Life Science and Industrial Applications of the AXIMA ResonanceHigh Vacuum MALDI-QIT-TOF Mass Spectrometer

AXIMA Resonance

MO358

Glycomics

Proteomics

Top-Down Proteomics

Post-Translational Modifications

Non-Enzymatic / Chemical Modifications

Lipidomics

Small Molecules

Polymer Analysis

Ac

Me

MALDI-QIT-TOF MSHigh performance as standard.. .

. . .Results delivered

The AXIMA Resonance

Introduction

The AXIMA Resonance is a versatile MALDI massspectrometer employed in a broad range of biologicaland organic analytical applications. Its main advantagesare:

• True high-vacuum MALDI ion source

• Quadrupole ion trap with MSn capability for structuralelucidation

• High mass resolution and mass accuracy independentof the number of cycles during MSn experiments

• Ability to isolate parent ions with isotopic resolutionover a wide mass range - up to 1000 FWHM

• Variable energy CID control during acquisition

• Outstanding sensitivity - uncompromised design, toensure highly efficient trapping functionality

• Low sample consumption - allowing many more MSn

experiments to be performed on the same spot

• Variable repetition rate N2 laser

• Manual or fully automated operation allowing theseamless analysis of few or many samples as required

• Dedicated software solutions for a wide range ofapplications

Given these advantages, the AXIMA Resonance is ideallysuited for high-end proteomics studies as well as lessconventional studies with polymers, lipids and smallmolecules. The purpose of this document is to give aninsight into the use of the AXIMA Resonance in theseapplications.

5AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSGlycomics

Glycomics

Classical glycan and glycopeptide analysis using MS2 and MS3

AXIMA Resonance, an ideal solution for carbohydrate research

4 Glycomics

Glycosylation is one of the major post-translational modifications (PTM) and has significant effects on protein folding and

ultimately on protein activity. Identification of the glycan structure forms much of the challenge in this research field. However, of

equal importance is the determination of the point of attachment to the protein. The AXIMA Resonance offers the advantage of

being able to address both of these challenges.

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.26

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3390

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1687

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1920

.55

3067

.70

2498

.48

Pep

Pep

Pep

Pep

Pep

Pep

Pep

PrecursorPep

-18

-18

-18

-18

-18-18

-18-18

203 203 203162 162+18162162162

Pep

120

-18

Pep-18

b17b16

y10

b12-17

1376

.23

y11 y12

y9

1000

.32

y8y7

Ser Cys Glu Thr Val

0,2X

+b12-18

MS3MS3

Figure 1: MS2 spectrum of a human transferrin glycopeptide [M+H-2Sia]+ (m/z 4139) that has already lost twosialic acid moieties. The spectrum exhibits the fragmentation of the glycan part attached to the peptide.

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x20 x50

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.13

765.

08

1275

.12

913.

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.14

856.

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1541

.23

2582

.68

1258

.11

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.20

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1861

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1936

.56

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2162

.32

2334

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.32

y9

y8y7y6y5

b 5-1

7

b 6-1

7b 7

-17

b 8-1

7

b 9-1

7

b 11-

17

b 12-

17

+b12-18

+y13y12y11y10 +y14 +y15 +y16

2197

.45

+y17 +y18 +y19

+y20-17

Precursor+Pep-17

Gln+Gln

0,2X

GlnHisLeuPheGlySer+AsnValThrAspCysSerGlyAsn

Figure 2: Positive ion MS3 spectrum of the product ion at m/z 2701 (from the MS2 analysis of m/z 4139 ion),exhibiting fragmentation of the peptide backbone. The detection of specific fragments allowed the identificationof the glycosylation site.

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1069

.24

548.

79

420.

81

913.

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93

1052

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618.

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401.

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856.

14742.

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914.

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941.

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1082

.16

y8y7y6y5

781.

11

y4y3 Precursor

CysSerGlyAsnPheCys

574.

99

725.

05

b7

986.

13

b8

748.

11

b4 b5 b6

ArgPheLeuCysPhe

Carbamidomethyl+SH

Figure 3: Positive ion MS3 spectrum of the product ion at m/z 1160, exhibiting fragmentation of the peptidebackbone. The peptide sequence is completely assigned.

603-QQQHLFGSNVTDCSGNFCLFR-623

MS2

m/z 2701m/z 4139603-QQQHLFGSNVTDCSGNFCLFR-623

17 MS3

603-QQQHLFGSNVTDCSGNFCLFR-623m/z 1160m/z 4139

CSGNFCLFR

MS2 MS3

603-QQQHLFGSNVTDCSGNFCLFR-623 m/z 4139

7AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSGlycomics6 Glycomics

Biosimilars

Accurate Glycan Analyser, a one-stop platform for smarter carbohydrate research

Accurate Glycan Analyser is a software-directed multistage MALDI mass spectrometric approach using the unique MSn capability

offered by the AXIMA Resonance and a populated database of real MS and MSn glycan spectra, guiding the user through the

process of structural analysis of glycans without requiring an expert level of knowledge.

Unlike others, the Accurate Glycan Analyser database is populated with glycans of high biological relevance, that have been

enzymatically generated using the extensive glycogene know-how from the Japanese National Institute of Advanced Industrial

Science and Technology. This recombinant technology-based approach is unique in this field and provides the researcher with a

database of considerable added value.

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1358.58

1359.59

1077.42

1078.43

1376.60874.32

712.25507.24 1375.48849.37 1010.35 1215.51374.01

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712.261077.42

993.42

1078.42

994.43388.13

874.32713.27

550.211358.60915.35 1196.52347.08 712.61

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1663.65

2028.79

2319.89

1257.48 2341.861954.75 2610.98

2654.952961.992245.85

1501.601970.70 2275.90 2946.06

2626.94

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1214.49 1579.64

1335.53

2054.78 2733.022419.921741.69

2106.84 2367.86 3046.092754.99

2076.772441.90

3068.062715.00

Figure 4: Overview of the Accurate Glycan Analyser - AXIMAResonance workflow applied to PA-labelled glycans released fromfetuin. At the MS level, potential glycan candidates are proposed.The software then suggests MS2 precursor masses that can beanalysed to confirm the proposed glycan structure. In some casesMS2 alone is not sufficient to unequivocally identify a single glycancandidate, and thus the Accurate Glycan Analyser softwareproposes further MSn precursor candidates (up to MS4). For thefetuin example shown, MS3 was necessary to unambiguouslyidentify the glycan structure.

MS

MS2

m/z 1376

MS3

m/z 1358

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2 a6 b 4 b 4

a 3

b 4 b 4

a6 b 4 b 4

a 3

OLIGO_ID Structure

ONG-00001d

ONG-00001c

ONG-0000b7

Job ID: 226675355Create Date: 2011-02-28 22:22m/z of precursor ion: 1376.535

Candidate in DB:

ONG-00001dONG-00001cONG-0000b7

Threshold

Score

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2 a6 b 4 b 4

a 3

b 4 b 4

a6 b 4 b 4

a 3

OLIGO_ID Score Structure

ONG-00001d 27.3159844

ONG-00001c 20.6774102

ONG-0000b7 12.6072472

Search resultMS/MS search result m/z of precursor ion: 1376.535

ONG-00001dONG-00001cONG-0000b7

Threshold

Score

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2 a6 b 4 b 4

a 3

b 4 b 4

a6 b 4 b 4

a 3

OLIGO_ID Score Structure

ONG-00001d 462.8711705

ONG-00001c 26.0603462

ONG-0000b7 5.3392022

Search resultMS/MS/MS search result m/z of precursor ion: 1376.535/1358.582

3 candidateglycans

2 candidateglycans

glycan structureconfirmed

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2a

6

b 4 b 4

a6 b 4 b 4

a 3

b 4 b 2a

6

b 4 b2

b 4 b 2

a6 b 4 b 4

a 3

b 4 b 2a

6

b 4 b 4

a6 b 4 b 4

a 3

b 4 b 2a

6

b 4 b2

b 4 b 4

a6 b 4 b 4

a3

b 4 b 2

a6

b 4 b2

b 4 b 6

b 4 b 4

a6 b 4 b 4

a3

b 4 b 2

a6

b 4 b2

b 4 b 6

b 4 b 4

a6 b 4 b 4

a3

b 4 b 2

a6

b 4 b2

b 4 b 6

1x

1x

2x

2x

2x

3x

2x

3x

4x

Identified glycan structures

Recombinant

Plasma-derived

Factor IX

Recombinant

Plasma-derived

AccurateGlycan

Analyser

Human Factor IX (FIX), a single-chain glycoprotein, plays an important role in the intrinsic blood coagulation pathway and is used

as a treatment for the bleeding disorder haemophilia B. There are two known glycosylation sites (Asn-157 and Asn-167).

The production of therapeutically active recombinant and plasma-derived FIX is approved and regulated by US and European

agencies. The current regulatory levels of quality control require meticulous characterisation of the glycosylation pattern. However,

due to the branched nature of glycans, the unambiguous identification of their structure is not trivial. The Accurate Glycan

Analyser - AXIMA Resonance platform facilitates this identification.

Figure 5: Summary of the identified glycan structures observed inplasma-derived and recombinant FIX using the Accurate GlycanAnalyser software in combination with MSn analyses. Significantdifferences were observed, in particular the extent of fucosylationand the core structures of the identified glycans.

PA-labelled

MS (+78 Da)

9AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSProteomics8 Proteomics

Proteomics

Classical Proteomics

AXIMA Resonance, designed with sequencing and structural characterisation in mind

Peptide mass fingerprint (PMF) and MS2 ion searching are widely used to enable high throughput protein identification. The

example shown here illustrates the suitability of the AXIMA Resonance for classical proteomics applications. The proteins retained

during a novel immunoadsorption therapy for rheumatoid arthritis (schematic 1) were studied in detail, combining 1D gel

electrophoresis, PMF and MS2 analysis.

Schematic 1: Treatment schematic of Staphylococcal Protein-A basedimmunoadsorption therapy. The patient plasma is separated from the redblood cells, passed through the immunoadsorption column, recombinedwith the blood cells and returned to the patient. Immobilised Protein-Abinds immunoglobulins and attached proteins (antigens).

Patient 1 Patient 2

1

23

45

6

Figure 6: 1D SDS-PAGE of the proteins retained on column during immunoadsorptiontreatment of rheumatoid arthritis patients 1 and 2. Marked bands were excised andsubjected to in-gel digestion with trypsin.

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1682.81

1124.65

1520.69

1490.72

1108.55

943.45

1567.761301.681009.62

1566.68982.50 1283.621873.931580.76

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508.02

1247.64

508.30

491.00

1472.72

1474.72623.01

939.52361.03 751.28 1446.671229.64

1013.46

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its 45

40353025201510

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Band 1

IGHG1

Band 2

CO3

Band 3

IGHG1

Band 6

APOA1

Band 5

APOE

Band 4

HRG

GC1

APOA1

Figure 7: PMF mass spectrum of the tryptic digest of gel band 4 (left) and MS2 analysis of m/z 1490 with individual Mascot MS2 Ion Search(right) result.

Figure 8: Summary of Mascot Ion Search results for all selected gel bands analysed for each patient. Matches to apolipoprotein A1(APOA1), apolipoprotein E (APOE) and histidine-rich glycoprotein (HRG) were identified. Lipoprotein A1 has been reported in theliterature to be present at an increased level in rheumatoid arthritis patients, however, little is understood of its biological role ininflammatory disease or the biological and therapeutic significance of the partial removal of these proteins from the bloodstream ofrheumatoid arthritis sufferers.

MS2

of1490

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00 25 50 75

Probability Based Mowse Score

HRG_human

11AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSProteomics10 Proteomics

Microbial Proteomics

Expanding rapid microbial identification through targeted proteomics

Mass spectrometric microbial identification is generally obtained using straightforward fingerprinting methods (eg: using the

Saramis software on the AXIMA IDplus platform). However, the investigation of differences at the fingerprint level using MS2

methods can provide more valuable and detailed information regarding the bacterial cells investigated.

The emergence of multi-drug resistant (MDR) enterobacteriaceae that can produce extended-spectrum beta-lactamases (ESBLs) is

currently a major worldwide concern. These enzymes confer resistance to a wide spectrum of beta-lactam antibiotics currently

used as first-line empirical therapy in the management of gram negative bacterial infections.

Significant differences in the microbial fingerprint were observed between ESBL- and non ESBL-producing E. coli. In particular, two

peaks at m/z 2341.3 and m/z 3790.0 dominated the fingerprint from ESBL-producing E. coli. These potential biomarkers were

selected for more in depth analysis by high resolution MS2.

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1314.77

2324.30

1623.96

2331.221114.68

700.37 1540.86 1868.09

1442.86 2195.261167.701943.07

1651.90 2289.271338.77

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3747.61

1570.88

3729.622206.15

3767.571707.92 2348.211160.62

3149.43 3754.621773.93 2260.17 2724.30

912.54 3216.491354.80

Figure 9: MS2 mass spectrum of m/z 2341.

Figure 10: MS2 mass spectrum of m/z 3790.

Figure 11: The MS2 data were searched using Mascot Ion Searchagainst all E. coli proteins in the NCBI database. Both peptidesmatched with high confidence to the same protein: acid shockprotein precursor.

Nu

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AAKKHHKNAKAEQKAPEQKAQ (m/z 2341)

gi | 91210807 Mass: 11571 Score: 71 Queries matched: 1acid shock protein precursor [ Escherichia coli UTI89]

gi | 91210807 Mass: 11571 Score: 91 Queries matched: 1acid shock protein precursor [ Escherichia coli UTI89]

AETATTPAPTATTTKAAPAKTTHHKKQHKAAPAQKAQ (m/z 3790)

Culture

Saramis

Axima IDplus

Protein characterisation

Biomarker discovery

Strain identification

Culture

A i ID l

Biomarker discov

Saramis Strain identificati

MSn

13AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSNon-Enzymatic / Chemical Modifications12 Top-Down Proteomics

Non-Enzymatic / Chemical Modifications

Thinking outside the box...detecting protein modifications that are not in the database

Glycation is the non-enzymatic modification of a protein with a sugar molecule. Known as the Maillard reaction, it is the result of

the condensation between the reducing end of a sugar and the reactive amine group of a protein. Unlike the enzyme-controlled

glycosylation process, glycation is haphazard and tends to impair the function of the modified protein. Detailed characterisation of

glycation products is therefore of high importance in the physiology and pathology of human diseases such as the chronic vascular

complications of diabetes. It is also, however, relevant to the field of food processing.

In this study human serum albumin (HSA) glycation was investigated.

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1467.84

1181.62

962.58

927.49 1311.73

875.51 1623.791197.64

810.42 1449.811853.89

2217.14 2489.24

Glycated peptide

Figure 15: HSA was incubated with glucose for three weeks, digested with trypsin and the PMF was compared tothat of unmodified HSA. Several differences between native and glycated HSA PMF were observed. These wereanalysed with MS2, eg: MS2 of m/z 1181 (inset).

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1061.55

1145.56

1061.96

588.31381.19 774.40 909.43

Figure 16: Glycation is a labile modificationunder UV MALDI conditions and in MS2

produces a distinctive neutral loss patternindicative of the modification (loss of 36,120 and 162 Da from the precursor). Usingthe PTMFinder software, the specific MS2

neutral loss signature for glucose wasscreened against glycated HSA digestiondata. The screenshot illustrates thePTMFinder results highlighting the neutrallosses on the spectrum.

Top-Down Proteomics

Mapping intact proteins by combining In-Source Decay and MSn

In-Source Decay (ISD) is a widely accepted technique in the sequencing of whole proteins by mass spectrometry, commonly referred

to as top-down proteomics. Of particular importance is the characterisation of the protein N-terminus. Top-down sequencing using

the AXIMA Resonance is achieved by a novel approach combining ISD and pseudo MSn analyses in an ion trap. This has the

advantage of desorbing large proteins and maintaining high resolution on the selection and detection of fragment ions.

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m/z

X5

1521.78

c33

c32c31c30c29c28c27

c26c25

c24c23

c22

c21c20

c19c18

c17

c16

c15

c14

c13

c12c11

c10

c9

b13

b12

b11b10b9b9b8

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* * * *

b12

b13

b17b16

c21

b12-NH3b12-H2O

b13-NH3b13-H2O

c21-NH3c21-H2O

[M+H]+

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m/z

X5 X5

1406.69

y7

y6y5y4

a7

y9y10

y11

b8 b9

b10

*

b12

Figure 12: AXIMA Resonance ISD spectrumof HSA protein with its correspondingsequences. HSA protein gave a regularc-ion series from c7 to c33 and some b- anda-ion series. Observed mass resolution wasgreater than 11 500 FWHM for the c33 ion.

Figure 14: Pseudo MS4 of the b13-ion(product ion of the c22-ion, m/z 1521.75).Many species were identified: b8-b10, b12,y4-y7 and y9-y11. Mass resolution wassimilar to the pseudo MS3 spectrum.

Figure 13: Pseudo MS3 of the c22-ion(m/z 2540.30). The two main speciescorrespond to b12 and b13 ions withtheir neutral losses of NH3 and H2O.Other species are annotated: b16, b17and c21. The observed mass resolutionsfor b13 ion, b13- NH3 and b13- H2Oexceeded 9 000 FWHM. The asterisks “*”mark internal fragments.

ISD of human serum albumin provided a significant portion of the N-terminal sequence.

Pseudo MS3 and pseudo MS4 fully characterised the N-terminus of the protein.

D A H K S E V A H R F K D L G E E N F K A L V L I A F A Q Y L Q Q C . . . . .

a9

b8 b10 b12 b16 b17b13b11b9b7 a24 a25a22a19

c7 c13c9 c12c10 c11 c14 c16 c18 c22c17 c21c19 c24c25c26 c27c23c20c15 c28 c29 c30 c31 c32

D A H K S E V A H R F K D L G E E N F K A Lb12 b16 b17b13 c21

D A H K S E V A H R F K Db12b9b8 b10

y11 y9y10 y6 y4y5y7

PTMFinder is Shimadzu proprietary software that uses data mining to investigate protein modifications, including hypothetical and

novel modifications. PTMFinder software offers the possibility of screening large MS2 datasets for specific peptide modifications.

15AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSPost-Translational Modifications14 Post-Translational Modifications

Post-Translational Modifications

MSn capability with high mass accuracy - an ideal alliance for post-translational modification analysis

Collagen is a highly abundant protein in mammals and constitutes the main protein within connective tissue, tendons, ligaments

and skin. Hydroxylation is a common post-translational modification of collagen formed through the reaction of proline amino acid

residues with prolyl hydroxylase. Alpha-1 type 1 collagen was investigated through enzymatic degradation and MSn mass

spectrometry. Several trypsin-digested peptides were found to contain multiple hydroxylation sites due to the prevalence of proline

residues. Mass spectrometric elucidation of the exact location of hydroxylation sites was performed using the AXIMA Resonance

mass spectrometer combining MS2 and MS3 for hydroxyproline (HyP) identification.

113.11

113.09

113.07

113.05

113.031 2 3 4 5 6 7 8 9 10 11

analysis

m/z

measured HyP residue mass measured Leu residue mass

Figure 17: As HyP and Leu/Ile are isobaric in nominal mass(113.04768 and 113.08406 Da respectively), very high accuracy wasnecessary in order to specifically determine HyP sites. The accuracyof the AXIMA Resonance is sufficient to differentiate HyP fromLeu/Ile (0.03638 Da) as illustrated in the statistical graph ofmultiple measurements of HyP (blue trace) and Leu/Ile (red trace)within HyP-Bradykinin and LHRH peptide sequences respectively.

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X10 X50

1604.7668

1586.7616

980.4747

1560.7412

789.34151142.5249

1944.88362349.1063

691.3523 2156.9919

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1251.6394

1234.6227

1191.60751138.5986

1233.62501173.60041148.5697 1207.6204

1171.5400 1247.64581145.5578

113.0408HyP

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1379.7022

1450.7406980.5212

1451.7459

920.4803550.29981381.7142

806.4096533.2797 1543.77721063.5568

766.4030463.2315 1322.6848

Figure 18: MS2 spectrum of the proteolytic peptide GSPGADGPAGAPGTPGPQGIAGQR ((M+H)+ = 2393 Da) from ITRAQ modified collagenalpha-1(I). The localisation of the HyP residue was demonstrated by a shift in the predicted y-ions (highlighted in yellow).

Figure 19: The dominant fragment ion at 1604 Da was selected for MS3 analysis and this confirmed the location of the HyP residue.

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449.2304

431.2316

362.2085 771.3397

341.1123450.2256

201.1355 674.3355305.1973522.2730 774.3169421.2339

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60

80

100

%Int.

200 300 400 500 600 700 800 900 1000

m/z

380.1892

522.3017

550.2992420.1963

283.1367 436.2226

601.3381 806.3347431.2403963.4196

269.1571523.3086

964.4278695.3294 823.3598300.1692507.2666

Figure 20: Combination of the MS3 data for the fragment ions 980 Da (A) and 789 Da (B) with that of 1604 Da (Figure 19) provided thecomplete amino acid sequence for this peptide.

A B

Figure 21: The MS2 data were interrogatedusing PTMFinder software. Herehydroxyproline was defined as a possiblemodification of proline (ΔM = 15 Da) andthe MS2 of m/z 2393 was screened. A singleHyP modification was correctly identifiedat amino acid position 12 out of thepossible 5 proline sites within the peptide.

b Seq y362.2341 G449.2661 S 2031.9843546.3189 P 1944.9522603.3404 G 1847.8995674.3775 A 1790.8780789.4044 D 1719.8409846.4259 G 1604.8140943.4786 P 1547.7925

1014.5158 A 1450.73971071.5372 G 1379.70261142.5743 A 1322.68111255.6220 HyP 1251.64401312.6435 G 1138.59631413.6912 T 1081.57491510.7439 P 980.52721567.7654 G 883.47441664.8182 P 826.45301792.8767 Q 729.40021849.8982 G 601.34161962.9823 I 544.32022034.0194 A 431.23612091.0408 G 360.19902219.0994 Q 303.1775

R 175.1190

b Seq y98.0600 P

155.0815 G 883.4744252.1343 P 826.4530380.1928 Q 729.4002437.2143 G 601.3416550.2984 I 544.3202621.3355 A 431.2361678.3570 G 360.1990806.4155 Q 303.1775

R 175.1190

b Seq y362.2341 G 503.2096449.2661 S 446.1882546.3189 P 359.1561603.3404 G 262.1034674.3775 A 205.0819

D 134.0448

17AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSSmall Molecules16 Small Molecules

Small Molecules

MSn, maximising structural information for small molecules

Anthocyanins belong to the flavonoid group of polyphenolic compounds and have been investigated for multiple health benefits

related to their anti-oxidant nature.

R2

HO

OH

OH

R3

OR2

OOH

OHO

OHO

OCH3

OHO

Anthocyanin R1 R2 R3 Phenolic acid esters

Pelargonidin (Pg) H H

Cyanidin (Cy) OH H

Peonidin (Pd) OCH3 H

Delphinidin (Dp) OH OH

Petunidin (Pt) OCH3 OH

Malvinidin (Mv) OCH3 OCH3

p-coumaroyl

caffeoyl

feruloyl

sugarmoiety

Figure 22: Basic structure of an anthocyanin. Many forms exist, the six most common core structures are shown in the table. The sugarmoiety varies between mono-, di- and tri-saccharide units with various phenolic acid ester groups.

0

20

40

60

80

100

%Int.

400 600 800 1000 1200

m/z

X5 X10 X5 X20

271.29519.37

271.60

520.23

741.19

273.25475.26

989.09

771.19

293.30

1019.08

522.43400.29757.18648.22

903.151151.02

1

2

3

45

6

7

8

9

10

11

12

Figure 23: The MS spectrum of red radish extract contains acomplex mixture of anthocyanins.

0

50

100

%Int.

300 400 500 600 700 800 900 1000

m/z

X5 X5

271.03

519.11

771.23

475.13

433.10

271.03

271.06

271.26

741.26

519.16

757.28

793.27523.19433.15

Figure 24: MS2 analysis of three of the radish anthocyanins (m/z 989 (A), m/z 1005 (B) and m/z 1019 (C)) indicatesthat the fragmentation pathway mainly consists of the loss of the sugar moieties: loss of malonyl (86 Da), of5-(malonyl) glucoside (248 Da), of the modified diglucosides at position 3, of both the modified diglucosides andmalonyl and finally complete loss of all the sugar moieties to form the Pg-aglycone.

0

20

40

60

80

100

%Int.

120 140 160 180 200 220 240 260 280

m/z

X20

271.03

120.96

173.01 197.01145.01

225.01

169.01

*

*

**

*

*

*

*

**

*

**

*

*

Figure 25: MS3 analysis of the Pg-aglycone fragment ion (m/z 271). Each of the peaks annotated with *corresponds to a known fragment of the Pg-aglycone structure. Fragmentation proceeds through loss ofwater (18 Da), loss of CO (28 Da) and loss of acetyl (42 Da).

1 pelargonidin-aglycone

2 pelargonidin-3-glucoside

3 pelargonidin-3-glucoside acetylated

4 pelargonidin-3-(malonyl) glucoside

5 pelargonidin-3-(p-coumaroyl) diglucoside

6 pelargonidin-3-(caffeoyl) diglucoside

7 pelargonidin-3-(feruloyl) diglucoside

8 pelargonidin-3-(p-coumaroyl) diglucoside-5-glucoside

9 pelargonidin-3-(feruloyl) diglucoside-5-glucoside

10 pelargonidin-3-(p-coumaroyl) diglucoside-5-(malonyl) glucoside

11 pelargonidin-3-(caffeoyl) diglucoside-5-(malonyl) glucoside

12 pelargonidin-3-(feruloyl) diglucoside-5-(malonyl) glucoside

C

B

A

19AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSPolymers18 Polymers

Polymers

Broadening horizons... compositional analysis of synthetic polymers

Fatty alcohol alkoxylates are used as non-ionic surfactants in home and industrial cleaning agents. Characterised by important

properties such as foam suppression, foam control and wetting effects, fatty alcohol alkoxylates are synthesised by reaction of

fatty alcohols with alkoxides such as ethylene oxide and propylene oxide amongst others. Alkoxylates are also relevant in a broad

range of chemical industrial applications where they are used as dispersal agents and emulsifiers.

MS and MS2 were used to characterise the fatty alcohol alkoxylate polymer shown in schematic 2.

OO

n

H

Schematic 2: Structure of the fatty alcohol alkoxylate polymers analysed.

n=4 n=6 n=8 n=10 n=12 n=14

[C8-O-(EO)n-H + Li]+

0

20

40

60

80

100

%Int.

300 350 400 450 500 550 600 650 700 750 800 850 900

m/z

445.37

489.40

533.43357.31

577.46446.38

621.48489.69

358.32 622.48 709.55446.66 797.60553.38 885.70

Figure 26: AXIMA Resonance mass spectrum of the ethoxylated octanol. The peaks represent (M+Li)+ ions. The mass distribution in thespectrum is asymmetric due to the low degree of ethoxylation in this product.

[HO-(EO)n-H + Li]+ [C8-O-(EO)n-H + Li]+

0

20

40

60

80

100

%Int.

100 150 200 250 300 350 400 450

m/z

445.34

201.12245.15

289.17 333.20157.07 377.24

401.32269.22 313.26357.28

227.13 446.34183.09139.05 334.20271.16 402.30

226.16 446.65

Figure 27: A key advantage of the AXIMA Resonance system is the ability to control the relative collision energy which results inhigh-quality CID-MS2 spectra. This is the MS2 spectrum of the lithium adduct precursor ion m/z 489 clearly showing the fragment series[HO-(EO)n-H+Li]+ and [C8-O-(EO)n-H+Li]+ (C8 = C8H17 and EO= CH2CH2O).

OO H

Li

m

+ + (OCH2 CH2)CH2CH OHO n

-C2H4

OO

CH

CH2

CH2

OH

Lim

OOHH2C

n

+

Schematic 3: A theory for the fragmentation of the ethoxylated fatty alcohols is based onthe rearrangement reaction in which the lithium ion is attached in a transition state to theterminal oxygen. In the next step the elimination of ethylene takes place.

octanol-ethoxylate

20 Lipidomics 21AXIMA Resonance

High Vacuum MALDI-QIT-TOF MSReferences

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Revolutionising lipid research with mono-isotopic precursor ion selectionand MSn capability

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0

10

20

30

40

50

60

70

80

90

100

%Int.

720 740 760 780 800 820 840 860 880

m/z

758.63

782.65

786.68760.66

780.62808.65

806.62

881.82

Phospholipids

Triglycerides

879.81

Figure 28: Representation of a typicalMALDI mass spectrum of a crude mixtureof lipids derived from human plasma.Plasma lipid extracts in MALDI-TOFMSexhibit mostly phospholipids andtriacylglycerols (triglycerides or TAG).Depending on the experimental conditions(matrix used, solvent, cations, etc) it ispossible to selectively ionise certain classesof lipids individually from a mixture.In this case, the chosen conditions allowedfor the detection of mainly thephosphatidylcholine and TAG species.

0

10

20

30

40

50

60

70

80

90

100

%Int.

450 500 550 600 650 700 750 800 850

m/z

Loss of 16:0623.49

0

10

20

30

40

50

60

70

80

90

100

874 876 878 880 882 884 886m/z

597.48Loss of 18:1

879.74

[M+Na ]+599.48Loss of 18:2

Figure 29: The positive ion MS2 spectrumof a TAG (16:0, 18:2, 18:1) is shown. Thesodium adduct [M+Na]+ (m/z 879.74) wasdetected and selected for MS2 withisotopic resolution in order to perform theCID experiment. The product ion spectrumclearly exhibits the loss of the threedifferent fatty acid chains (loss of RCOOH)where RCOOH can be a 16:0, 18:1 or 18:2fatty acid.

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AX

IMA

Resonance

▼