ion fragmentation in mass spectrometry 01-16-07f.pdf · ion fragmentation in mass spectrometry ......

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S. Barnes - J. Prasain 01/16/07

Ion fragmentation in mass spectrometry

Stephen Barnes and Jeevan [email protected] [email protected]

4-7117 6-2612


Lecture goals• Value of fragmentation in determining

structure• How peptides fragment

– Interpreting the tandem mass spectrum• Automating identification of peptides from

their fragment ions– pros and cons

• Controlling fragmentation– Choice of ionization and fragmentation

methods

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Why ion fragmentation provides useful information

• Compounds can have the same empirical formula, i.e., the same molecular weight or m/z, but be different chemically.

• Breaking them into parts (fragmenting them) helps to identify what they are.

• Each of the following gives the same [M+2H]2+ ion– NH2VFAQHLK-COOH NH2VAFQHLK-COOH– NH2VFQHALK-COOH NH2VHLAFQK-COOH

• In proteomics we want to distinguish these peptides


What is MS-MS (tandem mass spectrometry)?

Analyzer 1 Analyzer 2Molecular ions selected ion

gas

3


Fragmenting a peptidex2

a2R1 O R2

| || |H2N--C--C--N+=C

| | |H H H

R3 O R4

| || |+O=C--HN--C--C--N--C--COOH

| | |H H H

a2x2

http://www.matrixscience.com/help/fragmentation_help.html

NH3+-CHR1-CO-NH-CHR2-CO-NH-CHR3-CO-NH-CHR4-COOH

R1 O R2

| || |H2N--C--C--N=C--C=O+

| | |H H H

R3 O R4

| || |H3N+--C--C--N--C--COOH

| | |H H H

b2 y2

y2

b2

R1 O R2 O| || | ||

H2N--C--C--N=C--C--NH3+

| | |H H H

R3 O R4

| || |+C--C--N--C--COOH| | |H H H

c2 z2

z2

c2


bn = [residue masses + 1]yn = [residue masses + H2O + 1]

Calculating expected b- and y-ion fragments

Alanine 71.037 Leucine 113.084Arginine 156.101 Lysine 128.094Asparagine 114.043 Methionine 131.040Aspartic acid 115.027 Phenylalanine 147.068Cysteine 103.009 Proline 97.053Glutamic acid 129.043 Serine 87.032Glutamine 128.058 Threonine 101.048Glycine 57.021 Tryptophan 186.079Histidine 137.059 Tyrosine 163.063Isoleucine 113.084 Valine 99.068

ADGTWLEVRb3 = ADG, 71.04+115.03+57.02+1= 244.09

y3 = EVR, 129.04+99.07+156.10+18+1= 403.21

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Identification of daughter ions and peptides - reading the sequence

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300mass0

100

%

y101100.61

y9987.52

b3375.21

b2262.12

234.13

y1175.12

y2274.19

y8916.49

parent681.45

y4487.32

y5602.32

y6730.43

b8875.42

y111247.69

y7859.45

b4446.23


Identification of daughter ions and peptide sequence

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300mass0

100

%

y101100.61

y9987.52

b3375.21

b2262.12

234.13

y1175.12

y2274.19

y8916.49

parent681.45

y4487.32

y5602.32

y6730.43

b8875.42

y111247.69

y7859.45

b4446.23

b ions 262 375 446 503 632 760 875 989 1088 1187 1343 N F L A G E K D N V V R

y ions 1361 1247 1100 987 916 859 730 602 487 373 274 175

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What’s in a peptide MSMS spectrum?

• In most cases, some, but rarely all, of the theoretic b- and y-ions are observed

• Besides b- and y-ions, other types of fragmentation can occur to form an and xnions, as well as also losing CO, NH3 and H2O groups

• Internal cleavage reactions can occur at acidic (Asp - Glu) residue sites


Immonium and Related Ions84.08 70.07

87.06 120.08 86.10 --- --- 102.05 101.11 88.04 87.06 72.08 72.08 87.09129.10 100.09

112.09N-terminal ionsa-NH3 ions --- 217.10 330.18 401.22 458.24 587.28 715.38 830.40 944.45 1043.52 1142.58 ---a ions --- 234.12 347.21 418.24 475.27 604.31 732.40 847.43 961.47 1060.54 1159.61 ---b-NH3 ions --- 245.09 358.18 429.21 486.23 615.28 743.37 858.40 972.44 1071.51 1170.58 ---b-H2O ions --- --- --- --- --- 614.29 742.39 857.42 971.46 1070.53 1169.59 ---b ions --- 262.12 375.20 446.24 503.26 632.30 760.40 875.43 989.47 1088.54 1187.61 ---

1 2 3 4 5 6 7 8 9 10 11 12H - N F L A G E K D N V V R

y ions --- 1247.67 1100.61 987.52 916.48 859.46 730.42 602.33 487.30 373.26 274.19 175.12y-NH3 ions --- 1230.65 1083.58 970.50 899.46 842.44 713.39 585.30 470.27 356.23 257.16 158.09

y-H2O ions --- 1229.66 1082.60 969.51 898.47 841.45 712.41 584.32- --- --- -- ---

Other ions observed in peptide fragmentation

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Towards automated MSMS sequencing

• The 2D-LC-ESI-MSMS method (MuDPIT) generates 50,000+ MSMS spectra for each sample

• If it takes 15 min to hand interpret one MS-MS spectrum, then it would take 12,500 hours to complete the analysis. For someone working 8 hours/day and a five-day week, this would be about 6 years!

• Using SEQUEST and MASCOT, methods were developed to use computer-driven approaches to analyze MSMS data


Issues in MS-MS experiment• At any one moment, several peptides may

be co-eluting• Data-dependent operation:

– The most intense peptide molecular ion is selected first (must exceed an initial threshold value)

– A 2-3 Da window is used (to maximize the signal)

– The ion must be in 2+ or 3+ state– Since the ion trap scan of the fragment

ions takes ~ 1 sec, only the most intense ions will be measured

– However, can use an exclusion list on a subsequent run to study minor ions

threshold

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The SEQUEST approach• Each observed MSMS spectrum has a corresponding molecular ion

[M+nH]n+. For ion trap data, ions are selected from the known or virtualproteome that are within 1 Da. These are then “fragmented” in silico to produce b- and y-ions and less abundant fragment ions.

• The cross correlations of the observed MSMS spectrum to each of the virtual MSMS spectra are calculated. The peptides are scored and the one having the highest score is deemed to be identified.


What has SEQUEST provided to proteomics?

• Initially, it seemed an awful lot! Typically, the “identified” proteins covered most of known biochemistry, so they satisfied everybody

• But the method obviously has limitations. There is redundancy - each protein yields multiple peptides

• The number of unique proteins was much less than the observed peptides

• Critically, it was missing controls

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SEQUEST sequencing

• Use of SEQUEST requires considerable computing power - if there are 500 possible peptides to compare, then examination of 50,000+ spectra would require 25 million correlations

• Data analysis is typically carried out using computer clusters to accelerate the analysis


More haste, less speed?

• Post analysis, the masses of the peptides triggering MS-MS are used to create a set of virtual peptides with masses within + 1 Da

• Predicted MS-MS are compared to the observed and the best fit is reported as a hit

• The abundance of these hits are plotted in the figure as closed circles

• Post analysis, the masses of the peptides triggering MS-MS are used to create a set of virtual peptides with masses within + 1 Da

• Predicted MS-MS are compared to the observed and the best fit is reported as a hit

• The abundance of these hits are plotted in the figure as closed circles

However, if the sequences of the peptides within + 1 Da are reversed in silico and their predicted MS-MS compared to the observed spectra, a similar histogram is obtained (open circles), but without the right side tail

A forced fit to a set of data will always come up with a match, but not necessarily the truth

A forced fit to a set of data will always come up with a match, but not necessarily the truth

Resing et al. (2004)

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Normal sequence

Reversed sequence

b ions 262 375 446 503 632 760 875 989 1088 1187 1343N F L A G E K D N V V R

y ions 1361 1247 1100 987 916 859 730 602 487 373 274 175

b ions 256 355 469 584 712 841 898 969 1082 1229 1343R V V N D K E G A L F N

y ions 1361 1205 1106 1007 893 778 650 521 464 393 280 133

So, b2corr = y2reverse - 18 and y2corr = b2reverse + 18

A reversed sequence has its problems as a control


How to improve MUDPIT• Reproducible column engineering

– Tandem columns, each built to separate, but high specifications

– Columns on a chip• More careful selection of the parent ion

– Accurate measurement of the peptide’s mass will eliminate many false peptides

– Accurate measurement of peptide fragments’masses

• Greater stringency in assessing score cutoff

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Let’s take a closer look at fragmentation

b3 ion - oxazaloneno b1 ion

Wysocki et al. 2005


Other amino acid fragment ions

Wysocki et al. 2005

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Detecting posttranslational modifications by MS

• A key issue is that the energy of ionization or the collisional process should not exceed the dissociational energy of the PTM

• MALDI-TOF MS with a N2 laser causes fragmentation of a nitrated tyrosine residue– Use ESI to make the molecular ion– Go to another wavelength

• O-glucosyl groups fragment more easily than the peptide to which they are attached– Use electron capture dissociation


Fragmentation of nitrated peptides in MALDI-TOF experiment

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ESI-tandem MS of a nitrated peptide


m/z400 500 600 700 800 900 1000 1100 1200 1300 1400

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

[Abs. Int. * 1000000]c V S S A N M

514.26180c 6

613.33054c 7

903.44322c 8

990.47346c 9

1061.51275c 10

1175.55852c 11

1306.59534c 12

c8 fragment with sugar attached

[M+2H]2+

Sequencing O-GlcNAc peptides by ECD FT-ICR-MSCasein kinase II - AGGSTPVSSANMMSG

H3N CH

C

R1 O

NH

CH

R2

C NH

O

CH

COOH

R3

c ion cleavageb ion cleavage

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Fragment ions of a small 5-mer peptide

Homework - write down the masses of the b and y ions

A Q Y E K

100 150 200 250 300 350 400 450 500 550 600 650

m/z

36

27

18

9

0

Rel

. Int

(%)

129

147

183

200

240

258

276363

403

439

492549

567

637

bn = [residue masses + 1]

yn = [residue masses + H2O + 1]


What is tandem mass spectrometry?

• Tandem mass spectrometry, abbreviated MS/MS, is any general method involving at least two stages of mass analyzers (one to pre-select an ion and the second to analyze fragments induced. This dual analysis can be dual in space, or dual in time. The most commonly used tandem mass spectrometry is the triple quadrupole.

Q1 Q2 Q3

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Metabolomics is an emerging “omics” science that determines the physiological status of a sample or tissue by comparing the concentration of small molecules (biochemicals) in a tissue or sample with a similar measurement in a control sample. Small molecules are a diverse group of natural and synthetic substances that generally have a low molecular weight (f. w. 150-3000).

Metabolomics and small molecules

Genistein(a plant secondary metabolite)

Triglycerides Taxol


Nomenclature: the main names and acronyms used in MS/MS

• Molecular ion: Ion formed by addition or the removal of one or several electrons to or from the sample molecules-Electron Impact (EI-MS). M + e- → M+• + 2e-

• Adduct Ion: Ion formed through interaction of two species and containing all the atoms of one of them plus one or several atoms of them (e.g. alkali, ammonium).

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Contd..

• Pseudomolecular ion: Ion originating from the analytemolecule by abstraction of a proton [M-H]- or addition of proton [M+H]+

• Precursor ion/parent ion: Ions undergoing fragmentation.

• Product ion/daughter ion: Ions resulting from parent/precursor ions.

• Neutral loss: Fragments lost as neutral molecules

• In positive ionization mode, a trace of formic acid is often added to aid protonation of the sample molecules; in negative ionization mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules. Proteins and peptides are usually analysed under positive ionization conditions and polyphenols and acids under negative ionization conditions. In all cases, the m/z scale must be calibrated.


Ways to approach predicting MS/MS

• Likely sites of protonation or deprotonation.• Likely leaving group.• Mobility of protons• Literature study

Fragmentation always follows the basic rules of chemistry

O

OOCH3

HO

OH

Biochanin A

Where are the sites of Deprotonation/protonation?What is the most likely leaving Group in this molecule?

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Applications of MS/MS

• Identification, characterization and authentificationof chemical components in a crude mixture

• Substructure analysis of unknown components

• Quantification of analytes in biological samples


Profiling taxoids metabolites in T.Wallichiana extract

Prasain et al. Anal Chem, 2001

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ESI-MS/MS spectra of taxoids (1-3). Peaks m/z 194 and 210represent the intact alkaloid side chain.


MS/MS precursor-scan spectra of typical alkaloid side chains to identify the basic taxoids compounds in an ethyl acetate

extract of T. wallichiana.

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600 620 640 660 680 700 720 740 760 780 800 820 840 860 880

605622

714

742

758 800

612 628 668 710

742

806.2 848 862

668684

726

744

758770 786

686 710

728

744770 786

800

A

B

C

D

Scaffold (m/z 309)

Side chain (m/z 210)



m/z

Inte

nsity

Comparison of precursor scan spectra obtained from thescaffold m/z 309 and side chain m/z 194, 210 and 252

Taxoids with scaffold m/z 309 and alkaloid side chains are shown by dashed lines


O

OH

O

O

O

OHOH

CH2OH

OH

O

OH

HO

O

OHOCH2OH

Puerarin; mol. wt. 416 Daidzin; mol. wt. 416

OHHO

MS/MS may help distinguish isomer structures

The Kudzu as a source of isoflavones

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O

OH

HO

O

OHO

HO

CH2OHOH

O

OH

O

O

O

OHOH

CH2OH

OH

PuerarinM/z 415

O- and C-glucosides fragment differently in ESI-MS/MS

Prasain et al. J. Agric. Food Chem. 2003

220 280 300 320 360 380 m/z0

%

0

100

%

255.050

256.057

297.043

267.037

268.041281.051

307.065321.046

363.046335.061351.044 381.055

100

240 260 340

[A]

[B]

-162 Da

Yo+

-120 Da

Daidzin m/z 415


Possible product ions of puerarin in ESI-MS/MSin negative ion mode

O

O-

HO

O

OH

O

O-

HO

O

O

O-

HO

O

HO

HO

O

O-

HO

O

OHOCH 2OH

OHHO

m/z 415

m/z 325

-90 Dam/z 295

-120 Da

m/z 267

-28 Da

Prasain et al. J Agric Food Chem. 51, 4213, 2003.

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m/z

100

50

0

50 100 150 200 250m/z

100

50

0

Rel

. Int

. (%

) 65 107

117

151

225

269

6391

107

133

159 180196

224

240

269O

OH

HO

OOH

Genistein

OOH

HO

OOH

Apigenin

O

O-

O-

149

Isomers like genistein and apigenin are readily separated by tandem mass spectrometry


M/z

100

50

0

50 100 150 200 250 300

100

50

0

63 107 139

167

183

195211 239

267

303

139

167

195

223251

287-36

-28

-28-28

-28

-36

-28

-28

O

OO_

HO

Cl

O

OO_

HO

Cl

OH

HCl loss (36 Da) is diagnostic for 3’-chloro derivative of genistein and daidzein in ESI-MS/MS

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100

50

0

Rel

. Int

. (%

)

50 100 150 200 250m/z

100

50

0

Rel

. Int

. (%

)

123

152

170213 229242

269

285

118 152169 197

214225

242

270

285O

OOH

H3CO

HO

O

OOCH 3

HO

OH

H+

H+

Biochanin A

Glycitein

[A]

[B]

Biochanin A and glycitein can be distinguished byESI-MS/MS in positive ion mode


Intensity of product ions indicates their stability -ions bearing aromatic ring are more intense

O

OH

HO

OHO

O

OH

OH

OH

OH

OH44

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Metal ion adducts are common in the positive ion mode

TIC

EMS (positive survey scan in trap)

EPI (Enhanced Product Ion Scan of m/z 579 in Trap)

Dimer [M+H]+Dimer [M+Na]+

Trimer


Comparison of product ions help elucidate the unknown structures

O

OOH

HO

O

HO

OH

OH

H2COH

+OH

150 200 250 300 350 400

100

%

255268

282

311

-120 431

Monohydroxylatedpuerarin

200 250 300 350 400

100

50

0

267 295

\

415

-120

Puerarin

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S. Barnes - J. Prasain 01/16/07m/z

100

50

0

100 200 300 400m/z

100

50

0.0

Rel

. Int

. (%

)253

109

161

224

253

271

433

433

180 Da

162 Da

Tandem mass spectrometry can provide crucialinformation on structure of unknown glycosides

O

O-

HO

O

O

OHO

OH

HOCH2OH

O

O-

O

O

OH

OHO

HO

HO CH2OH

162 Da

Possiblestructure

Possiblestructure

S. Barnes - J. Prasain 01/16/07m/z

0

300 400 500

100

50

0

433

447449

463

259291

433

449

100

50

479

Ions obtained from neutral loss scan of 162 [A] and 132 [B] In the EtOAc extract of cranberry

fresh fruits

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100 200 300 400m/z

100

50

0

Rel

ativ

e In

tens

ity (%

)

5985

113133 181 224

269

Glucuronide loss (176 Da)Da)

O

OHHO

OH

COOH

445

Product ion spectrum of genistein glucuronidein ESI-MS/MS

Glucosides/glucuronides conjugates are easily cleaved off by higher potential at orifice


Change in mass

Methylation 14Demethylation -14Hydroxylation 16Acetylation 42Epoxidation 16Desulfuration -32Decarboxylation -44Hydration 18Dehydration -18

Metabolic rxn

Change in mass is associated with possiblemetabolic reactions

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Ionization mo Scan

Glucuronides pos/neg NL 176 amuHexose sugar pos/neg NL 162 amuPentose sugar pos/neg NL 132 amuPhenolic sulph pos NL 80 amuPhosphate neg Precursor of m/z 79 Aryl-GSH pos NL 275 amuAliphatic-GSH pos NL 129taurines ps Precursor of m/z 126N-acetylcysteneg NL 129 amu

NL = neutral loss.

Conjugat

Characteristic fragmentation of drug conjugates by MS/MS


References1. Electrospray Ionization Mass Spectrometry by Richard B.

Cole.

2. Stefanowicz P, Prasain JK, Yeboah KF, Konishi Y. Detection and partial structure elucidation of basic taxoids from Taxus wallichiana by electrospray ionization tandem mass spectrometry. Anal Chem. 2001;73:3583-9.

3. Prasain JK, Patel R, Kirk M, Wilson L, Botting N, Darley-Usmar VM, Barnes S. Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites. J Mass Spectrom. 2003;38:764-71.

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Question-?What structural information do you get from the MS/MS Spectra shown below.?

ion fragmentation in mass spectrometry 01-16-07f.pdf · ion fragmentation in mass spectrometry ......

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