollens’ (Ag(NH3)2OH) or Benedict’s reagents (solution of

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copper (II) sulfate, sodium carbonate, sodium citrate dihydrate and 2,5-difluorotoluene) to reduce

either Ag+2 or Cu+2. If a sugar contains an acetal group then it cannot react with the Tollens’ or

Benedict’s reagents and it is called a non-reducing sugar.

While hexoses are the most abundant sugars, there are a number of monosaccharide sugar

derivatives that are naturally abundant and important. Some of these derivatives are

N-acetylneuraminic acid (sialic acid), α-D-acetylgalactosamine and α-D-acetylglucosamine.

These derivatives are found primarily in animals as the major components of glycoproteins and

glycolipids.

Disaccharides

Disaccharides, the next largest saccharide are formed when a hydroxyl group of one

monosaccharide reacts with the anomeric carbon of the other, Figure 1-6. The resulting bond is

known as an O-glycosidic linkage. When two cyclic hexoses come together, a glycosidic linkage

can occur at one of the five hydroxyl positions. This leads to numerous possible isomers with

various linkages. Disaccharides are composed of a non-reducing monosaccharide that is fixed in

the ring conformation and a reducing-monosaccharide that can interconvert between the α- and

the β-configuration. Therefore, in solution, there will be a mixture of the α- and β-configurations

of the reducing sugar of the disaccharide.

While most sugars have a common, non-systematic name, there is a systematic

nomenclature scheme for disaccharides. In it, the name of the first monosaccharide unit, its

anomeric configuration and then the linkage followed by the second monosaccharide unit is

given. For example, two glucose (Glc) units that are α- connected at the 1 and 6 carbon will be

named glucose α1-6 glucose (Glcα1-6Glc), for which the common name is isomaltose. For

larger oligosaccharides the nomenclature process is the same, but for each monosaccharide

attachment the linkage and anomeric configuration followed by the monosaccharide is given.

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For example a trisaccharide that has a glucose β-linked to carbon number 2 of a mannose (Man)

monosaccharide which is α-linked to carbon number 4 of another glucose unit would be named

Glcβ1-2Manα1-4Glc. When the anomeric carbons of both monosaccharide units are linked, the

anomeric configuration of each saccharide is given. For example, sucrose is a disaccharide when

the anomeric carbon of both the glucose and fructose (Fru) monosaccharide units are linked. For

this, the systematic name would be Glcα1-2βFru. Since both anomeric carbons are linked in

sucrose, it is a non-reducing sugar, unlike kojibiose (Glcα1-2Glc) and sophorose (Glcβ1-2Glc)

that are examples of reducing sugars.

Oligo- and Polysaccharides

Oligosaccharides are the next largest saccharide chains that consist of 3 to 10

monosaccharide units linked together. Sugars that contain more than ten monosaccharide units

are called polysaccharides. Oligo- and polysaccharides can be either homo- or

heter-oligosaccharides. Homo-oligosaccharides contain the same monosaccharide unit that

repeats, whereas heter-oligosaccharides contain different monosaccharide units linked together.

One homo-polysaccharide is starch, which can be found in foods such as potatoes. Starches

characteristically have α1-4 linkage between two glucose units.10 Other polysaccharides that do

not have this linkage, also known as non-starch polysaccharides, can be found in foods such as

bran, bananas and hazelnuts. Other common polysaccharides are cellulose and glycogen.

Cellulose is a polysaccharide that contains several hundreds to thousands of β1-4 linked glucose

units. It is the main component of the primary cell walls of plants and can be found in some

algae. Glycogen is a glucose-polysaccharide that has a lot of branching and most commonly

functions as short-term energy storage in animals.

Common oligosaccharide derivatives are those of N-acetyl hexosamines, primarily

N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc), Figure 1-7.11 The

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GlcNAc reducing end is linked to serine or threonine residues whereas the GalNAc reducing end

is linked to asparagines. N-acetylglucosamine is a component of chitin and GalNAc is the

terminal carbohydrate that forms the antigen of blood group A. N-acetylgalactosamine is also

the first monosaccharide unit that connects to serine and threonine in glycosylation and is

necessary for intercellular communication.

Polysaccharides and oligosaccharides are also known as glycans. Glycosylation is a

post-translational modification where oligo- and polysaccharides are linked to proteins and

lipids, forming glycoconjugates. Glycosylation is one of the most common post-translational

modifications for proteins and it is approximated that more than 50% of all proteins are

glycosylated.12 Linkages between a glycan and a protein form glycoproteins and those with

lipids form glycolipids.

The type of glycoprotein is determined by the linkage between the carbohydrates and the

protein. Glycoproteins can be O- or N-linked. While N-linked are linked by a chitobiose (dimer

of β1-4-linked glucosamine units) unit to an amide nitrogen of an asparagine residue, O-linked

are linked to the oxygen of a side chain of an amino acid.13,14 Typically the linkage is through a

serine or threonine residue. N-glycosidic bonds are found in all nucleotides (the resulting sugar

and nucleotide structures are called nucleosides, such as ribonucleic acid (RNA) and

deoxyribonucleic acid (DNA)). Unlike other oligosaccharides that are linked by oxygen bridges,

RNA and DNA are polyesters that are linked by phosphate bridges. DNA is the largest known

polymer with more than 1012 units found in human genes and the number of units found

decreases as one goes down the evolutionary chain.8 Another example of a polysaccharide with

N-linkages is chitin. Chitin is a naturally occurring polysaccharide, composed of β1-4-linked

N-acetyl-D-glucosamines, which is found in places like fungi and exoskeletons of arthropods

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such as crustaceans. The three classes of glycoproteins are: N-glycosyl protein, O-glycosyl

protein and N,O-glycosyl protein. Since multiple types of linkages (O- or N-linked) and

anomeric configurations are possible, it is no surprise that many different isomers are possible.

When attached to proteins (glycoproteins), oligosaccharides have been found to aid in a

plethora of functions in the human body including cellular recognition, signaling, receptor

binding and immune responses.15-17 They also serve to influence folding, biological lifetime and

recognition of binding partners for proteins.17 Carbohydrates are also involved in the glycosyl

phosphatidyl-inositol (GPI) anchor, by which proteins are attached to the plasma membrane and

the oligosaccharides are linked to lipids which are attached to cell membranes.18 In this process,

a glycolipid can be connected to the C-terminus of a protein during post-translation modification.

Since the biological role of oligosaccharides depends on the linkage, branching,

configuration and saccharide units, being able to distinguish and differentiate the smaller

mono- and disaccharides that compose larger oligosaccharides is very important. Due to the

various linkages (carbons 1-6 of each monosaccharide unit), anomeric configurations (α- or β-)

and monosaccharide units (any of the eight D-hexoses) there is a plethora of possible isomers,

which makes analysis of carbohydrates a very difficult task.

Differentiation of Mono- and Disaccharides

Glycans must be isolated and prepared for analysis. The preparation method can include

releasing the glycans, separating them and then finally analyzing them. Once separated common

methods for analysis have included nuclear magnetic resonance (NMR) and/or mass

spectrometry (MS). Figure 1-8 shows a schematic of the different methods used for separating

and analyzing saccharides.

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Separation of Oligosaccharides

Typically oligosaccharides can be released by several methods, either chemical or

enzymatic. Enzymatic methods use a specific enzyme to pick out a particular substrate from

mixtures.14 Enzymes, for example glycosidase and galactosidase, are used to remove specific

sugar residues sequentially from the non-reducing end. A chemical method for releasing glycans

is an alkali-borohydride treatment which then can be followed by hydrolysis, with the resulting

species then separated by high performance liquid chromatography (HPLC) and/or

gas chromatography (GC).19,20

Once released, the oligosaccharides can then be separated. Methods for determining and

separating mixtures of carbohydrates include thin layer chromatography (TLC), column

chromatography methods (including gas chromatography, liquid chromatography, gas-liquid

chromatography and high performance liquid chromatography) and capillary

electrophoresis (CE).13,14,21

Thin layer chromatography is a relatively cheap and inexpensive method for separating

analytes. Microcrystalline cellulose and silica gel are two typical solid supports. Cellulose

separation occurs by a liquid-liquid partition where the sugar of interest is distributed between

the mobile phase and the cellulose-bound complex in water. The separation occurs based on the

solubility of sugar in the eluent and how easily it can enter the solid support. Cellulose TLC has

the same chromatographic characteristics as paper TLC, but allows for shorter elution time and

increased sensitivity. Silica gel separation is similar to cellulose, but requires an additional

adsorption component, typically an inorganic salt (phosphate, bisulfate). Numerous solvents are

used to separate the various monosaccharides.

High performance liquid chromatography, gas-chromatography (GC) and

gas-liquid chromatography (GLC) can also be used to separate components of mixtures. All of

23

these methods require somewhat expensive equipment. High performance liquid

chromatography is typically preferred for monosaccharide mixtures, oligosaccharide analysis and

purification. Whereas GLC is limited to monosaccharide mixtures only, HPLC requires different

columns and various solvents are used to elute the mixture through the column. Typical columns

include sulfonated polymeric or amino-bonded silica columns. Typical solvents include

acetonitrile/water mobile phase. Gas liquid chromatography is a sensitive technique and allows

the analysis of sub-nanomolar amounts of carbohydrates.14

Capillary electrophoresis is a newer technique that yields results in relatively short times

and with high efficiency. To achieve electrophoretic separation, the two ends of the capillary are

submerged into two separate electrolyte reservoirs that contain a high voltage electrode. The

separation is due to the variation of molecular size and electric charge ratios of the sugars within

the mixture. This method does not require derivatization of the oligosaccharides and cannot be

used to identify and separate oligosaccharides that have the same degree of polymerization,

i.e. isomers.

Derivatization of oligosaccharides allows for them to be more volatile and therefore more

compatible with analysis methods such as mass spectrometry. One common derivative method

is hydrolysis followed by chromatographic separation.22,23 Besides hydrolysis, other common

methods used to derivative oligosaccharides are permethylation 24 and peracetylation.25

Permethylation has been shown to easily determine branching and interglycosidic linkages. It

also helps stabilize sialic acid residues in acidic oligosaccharides and in conjunction with

matrix-assisted laser desorption ionization (MALDI) has been shown to give more predictable

ionization than non-permethylated oligosaccharides.26 Two common methods for

permethylation are the use of dimethyl sulfoxide anion (DMSO-) to remove protons from the

24

analyte and replace them with methyl groups27 and the addition of methyl iodide to DMSO-

which contains powdered sodium hydroxide. This second method effectively replaces protons

with a methyl group at both oxygen and nitrogen sites in oligosaccharides.24

Analysis Methods

Once released and separated, the oligosaccharides can then be analyzed individually. One

past method for differentiation of isolated and separated carbohydrates is NMR spectroscopy.28-30

Over the past 25 years advances in NMR have allowed it to become suitable for structural

analysis of carbohydrates.31 Such advances include improvements in instrumentation, pulse

sequences, ability to interpret spectra, isotopic labeling of compounds and improvement in

molecular modeling. With the advances of technology, the ability and accessibility of these

techniques have become faster, better and more accessible. The improved coupling of molecular

modeling with NMR has provided the ability to determine primary structure and

three-dimensional structures of different biological molecules.31

While NMR has been used to study carbohydrate structures, including glycosidic linkages

of saccharide units, and has developed considerably in recent years, it still has several drawbacks

and areas in need of improvement. First, the sample size required for NMR analysis is relatively

large. Another major drawback is that data analysis can be complicated and time consuming.

Typical 1H NMR spectra can be used to give partial spatial arrangement, but due to the

incomplete separation of the proton resonance signals they cannot provide a lot of structural

information. Other types of NMR have been used in the past to analyze carbohydrates and

include 13C, 15N, 17O, 19F and 31P. The resolution and sensitivity of each method varies and

therefore different information can be ascertained by using each method. For example,

13C-NMR can give the information of the anomeric configuration of the carbohydrate residues.

It can also provide sequence information of the composite monosaccharides, their sequence and

25

the overall conformation of the carbohydrates. Another NMR method that improves the results,

but increases the complexity, of data analysis uses 2D- homonuclear correlation types of spectra

(2D-COSY) to assign resonances and give further structural information. Although these spectra

give more information, they do not provide monosaccharide sequence information because there

is an absence of coupling over the glycosidic linkage. For this, nuclear overhauser enhancement

spectroscopy (NOESY) or rotating-frame overhauser enhancement spectroscopy (ROESY) may

be used. While there is some success with these methods, the linkage is not always identified.31

Since carbohydrates are inherently flexible, in solution carbohydrates may undergo

alternations. Estimation of the solution structure required knowledge of the configuration of the

composing monosaccharides. Flexible motions of the whole molecule on a short time scale

involve fast vibrations at bonds and angles and on a longer time scale involve changes in the

dihedral angles. Therefore changing the relaxation time can help deduce the internal flexibilities

of carbohydrates in solution. As one can see, the data required for this type of analysis are

extensive and analysis can be extremely time-consuming.

Mass spectrometry is another very popular analytical technique that is used for gas-phase

analysis of carbohydrates. Several types of mass spectrometers have been used for analysis,

including Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), which will

be discussed further in chapter 2. Mass spectrometry has been shown to have 3 to 4 orders of

magnitude higher sensitivity than NMR.32 Mass spectrometry is highly sensitivity and can be

used in multi-step approaches to determine structural information. In order for analysis with

mass spectrometry to be done, one of several ionization methods can be used to introduce the

analyte of interest into the mass spectrometer.

26

Mass Spectrometry: Ionization Techniques

Both hard and soft ionization methods exist. Hard ionization methods are ones that result

in fragmentation and degradation of the sample during the ionization process, whereas soft

methods produce little or no fragmentation during the ionization process. One previous hard

ionization method widely applied is electron ionization (EI). In EI a beam of electrons is used to

excite and ionize a volatile analyte. A main drawback of EI is fragmentation of the sample

before detection.33 Soft ionization methods are currently preferred since they result in the

ionization with molecules of the sample remaining intact. Electrospray ionization (ESI) is the

most popular of the soft ionization methods. Several soft ionization techniques have been used

in the past for carbohydrate analysis and include fast atom bombardment (FAB),34-36 MALDI37

and ESI.38,39

In FAB, the analyte is mixed with a liquid matrix and is bombarded under vacuum with a

high energy beam of atoms. Fast atom bombardment results in the release of [M+H]+ or [M-H]-

ions which can then be analyzed.40 Analysis with FAB had several constraints including poor

ionization of neutral and basic oligosaccharides and restriction of analysis to relatively smaller

molecules. While basic oligosaccharides were ionized poorly with FAB, acidic oligosaccharides

produced stronger signals in the negative ion mode.35 When FAB was coupled with FTICR-MS,

extensive fragmentation, including cross-ring cleavages was seen.41,42

While FAB uses a liquid matrix, MALDI uses a crystalline matrix where the analyte of

interest is co-crystallized with the solid matrix molecules. A laser is focused onto the matrix

and its photon energy is absorbed by the matrix and the analyte of interest is released as charged

ions.43 While analysis with traditional MALDI is possible, analysis of smaller saccharide units is

a challenge because most peaks of the typical matrix are present in the range m/z <500, where

peaks due to smaller saccharides such as mono-,di- and trisaccharides are also found. Recently,

27

use of an acid fullerene matrix instead of the traditional matrix has allowed for disaccharides to

be successfully studied with MALDI.44 This approach needs to be developed more fully and

applied to other types of carbohydrates.

Electrospray ionization is the least energetic of these three gentle ionization techniques. A

primary benefit of ESI ionization is the absence of matrix peaks and therefore ease of analysis

for smaller mono-, di- and trisaccharides.33,45,46 In ESI, a solution of the analyte and solvent is

passed through a capillary with a high voltage (2 to 5 kV) applied to it.47 This process allows for

charged droplets to be formed. Once formed these charged droplets can then be transferred

(through differential pumping and ion optics) into a mass spectrometer and analyzed by mass

spectrometry. Electrospray ionization is versatile when it comes to carbohydrates since it can be

used to ionize both basic and acidic oligosaccharides. Since multiply charged ions are formed,

and mass spectrometers typically separate based on mass-to-charge ratio, ESI has virtually no

limit to the size of the ion that can be analyzed. This dissertation will concentrate on ESI since it

was used exclusively in the research to be reported.

Fragmentation Methods

Since isomers have the same mass and therefore cannot be differentiated by mass

spectrometry alone, differences in ion fragmentation can be used to distinguish isomers. To

obtain structural information, several fragmentation methods have been used. These methods

include electron capture dissociation (ECD), collision induced dissociation (CID) and infrared

multiple photon dissociation (IRMPD).

Electron capture dissociation uses low energy electrons to induce fragmentation of the

saccharide.6 It results in multiply, positively charged ions that can then be analyzed with a mass

spectrometer. Past research has included using ECD to do top-down analysis where a whole

protein is sequenced simultaneously. Also, O-glycosylation sites on proteins have been explored

28

using ECD.48 While ECD can be used for proteins and peptides, it has limited application to

oligosaccharides.

Another dissociation technique that is more applicable to oligosaccharides is CID. In

on-resonance or traditional CID, a neutral background gas is pulsed into the cell, analyte ions are

accelerated to higher kinetic energies, and collide with the introduced gas. These collisions

result in fragmentation of the analyte of interest.49 Another commonly used CID method in

FTICR-MS is sustained off-resonance irradiation collision induced dissociation (SORI-CID).50

In SORI-CID, ions are excited by an off-resonance frequency, causing their kinetic energies to

increase and decrease repeatedly with time, resulting in less-energetic collisions with background

molecules over a longer time period than with conventional CID. These collisions can

nonetheless result in fragmentation of an isolated ion of interest. Collision induced dissociation

of oligosaccharides results in fragments that can be used to determine stereochemistries, linkage

position and branching information.34,51,52 A disadvantage to SORI-CID with respect to

identification of oligosaccharide is that since SORI-CID is low energy, cross-ring fragmentations

are less likely than the fragmentation of the glycosidic linkage. Also, the ability to control the

energies of collisions is limited with CID. Due to the collisions and variance of energy, CID can

give different fragmentation than other dissociation methods.

One fragmentation method that gives similar and complementary fragments to CID, but

allows for finer control of the energy imparted to the system is IRMPD.53 IRMPD relies on

absorption of photons by one vibrational normal mode of trapped ions and the subsequent

redistribution of photon energy into other vibrational modes of the ions. This redistribution

occurs via intramolecular vibrational relaxation.54,55 If sufficient photons are absorbed without

excessive collisional or radiative relaxation, then the internal energy of the ion increases to a

29

level above the dissociation threshold, resulting in fragmentation. One advantage of IRMPD

over CID is that the power is only limited by the laser being used. Therefore, use of a tunable

laser gives finer control over the power imparted into the system. The theory and history of

IRMPD will be discussed in more detail in chapter 3.

A systematic nomenclature method has been developed for naming fragments of

carbohydrate ions. In this method, the fragments which contain a non-reducing end sugar are

labeled with uppercase letters sequentially starting with A, Figure 1-9.17 Those fragments that

contain the reducing sugar are labeled sequentially with letters from the end of the alphabet

(X, Y, Z). Ions formed by cleavage across a ring are A and X ions. The subscripts for these

fragments are given by assigning each ring bond a number and then counting clockwise.

Charged Ions

Since mass spectrometry only detects charged particles, metal ions have become a

common way to ionize neutrals and then detect the complexes formed with mass spectrometry.

Adduction of an alkali metal ion has been used with FAB, MALDI and ESI in both the positive

and negative ion mode.56-61

For fragmentation of a metal-attached ions two pathways predominate. The first type of

fragmentation is loss of the metal ion and the second type is fragmentation of the molecule into

smaller charged parts which often retain the metal ion. The fragmentation pathway that occurs

depends on the strength of the bonds of the adduction of the metal to the molecule, Figure 1-10.

If the binding energy of the metal ion is less than the dissociation threshold, then loss of the

metal will occur. This type of fragmentation is seen when large alkali metals are adducted to

molecules. This is because the binding energy of the larger alkali metals ions is lower than that

of smaller alkali metal ions.58 The opposite has been seen with the smaller alkali metal ions.

Since their binding energies are larger and thus metal ion dissociation is less likely, the result is

30

greater fragmentation of the molecules with the smaller alkali metal ions remaining attached to

the fragments. Cancilla et al. found that the relative binding energy for alkali metal ions is

Li+>Na+>K+>Rb+>Cs+.58 The stronger the binding energy, the more fragmentation that will be

seen with IRMPD since it is more likely the molecule will fragment before losing the metal.6

Xie et al. have compared the ability of CID and IRMPD to fragment alkali-adducted molecules

and showed that for smaller ions such as Li+ and Na+ both dissociation method yielded similar

fragments.62

Specifically, adduction of lithium to saccharides has been studied by Hofmeister et al.60 In

this research they determined that the lithium cation interacts with disaccharides through several

oxygen sites, including the glycosidic bond. This triple interaction leads to stronger binding and

therefore greater fragmentation is seen with IRMPD. The research performed in this dissertation

primarily used adduction of lithium ions and analysis in the positive ion mode.

In the negative ion mode, Cole & Zhu have shown that chlorinated species can be studied

conveniently.61 Formation of the chlorine adduct has proven successful for species that are

polar, neutral molecules or slightly acidic molecules that do not generate negative ions through

deprotonation. Therefore, chlorination has been shown to be one easy method for exploring ions

in the negative mode when addition of a strong base does not promote deprotonation.

While the addition of an appropriate salt can help facilitate the ESI process through

producing charged adducts, excessively high salt concentrations can cause background

interferences; therefore caution needs to be taken when using salts for the creation of ions.

These interferences can lead to signal suppression and the subsequent inability to detect the ions

of interest. The ease of the adduction of metals to create ions with oligosaccharides makes their

31

use with IRMPD a promising method to differentiate the sugars in both positive and negative ion

modes.

Objective of This Research

Since carbohydrates are biologically important, being able to differentiate both their

linkages and anomeric configurations can give valuable information. For this research,

FTICR-MS was used in conjunction with IRMPD to distinguish various mono- and disaccharide

ions in both the positive and negative ion mode. Fourier transform ion cyclotron resonance mass

spectrometry not only gives superior mass resolution and mass accuracy when compared to other

types of mass spectrometry, but it also allows for tandem mass spectrometric experiments to be

done in the same region of space (within the analyzer cell), thereby eliminating extra

instrumentation that is often needed with other mass spectrometers.63,64

Since IRMPD uses lasers to introduce photons, various lasers have been used in the past

including fixed frequency and wavelength-tunable CO2 lasers65-69 and free electron lasers

(FELs).55,70-72 Fixed frequency CO2 lasers produce photons at one wavelength (10.6 μm), thus

the information that can be obtained with them is limited. Free electron lasers, on the other hand,

have a large output wavelength range (5 to 250 μm) but these lasers are very expensive and

access to beam time is limited. Therefore, a less expensive alternative with at least a (limited)

range of wavelengths (9.2 to 10.6 μm) is the tunable CO2 laser that will be emphasized in this

research.

The objective of this research was to produce a method for discriminating between various

linked and anomeric configurations of mono- and disaccharides. While previous research done

by Polfer et al. with irradiation produced by a FEL had shown that the linkages and anomeric

configurations could be distinguished by wavelength-dependent ion fragmentation patterns, a

32

method to do so in more conventional (i.e. non-FEL equipped) laboratories had not been

demonstrated.73,74

In this research the anomeric configuration of mono- and disaccharides was determined by

examining the fragmentation patterns produced by IRMPD with a tunable CO2 laser in both the

positive and negative ion modes using FTICR-MS. While past methods have studied the

lithiated disaccharides in the positive ion mode with FEL irradiation, the negative mode of

mono- and disaccharides has not been explored. Therefore the fragmentation of

glucose-containing disaccharides, some of their specific fragment ions and some selected

monosaccharides was also examined in the negative ion mode at the Free Electron Laser for

Infrared eXperiments (FELIX) facility.

Overview

The next chapter will give a description of FTICR-MS. This description will include a

history as well as theoretical and practical aspects of FTICR-MS. Chapter 3 will discuss the

mechanism and theory of IRMPD. The types of lasers used for IRMPD will also be described in

this chapter. Chapter 4 is a detailed description of the procedure and apparatus used to

differentiate lithiated monosaccharides with a tunable CO2 laser at the University of Florida in

Dr. John Eyler’s laboratory. The results of this study will also be discussed. Chapter 5 will

discuss a method to determine both the linkage and anomeric configuration of lithiated glucose-

containing disaccharides in the positive ion mode with a CO2 laser. Chapter 6 will next describe

IRMPD fragmentation of deprotonated and chlorinated disaccharides in the negative ion mode

by wavelength-tunable CO2 laser. A description of the procedure and apparatus used for the

fragmentation of deprotonated disaccharides done at the University of Florida in Dr. David

Powell’s laboratory will also be given. Chapter 7 will give a detailed account of negative mono-

and disaccharides ions and some of their fragment ions explored at the FELIX facility. Finally, a

33

conclusion including a summary of the strengths and weaknesses of this work along with

proposed future work will be presented.

34

CHO

OHH

HHO

OHH

OHH

CH2OH

D-glucose

CHO

HHO

OHH

HHO

HHO

CH2OH

L-glucose

Figure 1-1. Fischer projection for D- and L-glucose.

O

H

HO

H

HO

H

OH

OHHH

OH

Alpha-D-glucose

1

23

45

6

Figure 1-2. Example of the numbering system for the carbons of monosaccharides. The carbons are numbered sequentially beginning with the anomeric (chiral) carbon.

CHO

OHH

OHH

OHH

OHH

CH2OH

CHO

HHO

OHH

OHH

OHH

CH2OH

CHO

OHH

HHO

OHH

OHH

CH2OH

CHO

HHO

HHO

OHH

OHH

CH2OH

CHO

OHH

OHH

HHO

OHH

CH2OH

CHO

HHO

OHH

HHO

OHH

CH2OH

CHO

OHH

HHO

HHO

OHH

CH2OH

CHO

HHO

HHO

HHO

OHH

CH2OH

D-Allose D-Altrose D-Glucose D-Mannose D-Gulose D-Idose D-Galactose D-Talose

Figure 1-3. Fischer projections for the D-hexoses of the aldose family. Isomers that vary in only one position are called epimers.

35

O

H

HO

H

HO

H

OH

OHHH

OH

O

H

HO

H

HO

H

H

OHHOH

OHA B

Figure 1-4. Anomers of D-glucose. A) Structure of the α-anomer of glucose, where the hydroxyl group on the anomeric carbon is in the axial position. B) Structure of the β-anomer of glucose, where the hydroxyl group on the anomeric carbon is in the equatorial position.

O

H

HO

H

HO

H

OH

OHHH

OH

O

CH

H

HO

H

HO

H

OHH

OHH

O

O

H

HO

H

HO

H

H

OHHOH

OH

OH

H

H

H OH

HO H

O

H

HOHO

H

OH

H

H OH

HO H

O

H

HOHO

O

H

H OH

HOHCH

O

H

HOHO

H

Figure 1-5. Inter-conversion of the ring structures for the 6-membered ring, pyranose, and the 5-membered ring, furanose, of D-glucose. Once the cyclic ring of the α-glucose opens, a nucleophilic attack results in the closing of the ring in the β-position.

36

O

H

HO

H

HO

H

OOHH

H

OH

O

H

H

HO

H

OHH

OH

O

H

HO

H

HO

H

H

OHH

OH

O

H

O

H

HO

H

OHH

OH

1

4

H,OH

H,OH

1 4

A

B

Non-reducing end

Reducing end

Non-reducing end Reducing end

Figure 1-6. Examples of disaccharides composed of two glucose (Glc) monosaccharides. A) Structure of maltose (Glcα1-4Glc) with an α-link between carbon 1 of the non-reducing sugar and carbon 4 of the reducing sugar. B) Structure of cellobiose (Glcβ1-4Glc) with a β-link between carbon 1 of the non-reducing sugar and carbon 4 of the reducing sugar.

NH

OH

OH

OH

OH

OO

N-acetyl glucosamine

NH

OH

OH

OH

OH

OO

n-acetyl galactosamine

A

B

Figure 1-7. Structures of two common oligosaccharide derivatives. A) Structure of N-acetylglucosamine. B) Structure of N-acetyl galactosamine. These derivatives are found linked to proteins and are biologically important.

37

Glycoconjugate

Chemical or enzymatic method

Released glycans

Separated glycans

Purification methods:-Gel filtration-Chromatography-Capillary electrophoresis

Derivatization:Methylation with CH3I and a strong base

Derivatization:Hydrolysis with a strong acid

DerivatizationHydrolysis with enzymes

Direct analysis:NMR and MS

Methylated saccharides Monosaccharides Smaller glycans

-Sequence-Glycosidic bond

conformation

-Enzymes-Methylation

-Sequence-Position-Glycosidic bond

conformation

-Type-Amount

-Glycosidic bond conformation

-CE-TLC-HPLC

Glycoconjugate

Chemical or enzymatic method

Released glycans

Separated glycans

Purification methods:-Gel filtration-Chromatography-Capillary electrophoresis

Derivatization:Methylation with CH3I and a strong base

Derivatization:Hydrolysis with a strong acid

DerivatizationHydrolysis with enzymes

Direct analysis:NMR and MS

Methylated saccharides Monosaccharides Smaller glycans

-Sequence-Glycosidic bond

conformation

-Enzymes-Methylation

-Sequence-Position-Glycosidic bond

conformation

-Type-Amount

-Glycosidic bond conformation

-CE-TLC-HPLC

Figure 1-8. Typical steps for analysis of glycans. Figure adapted from Valle, J. J. Ph.D., University of Florida, Gainesville, 2005.74

38

O

OHO

OH

OH

CH2OH

O

O

OH

OH

CH2OH

O

O

OH

OH

CH2OH

R

Y2

B1

Z2

C1

Non-reducing end Reducing-end

Y1

B2

Z1

C2

Y0

B3

Z0

C3

0,2A1

1,5X1

Figure 1-9. Fragmentation nomenclature for oligosaccharides. Figure adapted from Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227.17

E2

∆ E

E1

A+ M+

A1+ [A2+M]+

∆ E= E1-E2

M=Li, Na where E1>E2

A.

E1

∆ E

E2 A+ M+

A1+ [A2+M]+

∆ E= E2-E1

B.

M= K, Rb,Cs where E2>E1

E2

∆ E

E1

A+ M+

A1+ [A2+M]+

∆ E= E1-E2

M=Li, Na where E1>E2

A.

E1

∆ E

E2 A+ M+

A1+ [A2+M]+

∆ E= E2-E1

B.

M= K, Rb,Cs where E2>E1

Figure 1-10. Possible fragmentation pathways for fragmentation by IRMPD. A) Fragmentation pathway for smaller alkali ions. B) Fragmentation pathway for larger alkali ions. Figure adapted from Park, Y.; Lebrilla, C. B. Mass Spectrom. Rev. 2005, 24, 232-264.6

39

CHAPTER 2 FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is a powerful

analytical technique with a plethora of research applications. This chapter will discuss the

history of the technique, the apparatus used for it and examples of research done with

FTICR-MS.

History

Today’s current research with FTICR-MS first became possible with the invention of E.O.

Lawrence’s cyclotron in the 1930’s.75 Lawrence’s cyclotron accelerator was used to bombard

target compounds with ions of various masses. In 1932, Lawrence et al. demonstrated that an

ion moving perpendicular to an uniform magnetic field is restricted to circular, cyclotron motion

with an angular frequency given by the following equation:76

m

qBc . (2-1)

In Equation 2-1, ωc is the cyclotron frequency, q is the ion’s charge, B (in Tesla) is the magnetic

field strength and m is the mass of the ion. This motion is independent of the particle’s orbital

radius. The direction of this motion depends on the charge of the ion, with positive ions rotating

in one direction and negative ions in the opposite direction, Figure 2-1.

This theory was incorporated into Sommer, Thomas and Hipple’s Omegatron in the

1950’s,77 which was later developed into other instruments that were used to study ion-molecule

reactions.78 Then in the 1970’s, Comisarow and Marshall introduced Fourier transform methods

into ion cyclotron resonance (ICR) mass spectrometry to build the first Fourier transform mass

spectrometer (FTMS).79,80 The number of Fourier transform mass spectrometers and

40

applications using them has been increasing since the initial demonstration of the technique in

the 1970’s.

Apparatus

While several types of FTICR-MS instruments are available, all have the same general

components.78 These include the magnet, vacuum system, analyzer cell and a data system. A

schematic diagram of the components of a 4.7 T FTICR-MS system (minus the data system) is

shown in Figure 2-2.

Magnet

The first component is the magnet, most commonly either an electromagnet or a

superconducting magnet. Magnetic field strengths of electromagnets are below 3.0 T, normally

around 1.5 T. Superconducting magnets are generally available in field strengths of 3.0 to 9.4 T,

but higher field strengths such as 20 T have been used in FTMS instruments.78 As magnetic field

strength increases, both the mass resolving power and highest non-coalesced mass increase

(Figure 2-3). Therefore, as magnetic strength increases the ability to study higher masses with

more resolving power is possible. Also, with stronger magnetic fields, longer ion trapping times

are possible. Since the capabilities of the mass spectrometer increase with magnetic field

strength, typical mass spectrometers are designed using the strongest magnet available or

affordable.

Vacuum System

To avoid collisions of the analyte with other molecules in the cell, low pressures are

needed for optimal ion excitation and detection. For best results, background pressures in the

10-9 to 10-10 Torr range are typically used. In order to achieve these low pressures, a pumping

system is needed. Generally such a system will use mechanical pumps for rough pumping and

turbo-molecular pumps to achieve the low pressures needed for FTICR-MS. To allow the

41

coupling of ambient ionization techniques, there is normally a region where higher pressures are

pumped down by differential or rough pumps near the source. Normally a gate valve separates

the source region from the high vacuum region. Optics are used to guide ions from a high

pressure region (10-5-10-6 Torr) to a lower pressure region, as seen in Figure 2-2. Fourier

transform ion cyclotron resonance mass spectrometers often use valves to permit pulses of gas

(or air) to be leaked into the cell, allowing fragmentation methods like collision induced

dissociation (CID) to be performed.

Analyzer Cell

An analyzer cell is the next part of the instrumentation. Ions are stored, mass analyzed and

detected in the cell. The analyzer cell is where ions can also be isolated and fragmented in

tandem mass spectrometry (MSn). Since the cell is the heart of the mass spectrometer, having

the most efficient design is desired. While a number of designs have been proposed over the

years,64 two typically used cells are of cubic and cylindrical geometry (Figure 2-4). These cells

are both composed of six electrode plates, which perform one of three functions when voltages

are applied to them. The first type of plate, the trapping plate, holds ions in the cell in the

direction parallel to the magnetic field. The second type of plate, the excitation plate, excites the

trapped ions to larger radii. The last type of plate, the detection plate, detects the excited ions.

In cubic cells, the trapping plates sometime have small openings that allow externally produced

ions to enter the cell, where they can then be excited and detected. Cylindrical cells are typically

preferred since they are larger, conform more closely to the geometry of superconducting

magnets (with cylindrical bores) and therefore can hold more ions

Data System

The next component is the data system. The data system takes the signal induced by

excited ions on the detection plates and transforms it into useable information. The

42

instrumentation in this component includes a frequency synthesizer that generates the

frequencies used for exciting the ions, a delay pulse generator, broadband radio frequency

amplifier, fast transient digitizer and computer system.78 A computer coordinates all of the

electrical devices needed for the experimental process. User-friendly interfaces allow for ease in

use of the system by operators. With these interfaces, tandem mass spectrometry experiments

can be performed simply by changing and/or adding events in the experimental sequence. It

goes without saying that as technology improves the capability and ease of FTICR-MS

instruments will also improve.

Theory

Cyclotron Motion

The force of the magnetic field on an ion causes it to move in a circular orbit. As an ion

with a charge (q) moves in a magnetic field (B) and electric field (E), the Lorentz force causes

the ion to move in a circular orbit in a plane perpendicular to direction of the magnetic field

(Equation 2-2). It should be noted that the Lorentz force is dependent on the mass and velocity

of the ion.

BvqEqdt

vdmonacceleratimassForce (2-2)

The Lorentz force must be equal to the centrifugal force for the ion to experience circular

motion. The velocity of the ion in the x-y plane, a plane that is perpendicular to the magnetic

field (B), is denoted by vxy and the angular acceleration (dvxy/dt) is expressed as v2xy/r. In the

absence of an electric field, Equation 2-2 then becomes the following:

r

vmBqv xy

xy

2

. (2-3)

Rearranging Equation 2-3 and solving for r gives the radius of the cyclotron motion as:

43

qB

mvr xy

c . (2-4)

Substituting in the angular velocity (in rad/s) as ω= vxy/r, Equation 2-4 becomes:

mω2r = qBωr. (2-5)

Rearranging Equation 2-5 gives the cyclotron frequency, Equation 2-1:

m

qBc . (2-1)

Since ω=2π/t=2πf, the linear cyclotron frequency can therefore be given by:

m

qBf c

c

22 . (2-6)

For example, at 4.7 T a singly charged ion of m/q 349 will have a cyclotron frequency of

209.8 kHz:

kHz 8.20910673.13492

7.410602.1127

19

kguu

TC

.

Equation 2-6 shows that the cyclotron frequency is dictated only by the magnetic field

strength, the charge of the ion and the mass of the ion. This means that the cyclotron frequency

is independent of the ion’s velocity and therefore independent of the ion’s kinetic energy. Since

the frequency is independent of the velocity and kinetic energy, all ions with the same m/q ratio

will have the same cyclotron frequency. Using Equation 2-1 and Equations 2-3 to 2-6, the

typical frequencies calculated are from the kilohertz (kHz) to the megahertz (MHz) range. These

frequencies are detectable by most commercially available instrument electronics.64,78

Trapping Motion

The presence of a uniform magnetic field in the z-direction allows for unrestricted motion

along the z-axis and confines the motion of ion in the x-y plane. To prevent ions from escaping

along the z-axis, a trapping voltage, Vtrap, can be applied to the end-cap electrodes of the cell.

44

This trapping voltage leads to a three-dimensional quadrupolar potential, in the cell, in the

form:64,81,82

22

22

2rz

aVtrap(r,z)

. (2-8)

In Equation 2-8, Vtrap is the trapping voltage, r is the radial position of the ion in the x-y plane

and equals 22 yx , a is a measure of the trap size and γ and α are trap shape dependent

constants. Equation 2-8 can used to solve in terms of the z-motion of the ion, giving:

),,(2

2

zyxqdt

zdmFaxial (2-9)

Solving Equation 2-9, gives:

)2cos()0()( tvztz z . (2-10)

An ion at a particular z-position will oscillate with a given frequency that be found by

Equation 2-11:

23 10 x 2.21088

ma

zVv trap

z

. (2-11)

In Equation 2-10, vz is in Hz, Vtrap is in volts, a is in meters, m is in atomic mass units and z is in

multiples of elementary charge.

Magnetron Motion

Combination of the electric and magnetic fields creates a three-dimensional trapping

potential that allows ions to be stored and analyzed for extensive intervals of time (seconds).

Although the cyclotron and trapping motions are not coupled, their motions combine to induce a

third type of motion: magnetron motion. The trapping potential of Equation 2-8 also produces a

radial force with the equation:

45

ra

qVqEF trap

rradial 2)(

. (2-12)

This radial force acts upon the ions in an outward direction that opposes the inward Lorentz force

of the magnetic field. An equation related to the motion of an ion that is subjected to a static

magnetic field and three-dimensional axial quadrupolar potential is given when Equation 2-1 and

2-11 are combined to give Equation 2-13:

ra

qVrqBrmF trap

22

. (2-13)

Solving this quadratic equation for zero gives:

02

2 ma

qV

m

qB trap . (2-14)

The absence of the radius, r, in Equation 2-14, indicates that ω is independent of the radius.

Therefore, each ion motion frequency is independent of the ion position within the ion trap.

Solving Equation 2-14 for ω yields two natural rotational frequencies. The first frequency, ω+, is

given in Equation 2-15. This is the perturbed cyclotron frequency that is observed in the

presence of a direct current (d.c.) trapping potential. The second frequency, ω-, is shown in

Equation 2-16. This is the magnetron frequency which is a circular motion that is superimposed

onto the cyclotron motion.

222

22

zcc (2-15)

222

22

zcc (2-16)

The cyclotron frequency is far greater than both the magnetron and trapping frequencies.

Therefore, only the cyclotron frequency is used for ion detection.64,78

46

Basic FTICR-MS Operation and Data Acquisition

Due to the design of an FTICR mass spectrometer, the various experimental events occur

in the same region of space, namely the analyzer cell. A typical event sequence can be seen in

Figure 2-5. The basic events of a typical experiment are: ionization, delays, excitation, detection

and quenching ions from the cell.

The first step of the experimental sequence is quenching. Quenching empties the analyzer

cell of any ions that may have been present from previous experiments. These ions are typically

ejected along the z-axis of the cell by changing or removing voltages on the trapping plate.

Usually a quench pulse of about 1 millisecond gives ample time to empty the cell of all

unwanted ions.

The next step in the experimental sequence is ionization in which gas-phase ions are

produced. Ions can either be formed internally in or externally to the cell. Externally made ions

have to be transferred into the cell for analysis through the use of ion optics. Once inside the

cell, the ions are constrained to motion in the x-y plane by the magnetic field and are trapped

along the z-axis by a voltage (typically 0.5 to 5 V) that is applied to the trapping electrodes.

Both positive and negative ions can be trapped and analyzed within the ICR cell by simply

changing the polarity of the voltages applied to the trapping electrodes. Also, ions of a large m/z

range can be trapped in the cell, all of which oscillate at their own particular frequencies as

determined by Equation 2-6.

After ionization, a series of delays usually follow in the experimental sequence. Such

delays allow time for ion injection, ejection of unwanted ions and reaction of trapped ions with

neutral species or irradiation by laser sources. Thus, during these delays the ions can be

subjected to tandem mass spectrometry techniques such as introducing collision gases (for CID),

47

laser pulses (for IRMPD) or electrons (for electron capture dissociation) into the analyzer

cell.64,83,84

Excitation of the ions into larger, detectable radii is the next event in the experimental

sequence. In order to detect a wide m/z range of ions, a swept frequency approach can be used in

excitation. For this, a wide range of frequencies is applied sequentially to the excitation

electrodes. These frequencies create a short, high intensity, broadband radio frequency signal

also know as a chirp. When the frequency applied matches the cyclotron resonance frequency of

an ion, the ion absorbs energy and this results in the acceleration of the ion into a larger orbit.

Ions of the same mass to charge, once excited, move together in ion packets. The excitation

event is brief since if the ions are excited too much, their radii will become too large, causing

them to impact the analyzer cell walls and thus resulting in their loss.

Use of stored waveform inverse Fourier transform (SWIFT)85 or chirp excitation allows for

the undesired ions to be ejected from the cell while permitting the desired ions to remain in the

cell for detection. While a SWIFT uses a calculated and then synthesized waveform and a chirp

uses a high voltage swept r.f. signal, they both excite unwanted ions into an orbital radius that is

larger than the cell radius, causing ion-wall collision of these undesired ions.64,78 This effectively

eliminates the unwanted ions.

Since a chirp is a high voltage, short duration event, all the ions are both excited and

detected almost simultaneously. For example, a frequency synthesizer can sweep over

frequencies from 100 kHz to 10 MHz in roughly 1 millisecond. This sweep excites all the ions

with cyclotron frequencies in that range. The resulting time domain spectrum (Figure 2-6 A) is

very complex. To produce the mass spectrum, the time domain signal is mathematically

analyzed using a Fourier transform algorithm. This generates a frequency domain spectrum

48

(Figure 2-6 B), with all the individual ion frequencies being present. A calibration formula

derived from the cyclotron frequency equation allows the frequency domain to be easily

transformed into a mass spectrum (Figure 2-6 C). Since this approach excites and detects all

frequencies simultaneously, the acquisition time needed is far less than that of classical ICR in

which only one frequency at a time could be detected and analyzed. The time it takes to perform

an FT on the data is only hindered by the technology available; as the speed of computers

increases, so does the ability to do FT.

Once excited, the ion packets create an alternating current (image current) in the detection

plates, where the amplitude is related to the number of charges in the cell. This image current

gives FTICR-MS the unique ability to detect ions without destroying them. While FTICR-MS

uses the image current, all other mass spectrometers, excluding orbitraps, detect ions in a

destructive manner. Since the ions are not destroyed during detection in FTICR-MS, they

remain in the cell and can be re-measured and reacted further without having to produce more

ions. Also, since multiple frequencies are applied during the excitation step, FTICR-MS can be

used to detect ions of many different masses simultaneously. This also allows FTICR-MS to

have increased sensitivity and resolution.

The entire sequence can then be repeated as many times as wanted or needed. The scans

that are collected can be signal averaged. Signal averaging leads to spectra with better signal to

noise (S/N) ratios and improves the quality of the collected spectra. Other events can be added

into the sequence to allow for tandem methods such as CID or IRMPD to be performed on the

ions within the cell. The actual experimental sequence and length can vary depending on the

experiment.

49

Mass Resolution

One major advantage of FTICR-MS instruments is their superior mass resolution when

compared to other mass spectrometers. Mass resolution (m2-m1 ≥ Δm50% where m1 and m2 are the

closest masses that can be resolved) is defined as the point where one valley begins to appear

between peaks of equal shape and height and is separated by Δm50%.64 Both high mass resolving

power and high mass resolution can significantly improve the quality of the experimental data

obtained. As mass resolving power increases, the maximum number of components in a mixture

that can be resolved also increases. Therefore, it may be possible to distinguish and differentiate

different chemical components in a mixture without prior separation. Another advantage is that

high resolution can decrease peak width, thereby giving a more accurate mass determination.

Fourier transform ion cyclotron resonance mass spectrometry is capable of giving the highest

mass resolving power and highest mass accuracy (for all ions up to m/z 5000) of all mass

spectrometry methods.

High mass resolution requires that a long time domain signal, also known as the transient

response signal, be acquired. The mass resolution increases in direct proportion to the length of

the transient recorded. The number of data points collected during the experiment is set by the

user before the transient is collected. Figure 2-7 shows that as the number of data points

increases, the peak widths decrease and the resolution of the mass spectrum increases. However,

the number of data points that can be processed from a transient is limited. Thus far,

approximately 106 data points can be processed using commercially available data analysis

programs. The higher the number of data points, the more computer memory is needed.

Therefore as technology advances, larger numbers of data points can be taken. The number of

data points required for a desired transient length can be calculated by Equation 2-16,

50

S

NTacq . (2-16)

In Equation 2-16, Tacq is the transient duration, S is the sampling rate and N is the number of data

points collected. The transient collection rate is based on the sampling frequency used.

According to the Nyquist theorem, the sampling frequency must be at least twice the highest

frequency (determined by the lowest m/z) being recorded. Based on the number of points

collected, the maximum resolution that can be achieved is determined by:

2acqcTf

R . (2-17)

In Equation 2-17, R is the resolving power, fc is the cyclotron frequency and Tacq is the duration

of the transient. As seen in Figures 2-6 and 2-7, the transient signal decays over time. This

occurs as the collisions between ions and neutrals destroy the coherent ion packet within the

analyzer cell. Therefore, to reduce the possibility of collisions within the cell, all FTICR-MS

experiments are carried out in ultra-high vacuum.

Another aspect that can affect the resolution is space charging. Space charging is due to

repulsions between ions having similar charge. It is a consequence of Coulomb’s law and can be

described by the following equation:

)(2

'

r

qqkF . (2-18)

In Equation 2-18, F is the force between the two ions, k is a proportionality constant, q and q' are

the ion charges and r is the distance between the two ions. Space charging can affect mass

measurement accuracy and sensitivity.78,86-88 The greater the force, the more space charging,

thereby resulting in a decrease in resolution. Reducing the number of ions held within the cell or

introducing an internal calibrant can reduce the effect of space charging.89-91

51

Tandem Mass Spectrometry

One distinct advantage of FTICR-MS is the ability to perform multiple (tandem) mass

spectrometry (MS) experiments. For tandem MS, the precursor ion is excited and then

dissociated. The resulting product ions are then detected and analyzed. Tandem MS allows

more information than just the precursor mass spectrum of an ion to be obtained. For example,

isomers with the same mass can be identified by ratios of the relative percent abundances of

product ions.92-94 Since more steps are involved, tandem MS experiments are inherently more

complex than regular mass spectrometric experiments. Current software allows tandem

experiments to be performed by simply adding additional steps into the experimental sequence.

Unlike tandem MS performed on magnetic sectors or quadruple mass spectrometers, where

additional mass analyzers are needed for each additional step, FTICR-MS only needs additional

steps added to the experimental sequence. The experimental sequence can also be altered to

include isolation steps for the product ions. Once isolated, both the precursor and/or the product

ions, can be dissociated by either collision induced dissociation (CID), irradiation with a laser or

by electron impact (EI).64,78,95,96

Dissociation Techniques

Several dissociation techniques are employed in tandem mass spectrometry. These

methods include CID, surface induced dissociation (SID),97 electron capture dissociation

(ECD)98,99, ultraviolet photodissociation (UVPD)100, blackbody infrared dissociation (BIRD)101

and infrared multiple-photon dissociation (IRMPD).95,102

One of the most popular dissociation techniques for biological molecules is dissociation by

collision. This method involves the trapping and reaction of ions in the analyzer cell prior to

dissociation. Application of an excitation pulse ejects all the ions of higher and lower masses

than the previously selected and isolated precursor ion from the analyzer cell. Ejection of the

52

unwanted ions can also be configured to involve exciting the precursor ion into a larger radius

orbit and thus increasing the kinetic energy of the ion.78,103 The relationship between the kinetic

energy and the radius is shown in Equation 2-19.

m

rBqE

2

222

(2-19)

In Equation 2-19, E is the kinetic energy of the ion, q is the charge of the ion, B is the magnetic

field strength, r is the radius of the ion’s orbit and m is the mass of the ion. This mass selected

and kinetically energized ion undergoes collisions with a background gas or a neutral gas

(typically Ar) that is pulsed into the cell by a pulsed valve.49,104 As long as the pulsed gas does

not increase the pressure in the cell too much, the ions are retained and detected.

One disadvantage of traditional CID is that the product ions are formed away from the

center of the analyzer cell. The farther the ions are from the center of the cell, the more likely it

is that there will be a decrease in detection efficient and resolution. An alternative to traditional

CID is sustained off-resonance irradiation (SORI)-CID which does not have this disadvantage

and is less energetic than traditional CID.50,105,106

Another tandem mass spectrometric method which was used for the research reported in

this dissertation is IRMPD. Traditional IRMPD dissociation uses a fixed wavelength CO2 laser

(10.6 μm) to introduce photons and slowly heat the ions by increasing their vibrational energies,

thus resulting in dissociation of the ions within the analyzer cell. In IRMPD, the photons are

absorbed and their energy is redistributed internally until the dissociation threshold is met or

exceeded, resulting in the fragmentation of the precursor ion. Recently, tunable lasers, including

free electron lasers, have been used to fragment oligosaccharides and other biological

samples.70-73,107

53

Infrared multiple photon dissociation results in similar and/or complementary fragments to

those produced by CID. The benefit of IRMPD over CID is that a gas pulse is not required for

fragmentation. With no gas pulsing, there is no need for extra experimental time to reduce the

pressure in the cell before detection. The ability to manipulate ions and the convenience with

which photons can be delivered into the cell makes coupling IRMPD with FTICR-MS an

advantageous method.

Conclusions

Fourier transform ion cyclotron resonance mass spectrometry has become a very valuable

tool for bioanalytical studies including proteomics and glycobiology. The increased ability and

popularity of FTICR-MS is mainly due to the increased efficiency of and developments in

hardware and software technology. It offers higher mass resolution and mass accuracy that any

other type of mass spectrometry, thereby allowing superior mass assignment. Along with these

benefits, the ability to do tandem mass spectrometry in time rather than space makes FTICR-MS

superior over many other mass spectrometric methods. With improvements in data acquisition

and analysis technology, the power and ease of use of FTICR-MS also increases, making it an

even more valuable mass spectrometric tool for future research.

54

BB

+ -BB

++ --

Figure 2-1. Ion cyclotron motion. The ions move perpendicular to the magnetic field and the cyclotron motion is opposite for opposite charges. Figure adapted from Marshall, A. G.; Hendrickson, C. L. Int. J. Mass Spectrom. 2002, 215, 59-75.84

ZnSeWindow

Infinity Cell

FOCL2

PL9

FOCL1

Gate Valve

HVO

YDFLXDFL

PL4 PL2 PL1

Extract/Trap Plate

Hexapole

Skimmer

Modified HeatedMetal Capillary

Electrospray Tip

Ion Optic Lenses

Turbopump 1Turbopump 2

Atmosphere

Turbopump 3

4.7T Superconducting

Magnet

ZnSeWindow

Infinity Cell

FOCL2

PL9

FOCL1

Gate Valve

HVO

YDFLXDFL

PL4 PL2 PL1

Extract/Trap Plate

Hexapole

Skimmer

Modified HeatedMetal Capillary

Electrospray Tip

Ion Optic Lenses

Turbopump 1Turbopump 2

Atmosphere

Turbopump 3

4.7T Superconducting

Magnet

Figure 2-2. Schematic diagram of the components of a Bruker 4.7 T FTICR mass spectrometer. Shown are the different vacuum pumping regions, the ion optics, the cell and an electrospray source. Figure courtesy of Dr. Michelle Sweeney.

55

7 T9.4T

14.5T

21 T

0 21

-Mass resolving power-Highest non-coalesced mass difference

2107 T

9.4T14.5T

21 T

Magnet strength, B (tesla) Magnet strength, B (tesla)

-Upper mass limit-Number of ions capable of being trapped-Ion trapping period

7 T9.4T

14.5T

21 T

0 21

-Mass resolving power-Highest non-coalesced mass difference

2107 T

9.4T14.5T

21 T

Magnet strength, B (tesla) Magnet strength, B (tesla)

-Upper mass limit-Number of ions capable of being trapped-Ion trapping period

Figure 2-3. Figures of merit for FTICR-MS as a function of magnetic field strength. Adapted from Marshall, A. G.; Hendrickson, C. L.; Emmett, M. R.; Rodgers, R. P.; Blakney, G. T.; Nilsson, C. L. Eur. J. Mass Spectrom. 2007, 13, 57-59.108

Detection

Trapping

Excitation

B

Y

X

Z

Trapping

Detection

Excitation

A B

Detection

Trapping

Excitation

B

Y

X

Z

Trapping

Detection

Excitation

A B

Figure 2-4. Two of the typical analyzer cells used for in FTICR mass spectrometers. A) Schematic of a cubic cell. B) Schematic of a cylindrical cell. Both types of cells have three sets of plates that trap, excite or detect the ions within the cell. Figure adapted from Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35.64

56

Quench Ionization Excitation Detection

Time delays:InjectionEjectionReaction Repeat

Time

Quench Ionization Excitation Detection

Time delays:InjectionEjectionReaction Repeat

Time

Figure 2-5. General schematic of a typical experimental sequence. This sequence can be repeated as many times as needed.

Frequency (kHz)350300250200150100

Ab

un

dan

ce

400

350

300

250

200

150

100

50

Time150100500

Ab

un

dan

ce

0.0090.0080.0060.0050.0040.0030.001

0-0.001-0.003-0.004-0.005-0.006-0.008-0.009-0.01

A

m/z1,000900800700600500400300200

Ab

un

dan

ce

400

350

300

250

200

150

100

50

B

C

Frequency (kHz)350300250200150100

Ab

un

dan

ce

400

350

300

250

200

150

100

50

Time150100500

Ab

un

dan

ce

0.0090.0080.0060.0050.0040.0030.001

0-0.001-0.003-0.004-0.005-0.006-0.008-0.009-0.01

A

Time150100500

Ab

un

dan

ce

0.0090.0080.0060.0050.0040.0030.001

0-0.001-0.003-0.004-0.005-0.006-0.008-0.009-0.01

Time150100500

Ab

un

dan

ce

0.0090.0080.0060.0050.0040.0030.001

0-0.001-0.003-0.004-0.005-0.006-0.008-0.009-0.01

A

m/z1,000900800700600500400300200

Ab

un

dan

ce

400

350

300

250

200

150

100

50

B

C

Figure 2-6. Various domains and spectra obtained from an FTICR-MS experiment. A) The time domain transient response signal. B ) The frequency domain. C) The m/z domain.

57

m/z350349348

Ab

un

dan

ce

650

600

550

500

450

400

350

300

250

200

150

100

50

Frequency (kHz)350349348

Abundan

ce

450

400

350

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250

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50

128 K

Frequency (kHz)350349348

Abundan

ce

250

200

150

100

50

256 K

m/z

m/zTime9080706050403020100

0.005

0.004

0.003

0.001

0

-0.001

-0.003

-0.004

-0.005

-0.006

Time454035302520151050

0.005

0.004

0.003

0.001

0

-0.001

-0.003

-0.004

-0.005

Time150100500

0.0090.0080.0060.0050.0040.0030.001

0-0.001-0.003-0.004-0.005-0.006-0.008-0.009-0.01

m/z

512 K

m/z350349348

Ab

un

dan

ce

650

600

550

500

450

400

350

300

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50

Frequency (kHz)350349348

Abundan

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128 K

Frequency (kHz)350349348

Abundan

ce

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256 K

m/z

m/zTime9080706050403020100

0.005

0.004

0.003

0.001

0

-0.001

-0.003

-0.004

-0.005

-0.006

Time454035302520151050

0.005

0.004

0.003

0.001

0

-0.001

-0.003

-0.004

-0.005

Time150100500

0.0090.0080.0060.0050.0040.0030.001

0-0.001-0.003-0.004-0.005-0.006-0.008-0.009-0.01

m/z

512 K

Figure 2-7. Effect of number of data points acquired and Fourier transform on mass resolution. As the number of points increases, the peak width decreases and the resolution increases.

58

CHAPTER 3 INFRARED MULTIPLE PHOTON DISSOCIATION

Introduction

Several methods have been used to fragment ions in mass spectrometers. One of them is

the absorption of light, which can result in different reactions and fragmentation of ions. Two

types of light that have been used in the past for ion irradiation involve wavelengths in the

ultraviolet/visible (UV/vis) and in the infrared (IR) region of the electromagnetic spectrum.

Absorption of UV/vis light by the ions usually involves promotion to excited electronic states of

the ions, which either by direct dissociation or internal conversion to high vibrational levels of

the ground state gives rise to internal energies above the dissociation threshold of the molecule,

thereby resulting in fragmentation. Another source of light is infrared (IR) radiation, which can

result in fragmentation by inducing step-wise vibrational excitation of the molecule. Infrared

photons are less energetic (typically 0.001 to 1.7 eV/photon) than UV/vis photons (typically 2 to

approximately 8 eV/photon). Therefore, the number of IR photons needed for dissociation of a

molecule is greater than that needed with UV/vis radiation.

The invention of high-power IR lasers enabled IR light to be utilized for ion and neutral

dissociation. In the 1970’s, IR lasers were used to explore the dissociation of trapped

ions.53,109-111 These experiments and the others that shortly followed demonstrated the

phenomenon of infrared multiple photon dissociation (IRMPD) and have been reviewed in detail

by Eyler and Polfer.95,96 Such experiments include dissociation of large biomolecules that were

ionized by electrospray (ESI)45,112 and matrix assisted desorption ionization (MALDI).43,113

This chapter will discuss the mechanism by which IRMPD occurs and the various lasers

that can be used as IRMPD radiation sources.

59

Mechanism of Infrared Multiple Photon Dissociation

A polyatomic molecule or ion that is irradiated with infrared radiation can absorb the light

energy as photons. The energy of the photon is converted to vibrational energy within the

molecule or ion. Since energy is quantized, the energy absorption leads to a transition between

energy levels if the energy of the photon matches the difference between the energy level of the

molecule or ion. Figure 3-1 depicts a typical potential-energy well of a diatomic molecule.

Initially the mechanism of IRMPD was thought to proceed via a ladder-climbing mechanism in

which rotational levels compensated for the unequal spacing of the vibrational levels as one

ascends the energy well. In this mechanism, the addition of each photon sequentially between

energy levels in the potential-energy well caused the total vibrational energy of the ion to

increase as pictured in Figure 3-1. This is not the case and therefore, this step-ladder mechanism

cannot be the mechanism for IRMPD.

The actual mechanism of IRMPD involves the slow, sequential absorption of multiple

infrared photons. Once absorbed, the energy of each photon is internally redistributed until the

dissociation threshold is either met or exceeded, resulting in fragmentation of the molecule of

interest. A schematic view of this process can be seen in Figure 3-2. The first photon is

absorbed by the fundamental of the vibrational mode in question (vi =0 → vi =1). Redistribution

of the photon energy that is absorbed by a normal mode occurs by rapid intramolecular

vibrational relaxation (IVR), which distributes the photon’s energy to an assembly of other

vibrational modes within the molecule.54,114-116 If the IVR is rapid enough (ps to ns time scale),

the absorbing vibrational mode can be de-excited with enough time to allow the absorption of a

sequential photon. This process can be repeated many times until enough internal energy has

been gained by the molecule or ion so that the dissociation threshold is either met or exceeded

and the molecule or ion fragments. As the number of photons absorbed increases, so does the

60

internal energy of the molecule. After a few photons have been absorbed, the internal energy of

the ion is such that it enters a quasi-continuum. In the quasi-continuum, the vibrational levels are

closer together and are indirectly coupled to the absorbing mode, allowing for the absorption

efficiency of the molecule to increase. Once this point is reached, if there is both favorable

absorption strength and laser power then multiple absorptions (10 to 100+ photons) can occur,

leading to higher internal temperatures, thus allowing dissociation to occur more rapidly. This

mechanism has been used to explain the absorption of up to hundreds of photons for polycyclic

aromatic hydrocarbons (PAHs)117,118 and fullerenes.119,120

Lasers Used for IRMPD

R.C. Dunbar first performed an experiment demonstrating the photodissociation of gaseous

ions in an ICR cell in the early 1970’s.121 While these experiments were performed using a slide

projector as the light source and a coarse cutoff filter as the wavelength selector, many

technological advances since then have been incorporated into more complicated experiments

and, in particular, allow IRMPD to be performed quite routinely. One specific advancement is

the development of sophisticated lasers.

The first experiments with IRMPD used high-power continuous wave (cw)-tunable CO2

lasers, but there are several other types of lasers that can be used for irradiation. All lasers

consist of a gain medium that is contained within a highly reflective optical cavity.122

Amplification of the light occurs when photons are passed through the gain medium and

stimulated emission of radiation takes place. It is this amplification from which the laser gets its

power. At one end of the cavity there is a small opening or partially reflective mirror through

which a low percentage of the light can pass creating the output beam of the laser. While the

general idea of a laser is simple, there are several types of lasers each having its own

methodology for creating light. Such laser systems include optical parametric

61

oscillators/amplifiers (OPO/OPA), tunable CO2 lasers and free electron lasers (FELs). The

research reported in this dissertation used both a cw-tunable CO2 laser and a FEL.

The first type of laser used in this research was a continuous wave CO2 laser. Carbon

dioxide lasers use a gas gain medium which contains carbon dioxide, helium, nitrogen and

sometimes a small amount of hydrogen, water vapor and/or xenon.123 Typically, CO2 lasers are

electrically pumped by a gas discharge. Nitrogen molecules are excited by this discharge into a

higher vibrational level. Once excited, the nitrogen molecules transfer their energy to the CO2

molecules that collide with them. The helium in the mixture serves as a depopulator, lowering

the laser power and removing the heat. Non-tunable CO2 lasers typically emit at a wavelength of

10.6 μm and tunable-CO2 lasers have outputs in the region of 9.2 to 10.8 μm, which corresponds

to the stretching frequency of C-O bonds. The laser power available for CO2 lasers can be from

a few watts to several hundred watts. The benefit of CO2 lasers, especially tunable lasers, is that

they are affordable bench-top lasers. The major drawback to tunable-CO2 lasers is that only a

limited wavelength range can be explored.

Free electron lasers are laser systems that not only are large and complex, but also are

expensive.124-126 For this reason, FELs are generally located in national laboratories. The

amplification and wavelength range of FELs is achieved through the use of an undulator. In the

undulator, the placement of magnets with alternating polarities, as seen in Figure 3-3, allows free

electrons to be accelerated, resulting in the release of photons. The spacing of the magnets

within the undulator and the energies of the electrons dictates the wavelength of the photons

being released. The released photons results in coherent light that can then be used in various

ways. The irradiation is composed of 5 to 20 macropulses (composed of hundreds of high-power

micropulses spaced 1 ns apart).127 The macropulses are delivered to the user station at a rate of 5

62

to 10 Hz. While the power of each micropulse is in the MWatt range, each macropulse has an

average energy of ~30 to 50 mJ. The major benefit of FELs is that they allow access to

wavelengths (~5 to 250 μm), that correspond to most of the “chemically interesting” infrared

wavelength range, much wider than a CO2 laser can achieve. The major drawback of FELs is

that they are very costly and access to beam time is limited since they are generally housed in

national facilities with many users applying for beam time.

Several FELs have been coupled with Fourier transform ion cyclotron resonance (FTICR)

mass spectrometers. Examples include the Free Electron Laser for Infrared

eXperiments (FELIX)127 at the FOM-Institute for Plasma Physics Rijnhuizen in The Netherlands,

the Centre Laser Infrarouge Orsay (CLIO)71 facility in Orsay, France and the FEL at the Science

University of Tokyo (SUT).70 The research done in this dissertation with IR photons from a FEL

was performed at FELIX. Figure 3-4 shows a schematic of the laser instrumentation of the

FELIX facility. Two lasers, FEL-1 and FEL-2 give FELIX its wide wavelength range

capability.127 Accelerator 1 allows FEL-1 to access wavelengths from 25 to 250 μm. When the

two accelerators are used in conjunction with each other, FEL-2 can access wavelengths from 5

to 30 μm. The free electrons are accelerated to either 15 to 25 or 25 to 45 MeV by one or two

radio-frequency linear accelerators. An undulator is used, where the positioning of samarium-

cobalt permanent magnets tunes the wavelength of the laser beam. The resonator of the laser is

defined by two gold-plated copper mirrors. The FELIX is a pulsed laser composed of micro and

macro-pulses. The micropulses are spaced 1 ns apart and have a duration of 3 to 6 ps.

Macropulses are possible for a duration up to 20 μs at a rate of 5 Hz or 10 Hz.

All lasers have their benefits and drawbacks. For this dissertation the relatively

inexpensive and technically simple wavelength-tunable CO2 laser was used for a majority of the

63

work. Although access to FELIX beam time is limited, some shifts were available, so some of

the research reported here was also done in The Netherlands. Use of both a FEL and CO2 laser

gave a plethora of information that neither laser alone would have provided.

64

v=0

v=1

v=2

v=3

v=4

v=5

j=1j=0 v=0

v=1

v=2

v=3

v=4

v=5

j=1j=0

Figure 3-1. Energy potential well. As one moves up the well, the spacing of the vibrational levels decreases.

v=0 v=0

v=1

v=2

v=3

v=4

v=5

v=0

v=1

v=2

v=3

v=4

v=5

IVRIVR

Dissociation threshold

IVR

v=2

v=3

v=4

v=5

v=1

v=0 v=0

v=1

v=2

v=3

v=4

v=5

v=0

v=1

v=2

v=3

v=4

v=5

IVRIVR

Dissociation threshold

IVR

v=2

v=3

v=4

v=5

v=1

Figure 3-2. Depiction of the IRMPD mechanism in polyatomic molecules. One photon of IR radiation is absorbed, its energy is then distributed into an array of vibrational modes through IVR. This process is repeated until the dissociation threshold is met or exceeded, resulting in the fragmentation of the molecule.

65

N

N

N

N

N

N

N

S

S

S

S

S

S

S

Electron beam

Released photons

N

N

N

N

N

N

N

S

S

S

S

S

S

S

Electron beam

Released photons

Figure 3-3. Schematic of an undulator used for FELs. Here a beam of free electrons enters the undulator and magnets of alternating polarities forces the electrons to travel in an oscillating path, resulting in the release of photons. The photons combine coherently to give the final beam of light.

Electron Injector Accelerator 1 Accelerator 2

Undulator 1

Undulator 2

FEL 1

FEL 2

Electron Injector Accelerator 1 Accelerator 2

Undulator 1

Undulator 2

FEL 1

FEL 2

Figure 3-4. Layout schematic of FELIX. Two accelerators and FELs are used to give FELIX its continuous wavelength range. Figure adapted from Oepts, D.; van der Meer, A. F. G.; van Amersfoort, P. W. Infrared Phys. Technol. 1995, 36, 297-308.127

66

CHAPTER 4 DIFFERENTIATION OF MONOSACCHARIDES IN THE POSITIVE ION MODE BY

IRMPD WITH A TUNABLE CO2 LASER

Introduction

As described in chapter 1, monosaccharides are the smallest of all the sugar units and play

an important role in biological systems. Past mass spectrometric methods have used soft

ionization techniques such as fast atom bombardment (FAB), electrospray ionization (ESI) and

matrix assisted laser desorption ionization (MALDI) to analyze monosaccharides in the positive

ion mode.128-132 Gaucher and Leary showed that metal ions complexed with various

monosaccharides can be used to differentiate the anomeric configuration of monosaccharides.128

They used electrospray ionization (ESI) and collision induced dissociation (CID) to identify and

differentiate hexoses (glucose and mannose) that were derivatized with

zinc (diethylenetriamine). Other adducts that have been used for the differentiation of anomers

and include copper (II)131, ammonium133, lead134 and sodium135. All these past research methods

have utilized fragments produced by CID to distinguish the identity of the monosaccharides and

derivatives being studied.

Infrared multiple photon dissociation (IRMPD) is another fragmentation method that gives

different, but complementary fragments to those produced by CID. Jose Valle used irradiation

by a free electron laser (FEL) to fragment rubidium- and potassium-attached monosaccharides.

Monitoring the dissociation of the various ions over a range of wavelengths showed differences

in the IRMPD spectra which were used to differentiate positively charged saccharide isomers

and anomers.74

As described in chapter 3, line-tunable CO2 lasers, when compared to FELs, have a limited

wavelength range. Although the wavelength is limited, the cost of a line tunable CO2 laser is far

less than that of a FEL. Therefore, use of a tunable CO2 laser in this work makes the method

67

developed here more affordable and accessible to other laboratories. This chapter will discuss

IRMPD research done at the University of Florida on lithiated O-methyl-gluco- and

galactopyranoside monosaccharides anomers in the positive ion mode using a CO2 laser.

Procedure

Each of the monosaccharides was prepared at a concentration of 0.1 mM in 80:20

general-use grade methanol to MilliQ ultra-pure H2O solution containing 0.1 mM LiCl. The

monosaccharides used in these studies were obtained from Dr. Brad Bendiak at the Department

of Cellular and Structural Biology, University of Colorado Health Sciences Center. Their

structures are seen in Figure 4-1 and include α-O-methyl-glucopyranoside,

β-O-methyl-glucopyranoside, α-O-methyl-galactopyranoside and β-O-methyl-galactopyranoside.

A schematic of the instrumental set-up is shown in Figure 4-2. The lithiated

monosaccharides were ionized with a commercial ESI source (Analytica of Branford, Branford,

CT, USA). The capillary of this source has been user modified136,137 with a conical capillary138

inlet to increase ion introduction into the mass spectrometer. For these experiments, the capillary

was set to temperatures between 120 and 125°C. The flow rate for all experiments was 15 μL/hr.

All experiments were performed on a Bruker 47e Fourier transform ion cyclotron

resonance (FTICR) mass spectrometer (Bruker Daltonics; Billerica, MA, USA) with a 4.7 T

superconducting magnet (Magnex Scientific Ltd.; Abington, UK) and an InfinityTM cell

(Figure 4-2).139 Precursor ions were isolated using a stored waveform inverse Fourier transform

(SWIFT)85 and irradiated for 1 second with a Lasy-20G tunable CO2 laser (Access Laser Co.;

Marysville, WA, USA). This laser has a power range of 0-20 W with a wavelength range of 9.2

to 10.8 μm. Typical laser powers for the experiments described in this chapter were

approximately 0.7 W, but were occasionally as high as 3 W to overcome the effects of slight

laser misalignment.

68

To facilitate fragmentation and increase signal intensity, ions were accumulated in the

hexapole for a period of 1.0-1.5 seconds. This accumulation period was kept constant

throughout all experiments when comparing monosaccharide anomeric pairs. Irradiation with

the CO2 laser was facilitated by a mechanical mirror. When the mechanical mirror was in one

position, the laser beam was blocked and directed into a power meter. When the mirror was in

the other position, the beam was passed into the back of the FTICR cell through a ZnSe window.

No internal mirrors were used, therefore there was only one pass of the laser beam through the

ion cloud within the cell. The wavelengths used were determined based on the stability and

power stated in the laser manual, where wavelengths with excellent stability and high power

were chosen. Each day, two sets of twenty-five scans of 512 K data points were collected and

averaged at each wavelength. All experiments were repeated at least once, several days apart.

Significance of results reported here is based on the 95% confidence interval of the mean.140

Reproducibility

Since variations in the alignment of the laser and the abundance of ions within the cell

change the amount of fragmentation observed, a method for calibrating the overall power of the

laser beam actually irradiating the was needed. To ensure reproducibility, the total number of

ions and irradiation power were kept constant throughout the day. The laser power used daily

was found by determining the power needed to keep a ratio of 1.26 ± 0.04 for the m/z 127 to

m/z 201 fragment ions of lithiated β-O-methyl-glucopyranoside at an irradiation wavelength of

9.588 μm. Once the power needed for this ratio was found, it was used throughout the day. The

power was monitored for each experiment with a power meter and adjustments were made to the

CO2 control electronics as needed to keep the power constant.

Due to slight variations in laser alignment as well as the cell heating during the course of

the day, even with a daily calibration, some variance in the fragmentation was seen. Also, the

69

inherent fluctuation of ion production by the ESI process could have added to the observed

variances. The method of calibration described in the last paragraph was settled upon after

several other methods were tried. Preliminary attempts kept the percent dissociation of the

precursor ion (m/z 201) constant for β-O-methyl-glucopyranoside at 9.588 μm and then used that

laser power for the day. While both methods of calibration resulted in similar ratios for the

abundances of key fragment ions (mainly m/z 109 and m/z 127), using the ratio of m/z 127 to

m/z 201 daily gave a smaller relative error, better reproducibility and finer control over the

energy imparted to the system. The fragment ion at m/z 127 was used for the calibration because

its appearance and disappearance changed significantly when compared to that of the precursor

ion (m/z 201) as a function of laser power.

Results and Discussion

Methyl-glucopyranosides

Anomers of O-methyl-glucopyranoside were first studied. Since monosaccharides can

open up into the chain form and then close again, permitting interconversion between the

different anomers, use of O-methylated monosaccharides insured that the anomer under study

was locked in its closed conformation and therefore could not interconvert. The fragmentation

patterns were obtained by using a CO2 laser to irradiate both the α- and β- anomers of

O-methyl-glucopyranoside. At each wavelength the percent abundance of each fragment was

determined by the following formula:

100AbundancePrecursor AbundanceFragment

AbundanceFragment abundancepercent Relative

. (4-1)

This percent abundance was plotted for each fragment over the range of 9.2 to 10.8 μm as a

function of both mass and wavelength. The major fragments for both anomers were m/z 67, 81,

91, 97, 109, 127, 141, 151 and 169, as seen in Figure 4-3. The fragmentation patterns of the

70

α- and β-anomers proved to be different as seen in Figure 4-3. Specifically, the relative percent

abundance of key fragments such as m/z 91, 109 and 127 appeared to be significantly different

for the anomers. The abundance of m/z 109 was always higher for α-O-methyl-glucopyranoside

than for β-O-methyl-glucopyranoside. Also, the relative percent abundance for m/z 127 was

always significantly higher for the β-anomer than the α-anomer. These fragments are key

identifying fragments and allow for easy discrimination between the two anomers.

The IRMPD spectra of these ions showed that using disappearance of the precursor ion to

distinguish between the α- and β-anomer is impossible. As seen in Figure 4-4, there are several

wavelengths at which the percent abundances of the remaining precursor ion signal overlap for

these anomers. These wavelengths include 9.230, 9.250, 9.305, 9.473, 9.448, 9.520, 9.675 and

9.7 to 10.8 μm. The power needed to fragment β-O-methyl-glucopyranoside to obtain a ratio of

1.26 ± 0.04 for m/z 127 to m/z 201 at 9.588 μm, was far less than the power that was needed for

fragmentation of either anomer at wavelengths 9.7-10.8 μm. Figure 4-5 demonstrates the

fragmentation efficiency at the two wavelengths 9.588 and 10.611 μm. While fragmentation at

the higher wavelength is possible, it requires longer irradiation times and/or higher laser power.

Typical laser power needed to fragment at the lower wavelengths was approximately 0.70 W, but

to obtain any fragmentation at the higher wavelengths (on the same day with the same alignment

and number of ions in the cell) required more than 3 W.

Since the IRMPD depletion spectra of the precursor ion was found to be of little help in

distinguishing the anomers, comparing the percent abundances of m/z 109 and m/z 127 fragments

for O-methyl-glucopyranosides allowed for the different anomers to be distinguished, Figure 4-6.

The ratio of m/z 109 to m/z 127 was always higher for the α-anomer than for the β-anomer.

While the values changed slightly at the different wavelengths, the α-anomer always produced a

71

value that was greater than one and the β-anomer gave values that were less than one. The

greater abundance of the smaller fragments and the larger depletion of the parent ion indicate

that the α-anomer of O-methyl-glucopyranoside requires less energy for fragmentation than

β-O-methyl-glucopyranoside.

Unknown Study of Methyl-glucopyranosides

To demonstrate the ability to differentiate isomers of glucopyranosides by the developed

method, a single-blind test was performed for two samples each of α- and

β-O-methyl-glucopyranoside. Representative spectra taken at 9.588 μm are shown in

Figure 4-7 A and B. These unknowns were identified based on their fragmentation patterns.

Figure 4-7 A has a greater m/z 109 to m/z 127 and a relatively small amount of m/z 169 in

comparison to the unknown seen in Figure 4-7 B. Both methyl-glucopyranosides were positively

identified based solely on their spectra.

Methyl-galactopyranosides

This fragmentation procedure was then applied to another hexoses anomer pair, α- and

β-O-methyl-galactopyranoside. To compare the results of the O-methyl-glucopyranosides with

the O-methyl-galactopyranosides, a daily calibration with the β-O-methyl-glucopyranoside was

performed at 9.588 μm. This ensured that the same amount of power was used for each anomer.

For these studies, experiments were performed twice about a week and a half apart. Figure 4-8

shows the resulting fragmentation pattern of the O-methyl-galactopyranosides as a function of

wavelength and mass.

One key difference in the fragmentation patterns of α- and β-O-methyl-galactopyranoside

was the significantly greater abundance of the m/z 121 fragment ion for the β-anomer as

compared to that seen for the α-anomer. Also, the ratio of m/z 169 to m/z 151 proved to be

useful in the identification of the α- and β-anomers, as shown in Figure 4-9 for wavelengths 9.2

72

to 9.7 μm. The abundance ratio of m/z 169 to m/z 151 was always less than 5 for

β-O-methyl-galactopyranoside and was always greater than 5 for the α-anomer.

When comparing Figure 4-3 to Figure 4-8, several differences in the fragmentation

patterns of the methyl-gluco- and galactopyranosides are apparent. For example, m/z 109, 127,

151 and 169 are key fragment ions whose relative percent abundances vary for the gluco- and

galactopyranosides. Comparing Figure 4-3 to Figure 4-8, one can see that the abundances of

m/z 169 are greater for both methyl-galactopyranosides than for the methyl-glucopyranoside

anomers, while the abundances of m/z 109 and m/z 127 are lower for the

methyl-galactopyranosides than for the methyl-glucopyranosides. Using these differences in

fragmentation patterns the gluco- and galactopyranosides can be distinguished from each other.

Unknown Study of both Methyl-gluco- and galactopyranosides

To test the method described above for differentiating both α- and β- anomers of both the

gluco-and galactopyranosides, a single-blind study was performed. For this study, two samples

each of α-, β-O-methyl-glucopyranoside and α-, β-O-methyl-galactopyranoside were randomized

and their identity concealed. The unknowns were then analyzed individually at wavelengths of

9.230, 9.473 and 9.588 μm.

The identities of the unknown samples were then determined using the flowchart shown in

Figure 4-10. Similar spectra to those shown in Figure 4-7 A and B were obtained for α- and

β-O-methyl-glucopyranoside. Spectra obtained for the galactopyranosides are shown in

Figure 4-11 A and B. As displayed in Figure 4-11 A, there was a relatively small abundance of

m/z 109 and m/z 127 and a high abundance of m/z 169, identifying this unknown as one of the

galactopyranoside anomers. Comparing the spectrum in Figure 4-11 A to the spectrum in

Figure 4-11 B, there is a higher ratio of m/z 127 to m/z 109 and very little m/z 151. The lack of

m/z 121 and the ratio of 8.9 for m/z 169 to m/z 151 positively identify this unknown as

73

α-methyl-galactopyranoside. This identification was confirmed with the fragmentation seen

when the unknown was irradiated with a higher laser power. For the next unknown, whose

spectrum is seen in Figure 4-11 B, the ratio of m/z 169 to m/z 151 of 3 and the appearance of

m/z 121 made it clear that the identity of this unknown was β-O-methyl-galactopyranoside. All

of the eight unknown samples were correctly identified in a similar way.

In order to simulate a real-life laboratory environment, no calibration of laser power was

done before the unknown studies reported in the last paragraph. Although various laser powers

were used for the unknown studies, the precursor ion was never depleted. In general, higher

powers were needed for O-methyl-galactopyranosides since they fragmented less than the

O-methyl-glucopyranosides. For example, the methyl-glucopyranosides that produced the

spectra seen in Figure 4-7 A and B were both irradiated with 2.61 W and the

methyl-galactopyranosides that produced the spectra seen in Figure 4-11 A and B were irradiated

with 3.98 and 4.33 W, respectively.

Conclusions

Use of a tunable CO2 laser produced unique fragmentation patterns for anomers of

O-methyl-glucopyranoside and O-methyl-galactopyranoside over the wavelength range of 9.2 to

9.7 μm. Various fragment ions and their ratios were used to differentiate between the two sets of

monosaccharides (O-methyl-galactopyranoside and O-methyl-glucopyranoside) anomers. Since

only two monosaccharides were studied, future work should include other hexoses.

74

O

H

HO

H

HO

H

H

OHHOCH3

OH

O

OH

H

H

HO

H

OCH3

OHHH

OH

O

OH

H

H

HO

H

H

OHHOCH3

OH

O

H

HO

H

HO

H

OCH3

OHHH

OH

Alpha-methyl-glucopyranoside Beta-methyl-glucopyranoside

Alpha-methyl-galactopyranoside Beta-methyl-galactopyranoside

Figure 4-1. Structures of the O-methylated monosaccharides discussed in this chapter.

CO2 LaserM1

M2Salt

window

Power meter

Wavemeter

Mirror gate

M3

Laser table

M4

M5 Iris ZnSewindow

Source

4.7 T Bruker Mass Spectrometer

Infinity cell

CO2 LaserM1

M2Salt

window

Power meter

Wavemeter

Mirror gate

M3

Laser table

M4

M5 Iris ZnSewindow

Source

4.7 T Bruker Mass Spectrometer

Infinity cell

Figure 4-2. Experimental set up of the 4.7 T FTICR mass spectrometer. When the mechanical mirror M3 is down the laser beam passes through the ZnSe window into the cell. If the mechanical mirror up the laser beam is blocked from entering the cell and reflected into the power meter.

75

Figure 4-3. Wavelength-dependent fragmentation patterns for the lithiated O-methyl-glucopyranosides for wavelength from 9.2 to 10.8 μm. A) Lithiated α-O-methyl-glucopyranoside. B) Lithiated β-O-methyl-glucopyranoside.

76

Figure 4-4. Infrared multiple photon dissociation depletion spectra of the precursor ions (m/z 201) for both α- and β-O-methyl-glucopyranoside – lithium cation complexes.

77

m/z200190180170160150140130120110

Ab

un

dan

ce

20

15

10

5

201

169

151

141

127

109

A

111

m/z200190180170160150140130120110

Ab

un

dan

ce

20

15

10

5

201

169

151

141

127

109

A

m/z200190180170160150140130120110

Ab

un

dan

ce

20

15

10

5

201

169

151

141

127

109

m/z200190180170160150140130120110

Ab

un

dan

ce

20

15

10

5

201

169

151

141

127

109

A

111

m/z200190180170160150140130120110

Ab

un

dan

ce

45

40

35

30

25

20

15

10

5

201

169141

127109

B

m/z200190180170160150140130120110

Ab

un

dan

ce

45

40

35

30

25

20

15

10

5

201

169141

127109

B

Figure 4-5. Comparison of the fragmentation of β-methyl-glucopyranoside at wavelengths 9.588 and 10.611 μm. A) Spectrum obtained at 9.588 μm. B) Spectrum obtained at 10.611 μm. The laser power used was 2.21 W for 9.588 μm and 5.86 W for 10.611 μm. This demonstrates the difference in absorbance and fragmentation for the different disaccharides. Also it shows that conventional non-tunable CO2 lasers, with an output wavelength of 10.6 μm, are not optimal for fragmentation.

78

Figure 4-6. Relative percent abundance of fragment ions for both lithiated α- and β-O-methyl-glucopyranosides over the wavelength range from 9.201 to 9.675 μm. A) Relative percent abundance of product ion m/z 109. B) Relative percent abundance of product ion m/z 127. C) The ratio of m/z 109 to m/z 127.

79

m/z200150100

Ab

un

dan

ce

150140130120110100908070605040302010

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91

97

109

127141 151

169

201

A

79

81

m/z200150100

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150140130120110100908070605040302010

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91

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109

127141 151

169

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127141 151

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A

79

81

m/z200150100

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150140130120110100908070605040302010 67

91

97

109

127

141151

169

201B

7981

m/z200150100

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ce

150140130120110100908070605040302010 67

91

97

109

127

141151

169

201B

m/z200150100

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150140130120110100908070605040302010 67

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127

141151

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201

m/z200150100

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150140130120110100908070605040302010 67

91

97

109

127

141151

169

201

67

91

97

109

127

141151

169

201B

7981

Figure 4-7. Spectra of unknowns in single blind study of methyl-glucopyranosides at wavelength 9.588 μm. A) Spectrum of α-O-methyl-glucopyranoside. B) Spectrum of β-O-methyl-glucopyranoside.

80

Figure 4-8. Fragmentation patterns over the wavelengths from 9.2 to 10.6 μm. A) Lithiated α-O-methyl-galactopyranoside. B) Lithiated β-O-methyl-galactopyranoside.

81

Figure 4-9. Ratio of m/z 169 to m/z 151 for α- and β-O-methyl-galactopyranoside.

m/z 169 > m/z 91,109,127,151

yes no

α-O-methyl-galactopyranosideβ-O-methyl-galactopyranoside

α-O-methyl-glucopyranosideβ-O-methyl-glucopyranoside

> 5 < 5 > 1 < 1

m/z 169/151 m/z 109/127

α-O-methyl-galactopyranoside β-O-methyl-galactopyranoside α-O-methyl-glucopyranoside β-O-methyl-glucopyranoside

m/z 169 > m/z 91,109,127,151

yes no

α-O-methyl-galactopyranosideβ-O-methyl-galactopyranoside

α-O-methyl-glucopyranosideβ-O-methyl-glucopyranoside

> 5 < 5 > 1 < 1> 1 < 1

m/z 169/151 m/z 109/127

α-O-methyl-galactopyranoside β-O-methyl-galactopyranoside α-O-methyl-glucopyranoside β-O-methyl-glucopyranoside

Figure 4-10. Decision flowchart used to identify the different monosaccharide anomers.

82

m/z200150100

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50

45

40

35

30

25

20

15

10

5

67 91 109

111

127

141 151

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97

A

79

m/z200150100

Ab

un

dan

ce

50

45

40

35

30

25

20

15

10

5

67 91 109

111

127

141 151

169

201

97

A

79

m/z200150100

Ab

un

dan

ce

150

100

5067

91109

111

127

141

151

169

201

97121

B

8179

m/z200150100

Ab

un

dan

ce

150

100

5067

91109

111

127

141

151

169

201

97121

B

8179

Figure 4-11. Spectra of unknowns identified as galactopyranosides in single blind study

obtained at wavelength 9.588 μm. A) Spectrum of α-O-methyl-galactopyranoside. B) Spectrum of β-O-methyl-galactopyranoside.

83

CHAPTER 5 DIFFERENTIATION OF DISACCHARIDES IN THE POSITIVE ION MODE WITH A

TUNABLE CO2 LASER

Introduction

The structure of carbohydrates dictates their biological function. This includes the

monosaccharide sequence, the anomeric configuration and the linkage between the

monosaccharides within the oligosaccharides, which are the carbohydrate building blocks. Since

various linkages and anomeric configuration are possible, being able to differentiate the smaller

oligosaccharides that compose the larger carbohydrates is a complicated task. As described in

chapter 1, disaccharides are the smallest saccharide units that contain the glycosidic bond. The

anomeric configuration of this bond can play an important role in the function of the saccharide.

In the past, collision induced dissociation (CID)60,141 and infrared multiple photon

dissociation (IRMPD)62,73,142 have been used for fragmentation of saccharides, in particular

disaccharides. Past experiments by Polfer et al. examined fragmentation patterns of lithiated

disaccharides with the Free Electron Laser for Infrared eXperiments (FELIX) at the

FOM-Institute for Plasma Physics Rijnhuizen in the Netherlands.73 They found that isomeric

ions with various linkages had different fragmentation patterns. They also found that the

intensity ratio of specific fragments (m/z 169/187) was higher for β-anomers than for α-anomers

and may be used to differentiate the anomeric configuration of the disaccharides. Although the

fragmentation and some ratios were explored, there was no attempt to quantitatively ascertain the

anomeric configuration.

This chapter describes attempts to develop a method for the differentiation of lithiated

disaccharides. Wavelength-selective fragmentation of glucose-containing disaccharide anomers

was performed by IRMPD with a tunable CO2 laser, and differentiation of the disaccharides was

84

based on their fragmentation patterns and ratios of the relative abundances of specific fragment

ions.

Procedure

All work described in this chapter was performed with the instrumental set-up described in

chapter 4. Each of the disaccharides was prepared as a 0.10 mM solution which also contained

0.10 mM LiCl. To aid in ionization, the solvent was composed of an 80:20 ratio of general-use

grade methanol to MilliQ ultra-pure H2O. The disaccharides used in these studies were obtained

from Dr. Brad Bendiak at the Department of Cellular and Structural Biology, University of

Colorado Health Sciences Center, and were composed of two glucose units having varying

linkages and anomeric configurations: kojibiose (α1-2), sophorose (β1-2), nigerose (α1-3),

laminaribiose (β1-3), maltose (α1-4), cellobiose (β1-4), isomaltose (α1-6) and

gentiobiose (β1-6).

Fragmentation Study

For each lithiated disaccharide, the precursor ion (C12O22H11Li+, m/z 349) was produced by

electrospray ionization (ESI) and isolated via a stored waveform inverse Fourier

transform (SWIFT). The isolated precursor ion was irradiated for 1 second using a laser power

determined by a daily calibration involving the precursor ion of sophorose (β1-2). This ion was

fragmented to obtain an m/z 349:229 intensity ratio of 1.04 ± 0.16. The laser power required to

achieve this ratio was then used for the remainder of the experiments over the wavelength range

from 9.2 to 9.7 μm.

For each disaccharide, three data sets composed of 15 scans of 512 K points were collected

and averaged at each wavelength. To test reproducibility, the entire fragmentation pattern of

sophorose was obtained once on two separate days. Once fragmented, all the relative percent

abundances of the fragments were calculated and correlated with the disaccharide linkage.

85

Anomeric Configuration Study

After the fragmentation patterns corresponding to different linkages were determined,

experiments to differentiate the anomeric configuration for the pairs of anomers were performed

at 9.342, 9.473 and 9.588 μm. For these experiments, the laser power was adjusted to obtain a

peak height ratio of 1:2 for the isolated precursor ion (m/z 349) to a specific fragment ion

(m/z 169, 187 or 229) for different anomers, see Table 5-1. Abundance ratios for other peak

pairs were measured and correlated with the anomeric configuration.

Results and Discussion

Differentiation of Disaccharides

The wavelength-dependent fragmentation patterns for all of the disaccharides are shown in

Figure 5-1. These fragmentation patterns from 9.2 to 9.7 μm are similar to the patterns found by

Polfer et al. with a free electron laser.73 For example, fragmentation of 1-2 linked disaccharides

produces a major fragment of m/z 229, while the spectra of disaccharides with linkage 1-3 have

major fragments of m/z 169 and 331 and linkage 1-4 spectra have similar amounts of m/z 169

and 187, with very little m/z 229. Lastly, the spectra of disaccharides with linkage 1-6 have

fragments m/z 169, 187, 229, 259, 289, but very little 331.

While the fragments produced with the CO2 laser are identical to those produced with

FELIX, the relative percent abundances of each fragment are slightly different. Since the

relative abundance of the fragments is dependent on the amount of dissociation of the precursor

ion (m/z 349), variations in the laser power experienced by the ion clouds and the energy

imparted to the ions during electrospray could be causes of these differences. Also, differences in

the nature of the laser irradiation (continuous wave CO2 laser vs. several macropulses composed

of multiple high-power micropulses) could be the cause of some of the differences seen.127

86

As seen in Figure 5-1, fragmentation of the precursor ion over a range of wavelengths from

9.2 to 9.7 μm gave fragmentation patterns unique for each of the disaccharides. The wavelength

of fixed-frequency CO2 lasers (10.6 μm) was also explored, but very little (if any) fragmentation

was seen for the eight disaccharides. Either longer irradiation times and/or much higher laser

power were needed to see the little fragmentation that was observed. This result is consistent

with those found by Polfer et al. in which the fragmentation of the lithiated disaccharides

dramatically declined around 10.6 μm.73 Furthermore, this research shows that the typical

wavelength of non-tunable CO2 lasers is not optimal for differentiation of the various

disaccharides.

The flowchart in Figure 5-2 shows that the presence and absence of a certain fragment

makes determination of the different linkages possible. For example, if the dissociation

spectrum contains the fragments m/z 331 and m/z 289, but not the m/z 259 fragment, the linkage

can be identified as 1-4. Similar patterns can be specified for each of the different disaccharides

that were studied. While the linkage can be determined by the presence or absence of certain

fragments alone, more information is needed to identify the anomeric configuration.

Determination of the Anomeric Configurations

To determine the anomeric configurations, additional experiments were performed at three

wavelengths (9.342, 9.473 and 9.588 μm) in which the m/z 349 precursor ion was dissociated

with sufficient laser power to decrease its abundance to one half that of a fragment ion specified

in Table 5-1. Using this laser power, the ratios of the relative percent abundances of other

fragment ions were measured and used to identify the anomeric configuration, as shown in

Figure 5-3. For example, if the fragmentation pattern indicated that the linkage was 1-4, the

precursor ion was then irradiated to give a ratio of 1:2 for m/z 349 to m/z 187. The ratio of two

87

product ions (m/z 229 and 289) was then calculated and, based on the flowchart in Figure 5-3,

the anomeric configuration was determined.

The resulting ratios from the relative percent abundances of the specific ions provide a

method to differentiate all of the anomers at the various wavelengths. While only one

wavelength is needed to differentiate the anomers from each other, use of multiple wavelengths

confirms the results. For the 1-2 linkage disaccharides the ratio of m/z 187 to 229 was always

higher for kojibiose (α-linked) than for sophorose (β-linked). The ratio of m/z 169 to 187 was

always lower for nigerose (α-linked) than for laminaribiose (β-linked). For 1-4 linked

disaccharides, the ratio of m/z 229 to 289 is greater than 0.25 for cellobiose (β-linked) and less

than 0.25 for maltose (α-linked). Lastly, the ratio of m/z 169 to 187 is greater for

gentiobiose (β-linked) than isomaltose (α-linked) at all of the three wavelengths.

Differentiation of Unknowns

To make this work applicable to other laboratories, a method to determine both the linkage

and the anomeric configuration of the different disaccharides was explored. To test this

experimental procedure, one sample of each disaccharide was transferred to a coded vial to

conceal its identity. Each “unknown” was then tested on two separate days, and the identity was

predicted based on the methods described above and in Figures 5-2 and 5-3. Figure 5-4 shows

the results of the unknowns in comparison to the known samples analyzed previously. All error

bars shown are the 95% confidence interval of the mean for the experimental scans for a given

day. In all cases, the identity of the unknown was positively identified based on the ratios and

flow charts.

Some of the fluctuation and discrepancy between the ratios of the unknown and known

samples could be due to variation in the electrospray source. Since the laser power setting is

based on the ratio of the precursor ion to a fragment, slight changes in the abundance of the

88

precursor ion can cause the ratios of fragments to be affected. Even with the variation, the

results are such that the disaccharides were distinguished unambiguously.

Conclusions

This research demonstrated the use of a tunable CO2 laser to identify both the linkage and

the anomeric configuration of various lithium-attached disaccharides. When compared to FELs,

tunable CO2 lasers have a limited wavelength range, but this research showed that results

comparable to those from the broad output wavelength range of the FEL can be achieved in the

CO2 wavelength range of 9.2 to 9.7 μm. These results provide a method for differentiation of

isomers that is accessible and economically feasible for other laboratories.

89

Figure 5-1. Wavelength-dependent fragmentation for the various linked lithiated disaccharides. A) Kojibiose (α1-2). B) Sophorose (β1-2). C) Nigerose (α1-3). D) Laminaribiose (β1-3). E) Maltose (α1-4). F) Cellobiose (β1-4). G) Isomaltose (α1-6). H) Gentiobiose (β1-6).

90

Nigerose (α1-3)Laminaribiose (β1-3) Maltose (α1-4)Cellobiose (β1-4)Isomaltose (α1-6)Gentiobiose (β1-6)

Kojibiose (α1-2)Sophorose (β1-2)

Isomaltose (α1-6)Gentiobiose (β1-6)

m/z 289 observed?yes no

Maltose (α1-4)Cellobiose (β1-4)Isomaltose (α1-6)Gentiobiose (β1-6)

Nigerose (α1-3)Laminaribiose (β1-3)

m/z 259 observed?

Maltose (α1-4)Cellobiose (β1-4)

m/z 331 observed?yes no

yes no

Nigerose (α1-3)Laminaribiose (β1-3) Maltose (α1-4)Cellobiose (β1-4)Isomaltose (α1-6)Gentiobiose (β1-6)

Kojibiose (α1-2)Sophorose (β1-2)

Isomaltose (α1-6)Gentiobiose (β1-6)

m/z 289 observed?yes noyes noyes no

Maltose (α1-4)Cellobiose (β1-4)Isomaltose (α1-6)Gentiobiose (β1-6)

Nigerose (α1-3)Laminaribiose (β1-3)

m/z 259 observed?

Maltose (α1-4)Cellobiose (β1-4)

m/z 331 observed?yes noyes noyes no

yes noyes noyes no

Figure 5-2. Flow-chart depicting how linkage of the disaccharides was determined.

91

Table 5-1. Table of ratios used to determine the laser power used for fragmentation. Disaccharide Ratio used for standardizing

irradiation power Kojibiose (α1-2) Sophorose (β1-2)

1:2 m/z 349:229

Nigerose (α1-3) Laminaribiose (β1-3)

1:2 m/z 349:169

Maltose (α1-4) Cellobiose (β1-4)

1:2 m/z 349:187

Isomaltose (α1-6) Gentiobiose (β1-6)

1:2 m/z 349:169

Nigerose (α1-3)Laminaribiose (β1-3)

Kojibiose (α1-2)Sophorose (β1-2)

Isomaltose (α1-6)Gentiobiose (β1-6)

Maltose (α1-4)Cellobiose (β1-4)

m/z 187/229

> 0.1 < 0.1

Kojibiose Sophorose

m/z 169/187

> 5.0 < 5.0

m/z 229/289

> 0.25 < 0.25

m/z 169/187

> 1.0 < 1.0

Laminaribiose Nigerose Cellobiose Maltose Gentiobiose Isomaltose

Nigerose (α1-3)Laminaribiose (β1-3)

Kojibiose (α1-2)Sophorose (β1-2)

Isomaltose (α1-6)Gentiobiose (β1-6)

Maltose (α1-4)Cellobiose (β1-4)

m/z 187/229

> 0.1 < 0.1

m/z 187/229

> 0.1 < 0.1

Kojibiose Sophorose

m/z 169/187

> 5.0 < 5.0

m/z 169/187

> 5.0 < 5.0

m/z 229/289

> 0.25 < 0.25

m/z 229/289

> 0.25 < 0.25

m/z 169/187

> 1.0 < 1.0

m/z 169/187

> 1.0 < 1.0

Laminaribiose Nigerose Cellobiose Maltose Gentiobiose Isomaltose

Figure 5-3. Flow-chart showing ratios of peak heights and values used to determine anomeric configurations.

92

Kojibiose and Sophorose m/z 187/229

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Kojibiose9.342

Kojibiose9.473

Kojibiose9.588

Sophorose9.342

Sophorose9.473

Sophorose9.588

Sample

Rat

io

Knowns

Unknown trial 1

Unknown trial 2Rat

io

A Kojibiose and Sophorose m/z 187/229

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Kojibiose9.342

Kojibiose9.473

Kojibiose9.588

Sophorose9.342

Sophorose9.473

Sophorose9.588

Sample

Rat

io

Knowns

Unknown trial 1

Unknown trial 2Rat

io

A

Nigerose and Laminaribiose m/z 169/187

0

2

4

6

8

10

12

Nigerose9.342

Nigerose9.473

Nigerose9.588

Laminaribiose9.342

Laminaribiose9.473

Laminaribiose9.588

Sample

Rat

io

Known

Unknown trial 1

Unknown trial 2

Rat

io

B Nigerose and Laminaribiose m/z 169/187

0

2

4

6

8

10

12

Nigerose9.342

Nigerose9.473

Nigerose9.588

Laminaribiose9.342

Laminaribiose9.473

Laminaribiose9.588

Sample

Rat

io

Known

Unknown trial 1

Unknown trial 2

Rat

io

B

Maltose and Cellobiose m/z 229/289

00.10.20.30.40.50.60.70.80.9

Maltose9.342

Maltose9.473

Maltose9.588

Cellobiose9.342

Cellobiose9.473

Cellobiose9.588

Sample

Rat

io

Known

Unknown trial 1

Unknown trial 2Rat

io

CMaltose and Cellobiose m/z 229/289

00.10.20.30.40.50.60.70.80.9

Maltose9.342

Maltose9.473

Maltose9.588

Cellobiose9.342

Cellobiose9.473

Cellobiose9.588

Sample

Rat

io

Known

Unknown trial 1

Unknown trial 2Rat

io

C

Isomaltose and Gentiobiose m/z169/187

0

0.5

1

1.5

2

2.5

3

3.5

Isomaltose9.342

Isomaltose9.473

Isomaltose9.588

Gentiobiose9.342

Gentiobiose9.473

Gentiobiose9.588

Sample

Rat

io

Known

Unknown trial 1

Unknown trial 2Rat

io

D Isomaltose and Gentiobiose m/z169/187

0

0.5

1

1.5

2

2.5

3

3.5

Isomaltose9.342

Isomaltose9.473

Isomaltose9.588

Gentiobiose9.342

Gentiobiose9.473

Gentiobiose9.588

Sample

Rat

io

Known

Unknown trial 1

Unknown trial 2Rat

io

D

Figure 5-4. Bar graphs comparing ratios from knowns and unknown lithiated glucose-containing disaccharides at the wavelengths 9.342, 9.472 and 9.588 μm. A) Ratio of m/z 187/229 for kojibiose and sophorose. B) Ratio of m/z 169/187 for nigerose and laminaribiose. C) Ratio of m/z 229/289 for maltose and cellobiose. D) Ratio of m/z 169/187 for isomaltose and gentiobiose.

93

CHAPTER 6 IRMPD STUDIES OF NEGATIVELY CHARGED DISACCHARIDES WITH A TUNABLE

CO2 LASER

Introduction

The fragmentation of alkali-attached disaccharides73 and polysaccharides formed by

electrospray ionization (ESI) has been previously studied.57 The results showed that

disaccharides and polysaccharides with different linkages give different fragments and that the

wavelength-dependent fragmentation patterns can be used to identify the linkages in these

positively charged species. Both collision induced dissociation (CID),59,60,141,143,144 and infrared

multiple photon dissociation (IRMPD) using a free electron laser (FEL)62,70,73,74,142 have been

used to fragment saccharides. While adduction of a metal in both positive and negative ion

modes makes ionization of carbohydrates easier, when developing a general approach for

saccharide isomeric determination it would be simplest to analyze the saccharide without any

metal ions attached.

Past studies done in the negative ion mode by Jiang and Cole showed that the addition of

chloride yields a higher abundance of the precursor ion than that seen for the deprotonated

disaccharide, thus making it easier to isolate and fragment the precursor.145 Early studies of

chlorinated disaccharides such as sucrose were performed with fast atom bombardment (FAB)

and CID by Prome et al.,146 who found that the saccharides give characteristic fragment ions that

can be used for identification in the negative mode. Along with other fragments, they observed

that the fragmentation of the chloride-attached species resulted in loss of HCl to yield a high

abundance of deprotonated sucrose. More recent studies in the negative mode on the

fragmentation of 1-3, 1-4 and 1-6 linked glucose-containing disaccharides were conducted by

Zhu and Cole,147 who showed that it is possible to identify the linkage of the chlorinated

disaccharides by CID. They also found that the spectra of the deprotonated species are very

94

similar to the spectra obtained for the chlorinated species, further supporting the theory of HCl

loss prior to dissociation.147 Even more recent studies by Jiang and Cole have shown that CID

fragmentation of chlorinated disaccharides not only gives structural linkage information, but also

has the potential for anomeric discrimination,145 using the relative abundances of chlorinated

products vs. the non-chlorinated products. Of the 1-4 linked disaccharides explored by Jiang and

Cole, the ratio of chlorinated to non-chlorinated products was always higher for the α-anomer

than the β-anomer. They observed the opposite trend for 1-6 linked disaccharides.

This chapter reports studies of the fragmentation patterns of several deprotonated

glucose-containing disaccharides with various linkages and anomeric configurations obtained by

irradiation with a CO2 laser in the negative ion mode. The fragmentation patterns of chlorinated

disaccharides at selective wavelengths are also described.

Procedure

Deprotonated Disaccharides

All the fragmentation results for the deprotonated disaccharides discussed here were

obtained at the University of Florida in Dr. David Powell’s laboratory. A Bruker Bio-Apex II

Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with a 4.7 T magnet

(Bruker Daltonics, Billerica, MA) and an InfinityTM cell were used to analyze several glucose-

containing disaccharides including: kojibiose (α1-2), nigerose (α1-3), laminaribiose (β1-3),

maltose (α1-4), cellobiose (β1-4), isomaltose (α1-6) and gentiobiose (β1-6). All disaccharide

samples were provided by Dr. Brad Bendiak of the Department of Cellular and Structural

Biology, University of Colorado Health Sciences Center. The disaccharides were ionized with a

pneumatically-assisted Apollo external electrospray source (Bruker Daltonics, Billerica, MA).

For irradiation, the beam from a Lasy-20G tunable CO2 laser (Access Laser Co.; Marysville,

WA) was passed into the ICR cell through a KBr window located on the end of the FTICR mass

95

spectrometer opposite the end from which externally generated ions were admitted. To control

the irradiation time, a mechanical mirror was used to direct the laser beam. When not directed

into the cell, the laser beam was directed into a power meter head, allowing the laser power to be

monitored throughout the day. Figure 6-1 shows a general schematic of the FTICR-MS and laser

instrumentation used for these experiments.

Solutions of each disaccharide were prepared at a concentration of 0.1 mM. For

deprotonation of the disaccharides, several bases were tried, including sodium

hydroxide (NaOH), tri-ethylamine (N(CH2CH3)3) and ammonium hydroxide (NH4OH). Of the

bases used, NaOH gave the most stable signal with largest abundance of the deprotonated

disaccharide. Therefore, NaOH was used for all of the deprotonated disaccharide experiments.

Several solutions with concentration ratios from 0.1 to 1.0 mM NaOH to 0.1 mM disaccharide

were tried, and a concentration of 1 mM NaOH was found to give the best signal. For improved

ionization, all solutions were composed of 80:20 methanol:water made with general-use grade

methanol and MilliQ water. For all experiments, flow rates between 3 and 7 μL/min were used.

The flow rate was adjusted for each disaccharide to obtain the most stable and abundant signal.

All of the isolated disaccharide precursor ions (m/z 341) were irradiated with the CO2 laser

for one second. The wavelengths used were chosen based on specifications given in the laser

manual, so that only wavelengths with good or excellent power and stability were used. For each

wavelength, three experimental sets of 10 scans of 128 K points each were collected. Once the

precursor ions were fragmented, the relative percent abundance of each fragment was

determined.

Chlorinated Disaccharides

Experiments which studied the fragmentation of chlorinated disaccharides were performed

at the University of Florida in Dr. Eyler’s laboratory with the same instrumental set-up as

96

described in chapter 4. All samples were 0.1 mM solutions made with 1:1 LiCl:saccharide in

80:20 methanol:water. To obtain higher signal intensities, ions were accumulated for

1.5 seconds before being transferred to the analyzer cell followed by isolation, irradiation and

detection. All disaccharides were irradiated for 1 second with a Lasy-20 CO2 laser. The laser

power used was adjusted to create a peak height ratio of 1:1 for the isolated precursor ion

(m/z 377) to a specific fragment ion (m/z 161 or 179). For disaccharides kojibiose (α 1 -2),

sophorose (β 1-2), isomaltose (α 1-6) and gentiobiose (β 1-6), a ratio of 1:1 for m/z 377 to

m/z 179 was used. For nigerose (α 1-3), laminaribiose (β 1-3), maltose (α 1-4) and

cellobiose (β1-4), a ratio of 1:1 for the peak heights of m/z 377 to 161 was used. For each

linkage, the choice of fragment was based on greatest abundance and highest sensitivity relative

to the disappearance of the precursor ion peak.

Reproducibility: Deprotonated Disaccharides

To aid in reproducibility, a daily calibration was performed using isomaltose, which was

the first successfully detected deprotonated disaccharide. In this calibration, the laser power

needed to produce an m/z 179:341 abundance ratio of 1.19 ± 0.17 was determined daily. The

laser power was kept constant for all experiments performed on the same day.

Reproducibility: Chlorinated Disaccharides

To increase the reproducibility of these experiments, the laser power was adjusted to give a

1:1 ratio of the precursor ion to a specific fragment ion. The laser power was determined by

monitoring the fragment ion (either m/z 179 or 161) for each wavelength in the experiment.

Most of the variation seen in these experiments can be attributed to electrospray source

ionization and/or laser power fluctuations. Specific wavelengths were chosen to give a general

coverage of the full range between 9.2 to 9.7 μm. For time considerations and for simplicity of

the experiments, spectra at three wavelengths (9.342, 9.473 and 9.588 μm) were obtained.

97

Results and Discussion

Deprotonated Disaccharides

Figure 6-2 shows the percent abundance of the precursor ion of isomaltose following

IRMPD for the various wavelengths studied (i.e. a depletion spectrum). While the calibration

wavelength of 9.588 μm has a 95% confidence interval of the mean from experiments performed

on two separate days of approximately ± 2%, the maximum variation (at wavelength 9.657 μm)

was ± 26%. Figure 6-2 shows that the day-to-day variation of this method is larger than desired.

Some of the fluctuations causing daily variations could result from instability in the electrospray

source or slight variation of the laser power. Based on the variance of relative percent

abundance for the fragments, this proved to be a qualitative rather than quantitative method for

determining the linkage position of the disaccharides.

The relative percent abundances for the fragments are plotted as a function of wavelength

and mass in Figure 6-3, which shows that the major fragments produced for all of the

disaccharides, except for kojibiose, were m/z 179 and m/z 161. For kojibiose, the major

fragments seen were m/z 263 and 323, as well as minor contributions from fragments m/z 331,

281, 113 and 101. For both isomaltose and gentiobiose, the appearance of m/z 281 was unique

for and thus characteristic of the 1-6 linkage. Thus, 1-2 and 1-6 linked disaccharides can be

distinguished based on the presence of the m/z 281 fragment ion. Disaccharides with linkages of

1-3 and 1-4 gave only fragments at m/z 161 and 179. To distinguish the two linkages, the

abundance of m/z 161 and 179 for each linkage was explored further. For the 1-3 linked, the

ratio of m/z 161:179 was approximately 1:2, whereas the ratio was approximately 6:1 for the 1-4

linked disaccharides. Thus, within the wavelength range 9.2 to 9.7 μm, the linkage of

disaccharides can be identified from the fragmentation pattern.

98

Although the linkage position of the disaccharides could easily be determined based on the

appearance of certain key fragments, determining the anomeric configuration was problematic.

Since fragmentation of 1-3 and 1-4 linked disaccharides produces only m/z 161 and 179 ions, the

ratio of these two was calculated for each enantiomeric pair. The results for the 1-3 and 1-6

linked disaccharides are shown in Figure 6-4. Unfortunately, the ratios for each anomeric pair

were very similar and the identities of the anomers cannot be distinguished by this means.

Therefore more research in this area is needed, including greater fragmentation of the precursor

ion to determine if lower mass ions can be used to distinguish the anomers. Due to time

constraints and chlorine contamination, the spectrum of deprotonated sophorose was not

obtained in this study.

While the relative percent abundances of the fragment ions appear to vary from day to day,

the identities of the fragment ions from each disaccharide remained the same. Figure 6-5 shows

an example of the percent dissociation of the precursor ion from isomaltose and relative percent

abundances of the fragment ions at two different wavelengths (9.201 and 9.657 μm) on two

separate days. These differences, for example the relative percent abundance of the precursor

ion (m/z 341), could be due to the inherent fluctuation of the electrospray source and the laser

power. Since the degree of depletion of the precursor ion dictates the abundances of the

fragments produced, an increase in laser power results in greater abundances of the smaller m/z

ions due to multiple fragmentation.

Chlorinated Disaccharides

During the experiments on deprotonated disaccharides, the presence of chlorine hindered

detection of the deprotonated species. If chlorine was present in the cell and/or solutions, the

sugar would preferentially bind to Cl- rather than lose H+. To take advantage of this, several

99

experiments with the chlorinated disaccharides were performed in Dr. Eyler’s laboratory at the

University of Florida.

First, the fragmentation induced by the irradiation of the chlorinated disaccharides was

studied. Table 6-1 shows the fragments for each chlorinated disaccharide obtained at 9.588 μm

by varying the laser power to decrease the signal abundance of the precursor ion to a relative

percent abundance of 2.6 ± 1.6%, Figure 6-6.

Almost depleting the precursor ion allowed for more of the lower mass ions (for example

m/z 101) to be observed. By monitoring the fragment ions produced, it is possible to identify the

linkage. The presence of m/z 263 and 323 without m/z 281 indicates a 1-2 linkage. The absence

of m/z 263, 281 and 323 indicates a 1-3 linkage, whereas the presence of these three ions

indicates a 1-6 linkage. Lastly, the presence of only m/z 143, 161, 179 and 341 indicates a 1-4

linkage.

This study showed that the fragments produced by IRMPD are in fact different from those

seen in the CID studies of Zhu and Cole.147 The fragments produced for each fragmentation

method are compared in Table 6-2. First, for 1-3 linked disaccharides, the IRMPD spectra

contained lower mass fragment ions m/z 101, 119, 131 and 143 that were not present in the CID

spectra. For the 1-4 linked disaccharides, the higher mass ions of m/z 263 and 281 present in the

CID spectra were not present in the IRMPD spectra. For the 1-6 linked disaccharides, the

IRMPD produced the lower mass ions m/z 119, 131 and 143 that were not found in the CID

spectra and the higher mass ions with m/z 221 and 251 present in the CID spectra were not

present in the IRMPD spectra. These results indicate that, in general, the higher laser powers

used for IRMPD allow production of some lower mass ions that are not seen with CID, perhaps

by photodissociation of higher mass fragments.

100

The ratios of the relative percent abundances of specific fragment ions were obtained to

differentiate the anomeric configuration of disaccharides with the same linkage. For these

experiments, three wavelengths (9.342, 9.473 and 9.588 μm) were chosen for fragmentation.

Selecting only three wavelengths allowed for a (limited) range of wavelengths to be explored

while decreasing the experiment time. For these wavelengths, the laser power was adjusted to

obtain a peak height ratio of approximately 1:1 for m/z 161 to 377 (for nigerose, laminaribiose,

maltose and cellobiose) and m/z 179 to 377 (for kojibiose, sophorose, isomaltose and

gentiobiose). As has been discussed in earlier chapters, using such a ratio to “standardize” the

laser power allowed for improved day to day reproducibility compared to solely monitoring the

depletion of the precursor ion. Figure 6-7 shows the day to day reproducibility at the three

wavelengths for the isomaltose precursor and fragment ions. In comparison to the average

uncertainty of the precursor ion abundance found for the deprotonated isomaltose (± 15%) the

variation for chlorinated isomaltose precursor ion abundances over the three was only ± 2%,

indicating much better day-to-day reproducibility.

Similar to the results found with IRMPD of deprotonated disaccharides, m/z 161 and 179

were the major fragments produced by irradiation of the chlorinated disaccharides. The spectra

obtained for each disaccharide at each of the three wavelengths are shown in Figure 6-8. Except

for the 1-3 linked disaccharides, both the linkage and anomeric configuration can be determined

for all the disaccharides. For the 1-3 linked disaccharides, only the linkage could be determined.

The appearance of m/z 263 as the primary peak for both kojibiose and sophorose at 9.342 μm

makes this a useful wavelength for distinguishing the linkage and anomeric configuration of the

1-2 linked disaccharides. For nigerose and laminaribiose, the fragment ions produced were

mainly m/z 161 and 179, with the abundance of m/z 161 approximately 1.3 times that of m/z 179.

101

In contrast, the spectra of maltose and cellobiose have major fragments at m/z 161 and 179, but

the abundance of m/z 161 is approximately 6 (for maltose) and 8 (for cellobiose) times greater

than the abundance of m/z 179. While m/z 161 and 179 are the major peaks in the spectra for

gentiobiose and isomaltose, the presence of the fragment at m/z 281 in the spectra facilitates

identification of 1-6 linked.

The fragmentation patterns and specific ratios were used in a single-blind study where one

sample of each disaccharide was analyzed at wavelengths of 9.342, 9.473 and 9.588 μm. The

decision flow-chart used to identify the unknowns in the blind study is shown in Figure 6-9.

Analysis of the spectra shows that specific ratios of fragments can be used to differentiate

the anomeric pairs of the disaccharides, as shown in Figure 6-10. The ratio of m/z 263 to

m/z 179 fragment ion abundances of the 1-2 linked disaccharides shows differences at 9.342 and

9.473 μm, with this ratio greater for kojibiose than for sophorose at 9.342 μm and smaller for

kojibiose at wavelength 9.473 μm. To differentiate the 1-4 linked anomers, the data show that

the ratio of m/z 161/179 can be used. While all three wavelengths gave similar results, with the

ratio of cellobiose always being greater than that for maltose, 9.342 μm gave the greatest

difference with the smallest error bars (ratio of 6.5 ± 0.04 for maltose versus 8.1 ± 0.3 for

cellobiose). Lastly, the ratio of m/z 161 to 143 is always higher for gentiobiose than for

isomaltose. The average value for gentiobiose is 9.6 ± 1.1 vs. 3.1 ± 0.4 for isomaltose.

Unfortunately, more work is needed to differentiate the anomers of the 1-3 linked disaccharides.

Since the major fragments produced for the 1-3 linked disaccharides were m/z 161 and 179 and

the error bars for the ratios overlap at the three wavelengths, further fragmentation with more

power may be needed to distinguish the α- and β-anomers.

102

Identification of Fragment Ions

Because, the disaccharides used in these studies are all composed of two glucose units, it is

impossible to distinguish from which of the two glucose monosaccharides, reducing or

non-reducing, a fragment was produced without 18O labeling of the sugar at the reducing-end.

Figure 6-11 shows the possible fragment identities for fragments m/z 119,161, 179, 221, 251 and

289 based on 18O-labeling studies done by Hofmeister et al. in the positive ion mode60 and

Fang et al. in the negative ion mode.93 For the chlorinated disaccharides, the resulting fragments

are produced by the loss of HCl. Fang et al. found that fragments m/z 73, 89, 97, 119, 179, 251,

263 and 281 are produced from the non-reducing end. Since the reducing-end monosaccharide

can interconvert in solution, there is a mixture of both the α- and β-anomers for the reducing-end

monosaccharide. Therefore, being able to use the non-reducing fragments to identify the

anomers allows one to be certain of the anomeric configuration being identified.

Conclusions

These studies of the deprotonated and chlorinated disaccharides revealed that the

fragments produced by IRMPD over the wavelength range of 9.2 to 9.7 μm can be used to

identify the linkage positions of the glucose monosaccharides which comprise the disaccharides.

Identification of the anomeric configuration of the disaccharides is a more difficult task, but it

can be achieved for most of the anomeric pairs by calculating ratios of certain fragment ions for

the various linkages.

The similarity of results for the deprotonated and chlorinated disaccharides supports the

conclusions of Cole and co-workers that loss of HCl occurs before fragmentation.145,147 This

study also showed that IRMPD can be used in the negative ion mode to determine both the

anomeric configuration and linkage of chlorinated glucose-containing disaccharides.

103

Future research should include multiple fragmentations (hopefully produced using higher

laser powers) to see if depletion of the higher mass fragments results in a greater abundance of

the lower masses for the 1-3 linked disaccharides. It may be possible that ratios of these lower

masses can be used in the differentiation of the anomeric configuration. Therefore, future studies

should use an abundance ratio of the precursor ion to a fragment ion at all wavelengths to see

how the abundances of the fragment ions change. Also, since all the disaccharides studied here

were composed of two glucose units, the fragmentation of disaccharides composed of other

monosaccharide units such as mannose or allose should be tried.

104

Power-meter

CO2

laser

Ion optics

Pneumatically-assisted ESIInfinity cell

KBr window

Mirror

Mechanical Mirror

Bruker Apex II FTICR

4.7T magnet

Power-meter

CO2

laser

Ion optics

Pneumatically-assisted ESIInfinity cell

KBr window

Mirror

Mechanical Mirror

Bruker Apex II FTICR

4.7T magnet

Figure 6-1. Schematic drawing of the laser/mass spectrometer set-up used for the analysis of deprotonated disaccharides.

0

10

20

30

40

50

60

70

9.201 9.329 9.473 9.588 9.657

RP

W

Wavelength (μm)

Re

lati

ve P

erce

nt

Ab

un

da

nc

e

0

10

20

30

40

50

60

70

9.201 9.329 9.473 9.588 9.657

RP

W

Wavelength (μm)

Re

lati

ve P

erce

nt

Ab

un

da

nc

e

Figure 6-2. Relative percent abundance of the precursor ion (m/z 341) of isomaltose at selected wavelengths. Error bars show the 95% confidence interval of the mean based on data acquired on two separate days.

105

Figure 6-3. Wavelength-dependent fragmentation patterns for the various deprotonated disaccharides. A) Kojibiose. B) Nigerose. C) Laminaribiose. D) Maltose. E) Cellobiose. F) Isomaltose. G) Gentiobiose.

106

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

9.201 9.23 9.282 9.305 9.329 9.342 9.473 9.488 9.52 9.588 9.657

Wavelength

Rat

io

Nigerose

Laminaribiose

Isomaltose

Gentiobiose

Wavelength (µm)

Ra

tio

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

9.201 9.23 9.282 9.305 9.329 9.342 9.473 9.488 9.52 9.588 9.657

Wavelength

Rat

io

Nigerose

Laminaribiose

Isomaltose

Gentiobiose

Wavelength (µm)

Ra

tio

Figure 6-4. Ratio of m/z 161/179 for 1-3 and 1-6 linked disaccharides, showing that this ratio is not optimal for distinguishing the different anomers. Error bars are the 95% confidence interval of the mean for each ratio. The observed overlap of many of the error bars indicates that this ratio alone cannot be used to positively identify the anomers.

107

9.201

-10

0

10

20

30

40

50

60

70

101 113 115 119 125 143 161 179 221 251 281 309 311 323 341

m/z

Day 1

Day 2

9.657

-10

0

10

20

30

40

50

101 113 115 119 125 143 161 179 221 251 281 309 311 323 341

m/z

Day 1

Day 2

A

m/z

m/z

Rel

ati

ve P

erc

ent

Ab

un

dan

ceR

elat

ive

Per

cen

t A

bu

nd

ance

9.657 μm

9.201 μm

B

9.201

-10

0

10

20

30

40

50

60

70

101 113 115 119 125 143 161 179 221 251 281 309 311 323 341

m/z

Day 1

Day 2

9.201

-10

0

10

20

30

40

50

60

70

101 113 115 119 125 143 161 179 221 251 281 309 311 323 341

m/z

Day 1

Day 2

9.657

-10

0

10

20

30

40

50

101 113 115 119 125 143 161 179 221 251 281 309 311 323 341

m/z

Day 1

Day 2

9.657

-10

0

10

20

30

40

50

101 113 115 119 125 143 161 179 221 251 281 309 311 323 341

m/z

Day 1

Day 2

A

m/z

m/z

Rel

ati

ve P

erc

ent

Ab

un

dan

ceR

elat

ive

Per

cen

t A

bu

nd

ance

9.657 μm

9.201 μm

B

Figure 6-5. Comparison of the fragmentation patterns of deprotonated isomaltose on two separate days. A) The IRMPD spectrum of isomaltose at wavelength 9.201 μm. B) The IRMPD spectrum of isomaltose at wavelength 9.657 μm. Notice that the percent abundances of the precursor and fragment ions are different, but in general the same fragments are produced day to day.

108

Table 6-1. Major fragment ions observed for the chlorinated disaccharides when the precursor ion (m/z 377) was almost depleted by IRMPD at 9.588 μm. The solid boxes indicate the presence of a fragment ion of that m/z, whereas the stripped boxes indicate the absence of the fragment.

323

Gentiobiose (β1-6)

Isomaltose (α1-6)

Cellobiose (β1-4)

Maltose (α1-4)

Laminaribiose (β1-3)

Nigerose (α1-3)

Sophorose (β1-2)

Kojibiose(α1-2)

341281263179161143131119113101Disaccharide m/z 323

Gentiobiose (β1-6)

Isomaltose (α1-6)

Cellobiose (β1-4)

Maltose (α1-4)

Laminaribiose (β1-3)

Nigerose (α1-3)

Sophorose (β1-2)

Kojibiose(α1-2)

341281263179161143131119113101Disaccharide m/z

Table 6-2. Comparison of the fragments produced by CID and IRMPD for the chlorinated disaccharides. Fragments produced by both IRMPD and CID are coded in orange, by CID only in yellow, by IRMPD only in blue, and those not produced by either CID or IRMPD are coded in white.

1-6 linked

1-4 linked

1-3 linked

341323281263251221179161143131119113101m/z

Linkage

1-6 linked

1-4 linked

1-3 linked

341323281263251221179161143131119113101m/z

Linkage

109

m/z350300250200150100

Ab

un

dan

ce

200

150

100

50

377341323

263

179

161

143131

119

113

A

m/z350300250200150100

Ab

un

dan

ce

200

150

100

50

377341323

263

179

161

143131

119

113

m/z350300250200150100

Ab

un

dan

ce

200

150

100

50

377341323

263

179

161

143131

119

113

A

m/z350300250200150100

Ab

un

dan

ce

300

250

200

150

100

50377341323

263

179

161

143131119

113

101

B

m/z350300250200150100

Ab

un

dan

ce

300

250

200

150

100

50377341323

263

179

161

143131119

113

101

m/z350300250200150100

Ab

un

dan

ce

300

250

200

150

100

50377341323

263

179

161

143131119

113

101

B

m/z350300250200150100

Ab

un

dan

ce

160150140130120110100908070605040302010

377341

179

161

143

131119

113

101 251** **

C

m/z350300250200150100

Ab

un

dan

ce

160150140130120110100908070605040302010

377341

179

161

143

131119

113

101 251** **

m/z350300250200150100

Ab

un

dan

ce

160150140130120110100908070605040302010

377341

179

161

143

131119

113

101 251** **

C

m/z350300250200150100

Ab

un

dan

ce

400

350

300

250

200

150

100

50 341

179

161

143

131119

113

101 251*** 377

D

m/z350300250200150100

Ab

un

dan

ce

400

350

300

250

200

150

100

50 341

179

161

143

131119

113

101 251*** 377

m/z350300250200150100

Ab

un

dan

ce

400

350

300

250

200

150

100

50 341

179

161

143

131119

113

101 251*** 377

D

m/z350300250200150100

Ab

un

dan

ce

350

300

250

200

150

100

50377

341

179

161

143

E

m/z350300250200150100

Ab

un

dan

ce

350

300

250

200

150

100

50377

341

179

161

143

m/z350300250200150100

Ab

un

dan

ce

350

300

250

200

150

100

50377

341

179

161

143377

341

179

161

143

E

m/z350300250200150100

Ab

un

dan

ce

80075070065060055050045040035030025020015010050 377

179

161

143

F

m/z350300250200150100

Ab

un

dan

ce

80075070065060055050045040035030025020015010050 377

179

161

143

m/z350300250200150100

Ab

un

dan

ce

80075070065060055050045040035030025020015010050 377

179

161

143

F

m/z350300250200150100

Ab

un

dan

ce

400

350

300

250

200

150

100

50 281

377341323263

179

161

143

131119

113

101

*

G

m/z350300250200150100

Ab

un

dan

ce

400

350

300

250

200

150

100

50 281

377341323263

179

161

143

131119

113

101

*

G

281

377341323263

179

161

143

131119

113

101

*

G

m/z350300250200150100

Ab

un

dan

ce

80

70

60

50

40

30

20

10

377

341

281

323*263

179

161

143

131119

113

101

*

H

m/z350300250200150100

Ab

un

dan

ce

80

70

60

50

40

30

20

10

377

341

281

323*263

179

161

143

131119

113

101

*

m/z350300250200150100

Ab

un

dan

ce

80

70

60

50

40

30

20

10

377

341

281

323*263

179

161

143

131119

113

101

*

377

341

281

323*263

179

161

143

131119

113

101

*

H

Figure 6-6. Fragmentation spectra for the nearly depleted precursor ion (m/z 377) for the chlorinated disaccharides at 9.588 μm. A) Kojibiose. B) Sophorose. C) Maltose. D) Cellobiose. E) Nigerose. F) Laminaribiose. G) Isomaltose. H) Gentiobiose.

110

9.342

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

W av

RP Day 1

Day 2

9.342 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

9.473

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

m /z

RP

A Day 1

Day 2

9.473 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

9.588

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

m

RP Day 1

Day 2

9.588 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce9.342

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

W av

RP Day 1

Day 2

9.342 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce9.342

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

W av

RP Day 1

Day 2

9.342 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce9.342 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

9.473

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

m /z

RP

A Day 1

Day 2

9.473 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

9.473

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

m /z

RP

A Day 1

Day 2

9.473 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

9.473 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

9.588

0

5

10

15

20

25

30

35

71 73 81 83 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

m

RP Day 1

Day 2

9.588 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

9.588 μm

m/z

Rel

ativ

e P

erce

nt

Ab

un

dan

ce

Figure 6-7. Infrared multiple photon dissociation spectra for chlorinated isomaltose obtained at three wavelengths on two different days. Similar reproducibilities were observed for the other chlorinated disaccharides.

111

0

10

20

30

40

50

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Sophorose

9.342

9.473

9.588

0

10

20

30

40

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Laminaribiose

9.342

9.473

9.588

0

20

40

60

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Kojibiose

9.342

9.473

9.588

0

10

20

30

40

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Nigerose

9.342

9.473

9.588

0

10

20

30

40

50

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Maltose

9.342

9.473

9.588

0

10

20

30

40

50

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Cellobiose

9.342

9.473

9.588

0

10

20

30

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Isomaltose

9.342

9.473

9.588

0

10

20

30

40

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Gentiobiose

9.342

9.473

9.588

m/z m/z

Re

lati

ve P

erc

ent

Ab

un

da

nce

Kojibiose Sophorose

Nigerose Laminaribiose

Maltose Cellobiose

Isomaltose Gentiobiose

0

10

20

30

40

50

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Sophorose

9.342

9.473

9.588

0

10

20

30

40

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Laminaribiose

9.342

9.473

9.588

0

20

40

60

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Kojibiose

9.342

9.473

9.588

0

10

20

30

40

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Nigerose

9.342

9.473

9.588

0

10

20

30

40

50

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Maltose

9.342

9.473

9.588

0

10

20

30

40

50

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Cellobiose

9.342

9.473

9.588

0

10

20

30

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Isomaltose

9.342

9.473

9.588

0

10

20

30

40

71 73 81 83 87 89 101 113 119 125 131 143 161 179 251 263 281 323 341 377 379

Gentiobiose

9.342

9.473

9.588

m/z m/z

Re

lati

ve P

erc

ent

Ab

un

da

nce

Kojibiose Sophorose

Nigerose Laminaribiose

Maltose Cellobiose

Isomaltose Gentiobiose

Figure 6-8. Average fragmentation spectra for the disaccharides at 9.342, 9.473 and 9.588 μm. The major fragments observed for kojibiose and sophorose are m/z 179, 263 and 323, whereas the major fragments for nigerose, laminaribiose, maltose and cellobiose are m/z 161 and 179. The major fragments observed for isomaltose and gentiobiose are m/z 161, 179 and 281.

112

1-3 linked1-4 linked1-6 linked

1-2 linked

1-3 linkedNigeroseLaminaribiose

m/z 323?yes no

1-6 linked 1-3 linked1-4 linked

m/z 161 >>179?

1-4 linked

m/z 281?yes no

yes no

> 2 < 2

> 4.35 < 4.35

> 6.75 < 6.75

m/z 263/179 at 9.473 μm

α1-2Kojibiose

β1-2Sophorose

β1-6Gentiobiose

α1-6Isomaltose

m/z 161/143 at 9.342, 9.473 and 9.588 μm

m/z 161/179 at 9.473 and 9.588 μm

α1-4Maltose

β1-4Cellobiose

1-3 linked1-4 linked1-6 linked

1-2 linked

1-3 linkedNigeroseLaminaribiose

m/z 323?yes noyes noyes no

1-6 linked 1-3 linked1-4 linked

m/z 161 >>179?

1-4 linked

m/z 281?yes noyes noyes no

yes noyes no

> 2 < 2

> 4.35 < 4.35

> 6.75 < 6.75

m/z 263/179 at 9.473 μm

α1-2Kojibiose

β1-2Sophorose

β1-6Gentiobiose

α1-6Isomaltose

m/z 161/143 at 9.342, 9.473 and 9.588 μm

m/z 161/179 at 9.473 and 9.588 μm

α1-4Maltose

β1-4Cellobiose

Figure 6-9. Decision flow chart used to identify disaccharide samples with unknown identities in a single-blind study. Once the linkage is determined, the anomeric configuration of all the disaccharides (except 1-3 linked) can then be determined from ratios of specific fragment ions.

113

m/z 263/179

0

0.5

1

1.5

2

2.5

3

3.5

9.342 9.473 9.588

Wavelength

Rat

io

Kojibiose Day 1

Kojibiose Day 2

Kojibiose Unknown

Sophorose Day 1

Sophorose Day 2

Sophorose Unknown

m/z 161/179

0123456789

9.342 9.473 9.588

Wavlength

Rat

io

Maltose Day 1

Maltose Day 2

Maltose Unknown

Cellobiose Day 1

Cellobiose Day 2

Cellobiose Unknown

m/z 161/143

0

2

4

6

8

10

12

14

16

9.342 9.473 9.588

Wavelength

Rat

io

Isomaltose Day 1

Isomaltose Day 2

Isomaltose Unknown

Gentiobiose Day 1

Gentiobiose Day 2

Gentiobiose Unknown

Wavelength (μm)

Rat

io

m/z 263/179A

Wavelength (μm)

Rat

io

m/z 161/143C

Wavelength (μm)

Rat

io

m/z 161/179B

m/z 263/179

0

0.5

1

1.5

2

2.5

3

3.5

9.342 9.473 9.588

Wavelength

Rat

io

Kojibiose Day 1

Kojibiose Day 2

Kojibiose Unknown

Sophorose Day 1

Sophorose Day 2

Sophorose Unknown

m/z 161/179

0123456789

9.342 9.473 9.588

Wavlength

Rat

io

Maltose Day 1

Maltose Day 2

Maltose Unknown

Cellobiose Day 1

Cellobiose Day 2

Cellobiose Unknown

m/z 161/143

0

2

4

6

8

10

12

14

16

9.342 9.473 9.588

Wavelength

Rat

io

Isomaltose Day 1

Isomaltose Day 2

Isomaltose Unknown

Gentiobiose Day 1

Gentiobiose Day 2

Gentiobiose Unknown

Wavelength (μm)

Rat

io

m/z 263/179A

Wavelength (μm)

Rat

io

Wavelength (μm)

Rat

io

m/z 161/143C

Wavelength (μm)

Rat

io

Wavelength (μm)

Rat

io

m/z 161/179B

Figure 6-10. Comparison of various ratios used to determine the anomeric configurations of the chlorinated disaccharides. The unknowns that were identified in the single-blind study by the decision flow chart (Figure 6-9) are also shown. A) The ratio of m/z 263/179 for 1-2 linked kojibiose and sophorose. B) The ratio of m/z 161/179 for 1-4 linked maltose and cellobiose. C) The ratio of m/z 161/143 for 1-6 linked isomaltose and gentiobiose.

114

O

OH

OH

OH

O

OH

OH

O

HO

OH

H, OH

O

OH

OH

OH

O

HO

O

OH

OH

OH

O

HO

O

OH

OH

OH

O

OH

OH

HO

H, OH

H,OH

A

B

C

-HCl

-HCl

-HCl

179

161

221

119

251

161

179

281

O

OH

OH

OH

O

OH

OH

O

HO

OH

H, OH

O

OH

OH

OH

O

HO

O

OH

OH

OH

O

HO

O

OH

OH

OH

O

OH

OH

HO

H, OH

H,OH

A

B

C

-HCl

-HCl

-HCl

179

161

221

119

251

161

179

281

Figure 6-11. Identification of some of the fragment ions for the various linked disaccharides. A) Fragments produced by 1-2 linked disaccharides. B) Fragments produced by 1-4 linked disaccharides. C) Fragments produced by 1-6 linked disaccharides. Based on results from Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 5964-5970 and Fang, T. T.; Zirrolli, J.; Bendiak, B. Carbohydr. Res. 2007, 342, 217-235.60,93

115

CHAPTER 7 DIFFERENTIATION OF DISACCHARIDES IN THE NEGATIVE ION MODE WITH FREE

ELECTRON LASER INFRARED MULTIPLE PHOTON DISSOCIATION

Introduction

While tunable CO2 lasers have a limited wavelength range of 9.2 to 10.8 μm, the output

wavelengths produced by free electron lasers (FELs) span a much wider range. Past infrared

multiple photon dissociation (IRMPD) studies with a FEL on both mono- and disaccharides

involved positive ions formed by the adduction of metal ions.73,74 Polfer et al. found that

fragmentation of various glucose-containing lithium-attached disaccharides yielded unique

fragmentation patterns that were a function of both mass and wavelength. The work of Jose

Valle showed that the IRMPD spectra of various rubidium-attached O-methylated pyranosides

were unique for the various monosaccharides.74

The Free Electron Laser for Infrared eXperiments (FELIX) of the FOM-Institute for

Plasma Physics Rijnhuizen, The Netherlands is a user facility that began operation in the

1990’s.74,127,148 The components of FELIX, discussed in chapter 3, are housed in the basement of

the facility, and the laser beam is directed through evacuated beam tubes into different user

stations by pneumatically controlled mirrors. For the studies reported in this chapter, a

laboratory-constructed Fourier transform ion cyclotron resonance (FTICR) mass spectrometer,

Figure 7-1, was used.148 The FELIX laser beam passes into the back of the FTICR mass

spectrometer through a ZnSe window, where a mirror system creates multiple passes resulting in

higher laser fluence over the wavelengths of approximately 5.5 to 11.0 μm used for most

studies.

Procedure

All the experiments reported in this chapter were performed at the FELIX facility with the

help of Drs. Jos Oomens and Jeffrey Steill. First the fragmentation and IRMPD spectra of the

116

deprotonated precursor ions of several mono- and disaccharides were obtained. Next, the

IRMPD spectra and fragmentation patterns of the m/z 179 monosaccharide anion isolated from

the fragmentation of deprotonated disaccharides in the negative ion mode were obtained. Also,

the fragmentation and IRMPD spectra of various deprotonated monosaccharides were studied.

All ions were irradiated with FELIX for 2.5 to 3.5 seconds with a macropulse repetition rate of

5 Hz (for the disaccharide studies) or 10 Hz (for the monosaccharide studies). For the dual laser

experiments, a fixed-frequency CO2 laser was used to fragment a disaccharide to yield the

monosaccharide anion (m/z 179). The disaccharides were irradiated with the CO2 laser with a

power of 0.8 W for 0.35 seconds to produce m/z 179 as the predominant fragment ion.

All deprotonated disaccharide samples of O18-labeled kojibiose, sophorose, nigerose and

laminaribiose were prepared in 80:20 methanol (MeOH):H2O solutions at 1.0 mM disaccharide

and 1.0 mM NaOH concentrations. The solution of chlorinated sophorose was prepared in

8:2 MeOH:H2O with 1 mM concentrations of both sophorose and LiCl. All monosaccharide

samples were prepared with 1 mM saccharide and 1 mM NaOH in 80:20 MeOH:H2O, with the

exception of glucose, which was made at 2 mM glucose to 1.5 mM NaOH (deprotonated studies)

or 1.5 mM LiCl (chlorinated studies). Several concentrations of NaOH were tried, but the

concentrations that gave the best results were either 1.0 or 1.5 mM. Also, triethylamine was tried

as an alternative base, but deprotonation with NaOH provided the best signal for both the

disaccharides and the monosaccharides.

Results and Discussion

Disaccharides

The deprotonated disaccharides and their fragmentation patterns were studied first.

Fang et al. showed that fragmentation of a cross-ring cleavage product (m/z 221), itself produced

by CID from the deprotonated parent ion, was useful in the differentiation of disaccharides.93

117

The research here explored the possibility of using another fragment ion (m/z 179) to

differentiate the disaccharides. First studies of the fragmentation of the precursor ion of the

18O-labeled deprotonated disaccharides (m/z 343) were performed to confirm that the anion

monosaccharide fragment (m/z 179) is produced solely from the non-reducing end. This step

was necessary to determine if the monosaccharide anion could be used to differentiate the

disaccharides and anomeric configurations. For these experiments, two sets of 18O-labeled

glucose-containing disaccharide anomers, kojibiose/sophorose (1-2 linked) and

nigerose/laminaribiose (1-3 linked), were used. The spectrum of chlorinated disaccharide of

sophorose was also obtained to explore the effect of chlorine ion adduction on the fragmentation

of disaccharides.

The fragmentation patterns of deprotonated 18O-labeled kojibiose, sophorose, nigerose and

laminaribiose are shown in Figure 7-2. The fragments produced for the two anomer pairs vary

based on the linkage. For example, fragments m/z 101, 119, 143, 223, 265 and 325 are present

for the 1-2 linked (kojibiose and sophorose) but are not present for the 1-3 linked disaccharides

(nigerose and laminaribiose). Higher mass fragments are present in IRMPD spectra of the

1-2 linked disaccharides, but only the lower masses of m/z 59 and 97 are produced by the

IRMPD fragmentation of 1-3 linked disaccharides. The fragmentation patterns show that the

relative percent abundances of specific fragments are different for each anomer. For example,

the relative percent abundances of m/z 223 and 265 were higher for kojibiose than for sophorose.

The fragmentation patterns for both nigerose and laminaribiose contain m/z 59, 62, 69, 71, 89,

97, 113, 115, 161, 163 and 179, but fragmentation of nigerose produces a higher abundance of

m/z 97 while fragmentation of laminaribiose produced a higher abundance of m/z 59 and 62 over

the wavelength range of approximately 9.0 to 11.0 μm.

118

The IRMPD fragmentation spectrum of chlorinated unlabeled sophorose was also obtained

over the wavelength range of 5.5 to 11.0 μm. The fragmentation of the chlorinated disaccharide,

Figure 7-3, shows a pattern very similar to that of the deprotonated disaccharide, Figure 7-2 B, in

which m/z 323, 263 (265 for 18O-labeled sophorose) and 179 are the major fragments produced.

One difference is that the ion m/z 223 (221 for 16O-sophorose) fragment is produced by

irradiation of the deprotonated sophorose, but not the chlorinated sophorose. Comparing the

IRMPD spectra of the deprotonated and chlorinated sophorose parent ions, Figure 7-4, shows

that adduction of chlorine produces a similar spectrum, but that the overall spectral peaks are

sharpened.

Monosaccharide Anion Produced from Disaccharides

The absence of m/z 181 in the fragmentation patterns for both sets of disaccharide anomer

pairs confirmed that the fragment ion m/z 179 comes solely from the non-reducing end. Next,

the IRMPD spectra of the monosaccharide anion (m/z 179) produced from fragmentation of the

various disaccharides were obtained. For this, m/z 179 was produced by sustained off-resonance

irradiation collision induced dissociation (SORI-CID) and then by laser irradiation with a

non-tunable CO2 laser. The results of this study were compared to those obtained from the

irradiation of deprotonated glucose. As seen in Figure 7-5, the presence of a peak around

~1720 cm-1 for all of the IRMPD spectra is consistent with a characteristic C=O stretch of an

aldehyde. Since the IRMPD spectra of the m/z 179 fragment ion produced by CID and laser

irradiation both contain the aldehyde stretch, these results indicate that the monosaccharide anion

opens upon fragmentation. A possible schematic for this process is shown in Figure 7-6.

To confirm the ring opening, several deprotonated monosaccharides including allose,

galactose, glucose and mannose were irradiated with FELIX and their spectra were obtained. As

seen in Figure 7-7, all monosaccharides produced very broad spectra over the range of 1000 to

119

1800 cm-1. More importantly, the IRMPD spectra for all the deprotonated monosaccharides

contained a peak around 1720 cm-1, thereby indicating the ring opening and loss of anomericity.

To give further spectroscopic evidence for the ring opening, the IRMPD spectra of O-methylated

anomers of glucose were obtained. Methylation of the C-1 oxygen locks the conformation of the

anomer and thereby eliminates mutarotation. When comparing these spectra, Figure 7-8, to the

spectrum of deprotonated glucose (also seen in Figures 7-5 or 7-7), the O-methylated compounds

lacked the aldehyde peak at 1720 cm-1, thereby confirming the suspicion of the opening of the

ring.

Since the IRMPD spectra alone could not differentiate the monosaccharides, more

information was needed. Irradiation of the monosaccharides over the wavelengths 5.5 to

approximately 11 μm produced fragment ions with m/z 59, 71, 89, 101, 113, 119,143 and 161.

While all of the monosaccharides produced the same fragment ions, the relative percent

abundances of the fragments varied depending on the monosaccharide. For example, the percent

abundances of m/z 89, 101, 131 and 161 were the largest fragments of glucose, while fragments

with m/z 59, 71 and 101 were the most abundant for allose, galactose and mannose. Since the

monosaccharides used for this study were not methylated, they existed as a mixture of anomeric

configurations, and therefore anomeric configurations were not studied. Also, due to limited

time, the spectra of these monosaccharides were obtained only once.

Conclusions

The studies performed with FELIX on mono- and disaccharides confirmed that at least

some of the monosaccharide anions open upon deprotonation resulting in loss of the anomeric

configuration. These findings also indicate that the fragment ion m/z 179 may not be the best to

use when differentiating disaccharides. These results also demonstrated that the fragmentation

120

patterns for the various mono- and disaccharides in the negative ion mode are unique and depend

on both the linkage and anomeric configuration of the saccharide.

121

Figure 7-1. Schematic of the FTICR set-up at FELIX. Drawing courtesy of Dr. Jos Oomens.

122

O

OH

OH

OHO

HO

O

OH

OH

OH

18OH,H

-H+

325

265

223

179

143

119113

10189

179

223

m/z

179

m/z

Wavelength(µm)

Re

lati

ve P

erc

en

t A

bu

nd

an

ce

A B

325

265

223179

143119113

10189

O

OH

OH

OHO

HO

O

OH

OH

OH

18OH,H

-H+

223

O

OH

OH

OHO

HO

O

OH

OH

OH

18OH,H

-H+

325

265

223

179

143

119113

10189

179

223

m/z

179

m/z

Wavelength(µm)

Re

lati

ve P

erc

en

t A

bu

nd

an

ce

A B

325

265

223179

143119113

10189

O

OH

OH

OHO

HO

O

OH

OH

OH

18OH,H

-H+

223

O

OH

OH

OHO

HO

O

OH

OH

HO

18OH,H

-H+

Wavelength(µm)

161179

113 115

97

89

7159

161

179

113 115

97

89

7159

m/z m/z

Rel

ati

ve P

erc

en

t A

bu

nd

anc

e

163 163

179

163

179

163

C D

O

OH

OH

OHO

HO

O

OH

OH

HO

18OH, H

-H+

O

OH

OH

OHO

HO

O

OH

OH

HO

18OH,H

-H+

Wavelength(µm)

161179

113 115

97

89

7159

161

179

113 115

97

89

7159

m/z m/z

Rel

ati

ve P

erc

en

t A

bu

nd

anc

e

163 163

179

163

179

163

C D

O

OH

OH

OHO

HO

O

OH

OH

HO

18OH, H

-H+

Figure 7-2. Infrared multiple photon dissociation fragmentation patterns over the wavelength range of 5.5 to 11 μm for the deprotonated 18O-labeled disaccharides. A) Kojibiose. B) Sophorose. C) Nigerose. D) Laminaribiose.

123

341323

263179

161

143131

119

10189

m/z

Wavelength(µm)

Rel

ativ

e P

erce

nt

Ab

un

da

nce

O

OH

OH

OHO

OH

OH

O

HO

OH

H, OH

-HCl

341323

263179

161

143131

119

10189

341323

263179

161

143131

119

10189

m/z

Wavelength(µm)

Rel

ativ

e P

erce

nt

Ab

un

da

nce

O

OH

OH

OHO

OH

OH

O

HO

OH

H, OH

-HCl

Figure 7-3. Fragmentation pattern of chlorinated unlabeled sophorose.

0

0.1

0.2

0.3

0.4

0.5

0.6

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

cm

Dis

so

cia

tio

n y

e

O-18 Deprotonated Sophorose

Chlorinated Sophorose

cm-1

Dis

so

cia

tio

n Y

ield

0

0.1

0.2

0.3

0.4

0.5

0.6

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

cm

Dis

so

cia

tio

n y

e

O-18 Deprotonated Sophorose

Chlorinated Sophorose

0

0.1

0.2

0.3

0.4

0.5

0.6

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

cm

Dis

so

cia

tio

n y

e

O-18 Deprotonated Sophorose

Chlorinated Sophorose

cm-1

Dis

so

cia

tio

n Y

ield

Figure 7-4. Comparison of the IRMPD spectra for O18-labeled sophorose and O16-chlorinated sophorose.

124

Spectra of m/z 179

0

0.1

0.2

0.3

0.4

0.5

0.6

1000 1100 1200 1300 1400 1500 1600 1700 1800

Wavenumber

Dis

so

cia

tio

n y

ieDeprotonated glucosefrom kojibiose produced by SORI-CIDfrom kojibiose produced by CO2 laserfrom sophorose produced by CO2 laser

cm-1

Dis

soci

atio

n y

ield

Spectra of m/z 179

0

0.1

0.2

0.3

0.4

0.5

0.6

1000 1100 1200 1300 1400 1500 1600 1700 1800

Wavenumber

Dis

so

cia

tio

n y

ieDeprotonated glucosefrom kojibiose produced by SORI-CIDfrom kojibiose produced by CO2 laserfrom sophorose produced by CO2 laser

cm-1

Dis

soci

atio

n y

ield

Figure 7-5. Comparison of the IRMPD spectra of the monosaccharide anions (m/z 179) produced by deprotonation of glucose and by fragmentation of a disaccharide by SORI-CID and CO2 laser irradiation.

O

H

HO

H

HO

H

O-

OHHH

OH

O-

H

HO

H

HO

H

O

OHH

OH

Figure 7-6. Schematic of the possible mechanism leading to the opening of the monosaccharide anion ring.

125

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800

cm

dis

s

Allose

Galactose

Glucose

Mannose

Dis

soci

atio

n yi

eld

cm-1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800

cm

dis

s

Allose

Galactose

Glucose

Mannose

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800

cm

dis

s

Allose

Galactose

Glucose

Mannose

Dis

soci

atio

n yi

eld

cm-1

Dis

soci

atio

n yi

eld

cm-1

Figure 7-7. Infrared multiple photon dissociation spectra of various deprotonated monosaccharides. All the spectra are very broad with a peak around ~ 1720 cm-1, indicating the opening of the ring.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Dis

soci

atio

Alpha-O-methyl-glucopyranoside

Beta-O-methyl-glucopyranoside

0

0.1

0.2

0.3

0.4

0.5

0.6

820 920 1020 1120 1220 1320 1420 1520 1620 1720 1820

Disso

wav

e

Deprotonated glucose

α-O-methyl-glucopyranoside

β-O-methyl-glucopyranoside

Deprotonated glucose

cm-1

Dis

soci

atio

n yi

eld

Dis

soci

atio

n y

ield

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Dis

soci

atio

Alpha-O-methyl-glucopyranoside

Beta-O-methyl-glucopyranoside

0

0.1

0.2

0.3

0.4

0.5

0.6

820 920 1020 1120 1220 1320 1420 1520 1620 1720 1820

Disso

wav

e

Deprotonated glucose

α-O-methyl-glucopyranoside

β-O-methyl-glucopyranoside

Deprotonated glucose

cm-1

Dis

soci

atio

n yi

eld

Dis

soci

atio

n y

ield

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Dis

soci

atio

Alpha-O-methyl-glucopyranoside

Beta-O-methyl-glucopyranoside

0

0.1

0.2

0.3

0.4

0.5

0.6

820 920 1020 1120 1220 1320 1420 1520 1620 1720 1820

Disso

wav

e

Deprotonated glucose

α-O-methyl-glucopyranoside

β-O-methyl-glucopyranoside

Deprotonated glucose

cm-1

Dis

soci

atio

n yi

eld

Dis

soci

atio

n y

ield

Figure 7-8. Comparison of the IRMPD spectra for anomers of O-methyl-glucopyranoside to the spectrum of deprotonated glucose.

126

D

Wavelength(µm)

m/z

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

59

71

89

101

143 161

Wavelength(µm)

C

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

89

101

113 119131 161

m/z

A

Wavelength(µm)

m/z

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

5971

89

101

119

143161

m/z

B

Wavelength(µm)

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

5971

89

101113 143

131113

59 71143

131

113

119

119 131161

O

H

HO

OH

H

H

OHH

OH

OH,H

-H+

O

OH

H

H

HO

H

OHH

OH

OH,H

-H+

O

H

HO

H

HO

H

OHH

OH

OH,H

-H+

O

H

HO

H

HO

OH

HH

OH

OH,H

-H+

D

Wavelength(µm)

m/z

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

59

71

89

101

143 161

Wavelength(µm)

C

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

89

101

113 119131 161

m/z

A

Wavelength(µm)

m/z

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

5971

89

101

119

143161

m/z

B

Wavelength(µm)

Re

lati

ve

Per

cen

t A

bu

nd

an

ce

5971

89

101113 143

131113

59 71143

131

113

119

119 131161

O

H

HO

OH

H

H

OHH

OH

OH,H

-H+

O

OH

H

H

HO

H

OHH

OH

OH,H

-H+

O

H

HO

H

HO

H

OHH

OH

OH,H

-H+

O

H

HO

H

HO

OH

HH

OH

OH,H

-H+

Figure 7-9. Comparison of the fragmentation patterns of the deprotonated monosaccharides over the wavelength range of 5.5 to 11 μm. A) Allose. B) Galactose. C) Glucose. D) Mannose.

127

CHAPTER 8 CONCLUSIONS AND FUTURE WORK

Infrared multiple photon dissociation (IRMPD) was used in conjunction with Fourier

transform ion cyclotron resonance mass spectrometry (FTICR-MS) to obtain fragmentation

patterns of mono- and disaccharide isomers in both the positive and negative ion mode. The

fragmentation patterns and IRMPD spectra produced with tunable irradiation from both a

continuous wave, line-tunable CO2 and free electron laser (FEL) were used to differentiate

various lithiated, deprotonated and chlorinated mono- and disaccharides.

The major benefit demonstrated by this research was that an affordable and accessible

line-tunable CO2 laser can be used for the differentiation of isomers. The fragmentation patterns

produced over the wavelength range from 9.2 to 9.7 μm for lithiated mono- and disaccharides

were used to identify and differentiate both their linkages and anomeric configurations. Along

with showing that the output wavelength of fixed frequency CO2 lasers (10.6 μm) is not at all

optimal for differentiation of isomers, this research also demonstrated the benefits of using

multiple wavelengths from a tunable CO2 laser.

The first project of this dissertation showed that CO2 laser irradiation of

O-methyl-gluco- and galactopyranosides produced unique fragmentation patterns over the

wavelength range of 9.2 to 9.7 μm. The relative percent abundance of fragment m/z 169 could

be used to distinguish the glucopyranosides from the galactopyranosides. Furthermore, ratios of

the relative percent abundance of specific fragments (m/z 109/127 for the glucopyranosides and

m/z 169/151 for the galactopyranosides) were used to differentiate the anomeric configuration of

monosaccharide isomers. In both cases, the ratios of the specified fragment ions for the

α-anomers were larger than for the β-anomers. A single-blind study confirmed that the isomers

could be identified based on fragment abundances in conjunction with ratios of the relative

128

percent abundances of specific key ions. In this project, a method for differentiation that could be

useful to other researchers was developed.

In a second project, irradiation of lithiated disaccharide isomers with a line-tunable CO2

laser over the wavelength range of 9.2 to 9.7 μm produced fragmentation similar to that obtained

with a more expensive and complex FEL. The fragmentation patterns seen from 9.2 to 9.7 μm

could be used to differentiate the linkage, while ratios of specific ions were used to determine the

anomeric configurations. Fragmenting the precursor ion (m/z 349) with a laser to produce a

1:2 peak height of precursor ion to fragment ion (m/z 229 for 1-2 linked, m/z 169 for 1-3 and

1-6 linked and m/z 187 for 1-4 linked disaccharides) at wavelengths 9.342, 9.473 and 9.588 μm

allowed the eight isomers to be differentiated. Comparing ratios of the relative abundances of

other key fragments (m/z 187/229 for 1-2 linked, m/z 169/187 for 1-3 and 1-6 linked and

m/z 229/289 for 1-4 linked disaccharides) gave a method to differentiate the anomeric

configuration. Specifically, the ratios calculated for fragments from the β-anomers were larger

than the ratios obtained for the α-anomers for all except the 1-2 linked disaccharides.

The study of deprotonated and chlorinated disaccharides fragmented with a line-tunable

CO2 laser demonstrated that differentiation of the disaccharides in the negative mode is more

difficult than that involving lithiated disaccharides in the positive ion mode. The fragments

obtained from the dissociation of the deprotonated disaccharides were primarily m/z 161 and

m/z 179 and were similar to those obtained for the chlorinated species. While the linkage for

each deprotonated disaccharide could be determined based on the relative percent abundances of

the fragment ions, the anomeric configurations of the deprotonated ions were not determined in

this study. Fragmentation patterns were used to determine the linkage of the eight chlorinated

disaccharides studied. Also, comparison of specific ratios of the relative percent abundances of

129

specific fragment ions (m/z 263/179 for 1-2 linked, m/z 161/179 for 1-4 linked and m/z 161/143

for 1-6 linked) for the chlorinated disaccharides gave a method of discriminating the various

anomers.

Lastly, study of deprotonated disaccharides with a FEL gave spectroscopic evidence for

opening of the monosaccharide anion. An 18O-labeling study of the fragmentation of 1-2 and

1-3 linked deprotonated disaccharides confirmed that the monosaccharide anion (m/z 179)

produced over wavelengths 5.5 to 11.0 μm contained solely the non-reducing monosaccharide.

Multiple fragmentation methods, including sustained off-resonance irradiation collision-induced

dissociation (SORI-CID) and laser irradiation by a fixed-frequency CO2 laser were used to

fragment various disaccharides and isolate the m/z 179 anion. The IRMPD spectra for the

isolated m/z 179 fragment ion revealed a band corresponding to C=O aldehyde stretch around

1720 cm-1. This gave strong evidence for opening of the non-reducing monosaccharide ring and

subsequent loss of anomericity of the monosaccharide anion produced from the fragmentation of

the glucose-containing disaccharides. Furthermore, the IRMPD spectra of several other

deprotonated monosaccharides also contained this peak. Opening of the monosaccharide ring

and thereby loss of the anomeric configuration confirms the need for more information, such as

fragmentation patterns, to differentiate the monosaccharide anomers that compose larger

oligosaccharides when using the deprotonated forms of these compounds for analysis.

Only glucose-containing disaccharides were used in the research discussed in this

dissertation, therefore future studies should examine the fragmentation patterns of other hexose-

containing disaccharides. It may be possible, since the monosaccharides studied in this

dissertation gave unique fragmentation patterns, that disaccharides composed of different

monosaccharide units could also give unique fragmentation patterns that could be used for

130

isomeric differentiation. The patterns of these smaller saccharides could then be used to

differentiate the linkage and monosaccharide units within larger oligo- and polysaccharides.

Since the largest saccharide units studied here were the disaccharides, larger saccharide units

such as trisaccharides composed of various monosaccharide units should be studied. A major

limitation of this research was that only pure samples of each disaccharide were used. Since in

nature mixtures of anomers are often present simultaneously in solution, a method that can

determine the percentage of each anomer within a mixture of saccharides should be developed.

Lastly, an instrumental set-up that utilizes an optical parametric oscillator (OPO) in

conjunction with a FTICR mass spectrometer may be useful for studying various hexoses. The

use of an OPO allows access to the 2.28-4.67 μm wavelength range, which corresponds to the

O-H stretch region. The various O-H stretches could be helpful in differentiating anomers of

mono- and disaccharide

131

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BIOGRAPHICAL SKETCH

Sarah Elizabeth Stefan was born in 1983 to Robert and Elizabeth Stefan. She grew up in

Plymouth, Massachusetts, with her parents and four siblings. Sarah attended Plymouth public

schools for elementary through high school. She graduated in the top five of her high school

class in May 2001. She then attended Wheaton College, a small liberal arts college in Norton,

Massachusetts. Under the direction of Dr. Laura Muller, she worked on her undergraduate

honor’s thesis entitled Analysis of Fingerprint Residue via Infrared Microscopy. In May 2005,

she graduated summa cume laude and with chemistry departmental honors, receiving a Bachelor

of Arts degree in chemistry with a minor in American politics. After graduation, Sarah moved to

Gainesville, Florida, to begin her graduate studies in analytical chemistry at the University of

Florida. She then joined the group of Dr. John Eyler and began her work using infrared multiple

photon dissociation and Fourier transform ion cyclotron resonance mass spectrometry in the

differentiation of carbohydrates. She received her Doctor of Philosophy from the University of

Florida in the spring of 2009.