curs carbohydrate

Biological Mass Spectrometry: Applications in the Post-Genome Era

March, 2012 SNSB

7. CARBOHYDRATES/7. CARBOHYDRATES/GLYCOCONJUGATESGLYCOCONJUGATES

Plasma membrane

Ribosomes

Secretion being released from cell by exocytosis

PeroxisomeIntermediate filaments

Microtubule

Microfilament

Microvilli

Centrosome matrix

Centrioles

Mitochondrion

Lysosome

Cytosole

Rough ER

Golgi apparatus

Smooth ER

Nuclear envelope

NucleusNucleolus

Chromatin

Top fields in Systems Biology

Genomics

Proteomics

Glycomics

DNAs

Proteins

Carbohydrates

Cells communicateto each other by theircarbohydrate epitopes, covalently liked either to lipids or proteins

Definition

•Carbohydrates can be attached to another molecule (peptide, protein or lipid) forming what's called a 'glycoconjugate'.

Glycoconjugates

•There are three different types of glycoconjugate classes:

proteoglycans glycoproteins

glycolipids

Glycoproteins

Glycoproteins=proteins to which carbohydrates are covalently N- or O-linked. The predominant sugars found in glycoproteins are glucose, galactose, mannose, fucose, GalNAc, GlcNAc and NeuAc.

O-glycosylation N-glycosylation

GalNAc GlcNAc

N-GlycansGlycoproteins

GlycoproteinsN-Glycans

Glycoproteins

GlycoproteinsTypes of O-Glycans

GalNAc

Man

Fuc

GlcNAc

NeuAc

Glycoproteins

GalNAc

Man

Fuc

GlcNAc

NeuAc

Strategy for glycoprotein analysis Glycoproteins

Strategy for O-glycan analysisGlycoproteins

Strategy for N-glycan analysis Glycoproteins

Nomenclature for carbohydrate fragmentation*

* B. Domon, C.E. Costello, Glycoconjugate J. 5 (1988) 397-409.

non reducing end reducing end

O

O O

O

OH

O

OH OH OH

B1 B2C1

Y1 Z1

C2

Y2 Z2

B3

Y0

OH

OH

OH

OH

OH

OH

OH

O

O O

O

OH

O

OH OH OH

0,2 A1

1,5 X1 bond 4bond 0

bond 3bond 1

bond 2

bond 5

OH

OH

OH

OH

OH

OH

OH

0

12

3

45

Y4/B3

Z4/C3

2,5A12,4A2

1,5X1

B1

Y4

C1

Z4 Z3 Z2 Z1Y3 Y2 Y1

B2 B3 B4C2 C3 C4

0,2A3

Fragmentation scheme of linear oligosaccharides

ESI MS and tandem MS by CID conditions for detection and sequencing of glycoconjugates

neutral oligosaccharides sialylated oligosaccharides- positive ion mode detection - negative ion mode detection-pH < 7 - pH 7- cone voltage: 40-80 V - cone voltage: 20-50 V- capillary voltage: 900-1100V -capillary voltage: 800-1000V- collision energy: ~ 40eV -collision energy: 30-50eV

glycosaminoglycans- negative ion mode detection- pH 7- cone voltage: up to 20 V-capillary voltage: 600-800V-collision energy: 10-30eV

glycopeptides- negative ion mode detection- pH 7; pH < 7- cone voltage: 20-50 V-capillary voltage: 900-1100V-collision energy: 20-70eV

- negative ion mode detection- pH 7- cone voltage: 40-150 V-capillary voltage: 900-1200V-collision energy: 40-80eV

glycolipids

Solvent: MeOH; ESI tip: 1.50 kV; sampling cone: 30-45 V; acquisition 1 minaverage sample consumption: 0.2 pmols

nanoESI chip-QTOF MS of a complex mixture of sialylated O-GalNAc glycosylated peptides

NeuAc2Gal2GalNAc

NeuAc2Gal2

GalNAc-H2O

500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 12500

100

%

x4 x4 1087.39532.20

525.19

707.79

532.71

580.75

533.23

581.26

673.27581.77

1065.43

708.29

714.79

774.32

715.29

717.62

717.94

718.79

1051.42

775.33890.35

834.33776.32 871.36891.34923.87 964.39

1088.40

1088.90

1089.41

1095.40

1098.401136.421184.47

1263.44

NeuAcGalGalNAc

2-1-

1-

NeuAc2GalGalNAc-Ser

1-

NeuAc4Gal3GlcNAcGalNAc-Ser

NeuAc4Gal3GlcNAcGalNAc-Thr

2-

1- 1-autoMS/MS

NeuAc2GalGalNAc-Ser

2-

NeuAc2GalGalNAc-Thr

2-

NeuAcGal2GlcNAcGalNAc-Thr

NeuAc2GalGalNAc-Thr-Pro

NeuAcGalGalNAc-Ser

2-NeuAcGalGalNAc-Thr

1-

NeuAc2GalGalNAc-Thr

1-

NeuAc2GalGalNAc-Thr-Pro

NeuAcGal2GlcNAcGalNAc-Thr

2-

1-

NeuAc2Gal2GlcNAcGalNAc-Ser

2-NeuAc2Gal2GlcNAcGalNAc-Thr

1-

810 820 830 840 850 860 870 880 890 900 910 920 930 940 950 9607

100

%

890.35

834.33

830.32

829.65812.32826.33

812.66819.34

834.67

871.36

834.97

853.36839.33

839.67

841.99847.32

854.32

864.32

872.37

873.37

876.33879.33

891.34

923.87897.75

898.25

901.34

901.83916.84913.39

931.36

964.39

931.86

963.40942.34

935.35 956.36951.35

autoMS/MS

NeuAc2Gal3GlcNAc2GalNAc-Ser

NeuAc2GalGalNAc1-

2-

2-

3-

3- 1-

1-

2-

2-

2-

3-3-3-

2-

NeuAcGalGalNAc-Thr-Pro

NeuAcGalGalNAc-Thr-Pro-H2O

2-

Fuc4Gal2GlcNAc3

GalNAc -Ser +NaFuc4Gal2GlcNAc3

GalNAc -Thr +Na

Fuc4Gal2GlcNAc3

GalNAc -Thr +2Na

2-

Acquisition time 1 min; collision energy: 40 eV; average sample consumption: 0.2 pmols

AutoMS/MS of the doubly charged ion at m/z 897.79

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 15000

100

%

x2 x2 x4

290.10

751.88

609.17

291.13

483.18

424.15292.11

518.15 610.16

673.21

897.79

789.23

889.79

875.76

1214.38898.24

1081.34

1080.34

1082.31

1505.471215.40

1224.36

752.38

1-

1-1-

2-

1-

1-

2-

1-

1-

1-

1-

[M-2H] 2-

[M-2H] 2-

-H2O

2-2-

[M-2H] 2-

-CO2

B1 or B1ß

0,4A6/B3ß

Y1ß/B1

B5ß

C3ß

0,4A6/B1

0,4A6

Y5ß/B1 orY2 /B1ß

Y5ß or Y2

Y5ß or Y2

0,4A6/B2ß

NeuAc-Gal-GlcNAc-Gal- GlcNAc

GalNAc-ThrNeuAc-Gal

GT E T T S H S T P

z 6 z 4z 5z 8*

c4*

O3GalNAc 3Gal 1 O

Z 0

c5* c6* c7* c9*

Y 0Y 1 Z 1 [M+2H]2+

z4

z5

z6

[M+2H]•+/[M+H]+

~ x 10

c9*

c4* c5*

c6*

c7*

z8*

Z1

Y1

Y0Z0

Determination of glycosylation site by (-) ECD FTICR MS

Glycosylation site

Mucine type of O-glycosylation

Proteoglycans

What are proteoglycans?

•a special class of glycoprotein heavily glycosylated

•consist of a core protein with one or more covalently attached glycosaminoglycan chain(s) •the glycosaminoglycan chains are long, linear carbohydrate polymers negatively charged under physiological conditions, due to sulphate and uronic acid groups

Proteoglycans

Proteoglycan structure

C C CCCC

CC

CC CC

C CC C CC

C CC C CC

Decorin

Biglycan

Fibromodulin

Lumican

C

Leucine-Rich Repeat

Cysteine

Chondroitin/Dermatan Sulphate

N-Glycan

Tyrosine Sulphate

Keratan Sulphate

Proteoglycans with Leucine-Rich Repeats

Proteoglycans

Tentative Structure of Decorin

Proteoglycans

DCN

The GAG attachment site on Ser7 is in redred. The N-linked oligosaccharide attachment sites are in purplepurple.

Model of the decorin

D E C O R I N D E C O R I N

CC

CC CC

C

L e u c i n e - R i c h R e p e a t

C y s t e i n e

C h o n d r o i t i n /D e r m a t a n S u l f a t e

N - G l y c a n

O

HH

H

O H

H O H

C O O H

O OO

HH

HO H

H N H C O C H 3

C H 2 O S O 3 H

O

n

C H O N D R O I T I N 6 - S U L F A T E

O

HH

C O O HH

O H

H O H

O O

O

HH

HS O 3 H

H N H C O C H 3

C H 2 O H

O

n

D E R M A T A N S U L F A T E

H

Proteoglycans

Structure of the repeating disaccharide unitsStructure of the repeating disaccharide units of of the major glycosaminoglycansthe major glycosaminoglycans

Chondroitin 6-sulfate

Keratan sulfate

Heparin

Dermatan sulfate

Hyaluronate

NHCOCH3

NHCOCH3

NHCOCH3

NHCOCH3

NHSO3-

Proteoglycans

O

COOH

OH

OH

O

CH2OSO3H

NHCOCH3

OH

O

O

COOH

OH

OH

OH

OO

CH2OSO3H

NHCOCH3

OHOHO

O

COOH

OH

OH

O

CH2OSO3H

NHCOCH3

OHOHO

O

CH2OSO3H

NHCOCH3

OHOHO

COOH

OH

OH

OH

O

Condroitinază

Di-6S DDi-6S

Chondroitin lyases

Di-6S D Di-6S

ProteoglycansDepolymerization by lyases

Recognition specificity of chondroitin lyases can beused as a tool for identification of GlcA- and IdoA-

containing domains in CS-/DSglycosaminoglycan chains

chondroitinase AC chondroitinase B

...-GlcA-GalNAc(S)-GlcA-GalNAc(S)-IdoA-GalNAc(S)-...

Proteoglycans

How to...

-obtain high ionization yield in GAG analysis? -detect long and short chains in mixtures? -identify sulfation grade? -detect over- and under-sulfated regions? -obtain a high coverage on diganostic sequence ions?

Proteoglycans

Objectives of Mass Spectrometry

Proteoglycans

Problems associated with the ionization/sequencing of the GAG chains

•lability of the sulfate group

• in source loss of sulfate groups resulting in ions wrongly attributable to „non- or undersulfated species“ • overlapping of isobaric signals

• contradictory sequencing conditions required for correct structural analysis: cleavage of the glycosidic bond while keeping the sulfate group attached

Nanoelectrospray MS and tandem MS conditions for detection and sequencing of single components

in oligosaccharide mixtures

neutral GAGs - positive ion mode detection - negative ion mode detection- water/pH < 7 - water/pH 7- cone voltage: 40-80 V - cone voltage: up to 20 V- capillary voltage: 900-1100V -capillary voltage: 600-800V- collision energy: ~ 40eV -collision energy: 10-30eV

Proteoglycans

a) purification on DEAE AECb) β-eliminationc) depolymerization of GAG chains by lyasesd) separation of DS by GFCe) collection of fractionf) further separation according to the number of sulfatesg) mass spectrometric analysis

Strategy for the MS analysis of GAG chains

Proteoglycans

Strategy for decorin GAG analysis based on the recognition specificity of chondroitin lyases and chip ESI multistage MS

Proteoglycans

Chip ESI MSChip ESI MSnn

GAG chainGAG chainDecorinDecorin

B lyaseB lyase

AC lyaseAC lyase BB

AA

Fully automated (-) nanoESI chip HCT MS1

of decorin CS dissacharide obtained by GAG chain depolymerization using chondroitin B lyase

2000

4000

6000

8000

Intens.

458.11

480.22

[M-H]-

[M-2H+Na]-

200 300 400 500 600 700 m/z

2000

4000

6000

8000

Intens.

458.11

480.22

[M-H]-

[M-2H+Na]-

200 300 400 500 600 700 m/z

M=GlcA-GalNAc (1S)M=IdoA-GalNAc (1S)

nS =nSO3, number of sulfate groups

Conditions: Solvent: MeOH/H2O/ACN; acquisition time 3 min; Chip ESI: -0.55 kV; capillary exit: -50 V.

Proteoglycans

Intens.

200

400

600

800

1000

m/z

4,5-ΔGlcA-O-GalNAc

SO3

458.17

300.15

282.14

450400350300250200

202.13

175.08

157.06

220.12

378.16230.41

C1

[M-H]-

[M-H-SO3]-

Y1

Z1

Y1-SO3

Z1-SO3

B1

B1 C1

Y1 Z1

0

-

-

-

-

-

-

324.15

4,5 DIdoA-O-GalNAc

SO3B1 C1

Y1 Z1

Diagnostic for Diagnostic for sulfation at GalNAcsulfation at GalNAc

Fully automated (-) nanoESI chip HCT CID MS2

of the singly charged ion at m/z 458.17

Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.3-0.6 V

Proteoglycans

m/z

458.17

538.12

560.14916.89

661.51

[M-H]-

[MS-H]-

[MS-2H+Na]-

[2M-H]-

M=GlcA-GalNAc (1S)

MS=GlcA-GalNAc (2S)

m/z

458.17

538.12

560.14916.89

661.51

m/z

458.17

538.12

560.14916.89

661.51

[M-H]-

[MS-H]-

[MS-2H+Na]-

[2M-H]-

M=GlcA-GalNAc (1S)

MS=GlcA-GalNAc (2S)

nS=nSO3-number of sulfate ester groups


of decorin DS dissacharide obtained by GAG chain depolymerization using chondroitin AC lyase

Conditions: Solvent: MeOH/H2O/ACN; acquisition time 5 min; Chip ESI: -0.50 kV; capillary exit: -50 V.

Proteoglycans

Z1

538.11

458.15

342.09

282.07 300.08

m/z

255.11

202.09

175.06

[M-H]-

[M-H-SO3]-

Y1

C1-SO3

Z1-SO3

0,2X1

4,5-ΔGlcA-O-GalNAc

SO3 SO3C1

Z1Y1

C1

#

0

50

100

150

200

250

300

500 1000

- -

--

-

-

Diagnostic for Diagnostic for sulfation at GalNAcsulfation at GalNAc

Diagnostic for Diagnostic for sulfation at GlcAsulfation at GlcA


of the singly charged ion at m/z 538.11


Proteoglycans

Fig.4

460 480 500 520 540 560 580 600 620 640 660 680 700m/z0

100

%

x4458.02

585.03

517.38

511.38459.16

502.90

552.66

517.70

546.90

552.91

572.63

611.28

585.35

687.38

619.20

619.55647.40

647.91

679.37

687.87

711.89

712.92

533.82

611.604-

3-

3-

5-

4-

4-

3-

3-

4-

2-

3-

664.893-634.89

octa(4S)

hexa (5S)

deca(4S)dodeca(5S)

deca(5S)

octa(3S) octa(4S)

dodeca(4S)

hexa(3S)

deca(3S)

IdoA-GalNAc-[GlcA-GalNAc]n

S S

nanoESI MS of a depolymerized GAG mixture

chondroitin AC -lyase

...- G lcA -G alNA c (S)- G lcA -G alNA c (S)- IdoA - G alNA c (S)-...

C S/DS G A G chain

DECORI N DECORI N

CC

CC CC

C

L e u c in e -R ic h R e p e a t

C y s te in e

C h o n d r o it in /D e r m a ta n S u lfa te

N -G ly c a n

O

HH

H

O H

H O H

C O O H

O OO

HH

HO H

H N H C O C H3

C H 2 O S O 3 H

O

n

C H O N D R O IT IN 6 -S U L F A T E

O

HH

C O O HH

O H

H O H

O O

O

HH

HS O 3 H

H N H C O C H 3

C H 2 O H

O

n

D E R M A TA N S U LFA TE

H

depolym erization

RobotChip

Separation by SECChip MS analysis

Proteoglycans

collected

GFC elution profile ofGAG chain oligosaccharides after cleavage with lyase

Proteoglycans

[GlcA-GalNAc]4 (4S)

[GlcA-GalNAc]4 (4S)

[GlcA-GalNAc]4 (4S)

[GlcA-GalNAc]4 (3S)

Octasaccharide completelydesulfated (low abundant,4- at m/z 379.08)

Trace of [GlcA-GalNAc]5

(5S) (low abundant, 6- at381.52)

300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 6000

100

%

458.01

305.00

305.16

366.44

366.24

305.32 366.63

366.84

458.26

458.51

458.76

459.01

584.00

6-

5-

4-

3-

nanoESI chip MS of the collected octasaccharide fraction

Proteoglycans

[M-4H] 4-

380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 9400

100

%

x4x10 458.02

439.29379.16

418.75

584.35

516.96

458.27

510.96

476.18

553.06

611.21 917.22

687.21

617.34

647.88

687. 70

877.23

775.34

735.28

696.25

837.27

877.75

899.41

917.72

918.22

2-2-

5-

1-

3-

3- 3-

3- 3-

3-

2-

2-

2-

2-

2-

2-

2-

m/z

1- 2-

Y3(2S)

B4(2S)

B7(3S)

C7(3S)

Y7(4S)

B3(0S)

B8(3S)B8(4S)

C8(4S)

Y6(2S)

Y6(3S)

Y7(3S)

Z8(2S)

Y8(3S)

2-

B8(4S)

C8(4S)

Y7(2S)

Z7 (2S)

nanoESI chip MS/MS of the ion at 458.02

Proteoglycans

13

2

45

67

8 910

Time (min)0.00 6.5

Abs

orba

nce

-0.368

1.47110-3

11

CE/UV profile of a fraction collected from GFC

Proteoglycans

Proteoglycans

Introduction of on-line CE/ESI MS for GAGs

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00Time0

100

%

1

2

3

4

5 6

7

8

0.7539780

0.6735670

0.5328

673

0.2614588

1.4171933

1.3267933

0.9348

841

1.7588856

t-elution time 15 min after injection

n- number of scans

m/z of the most abundant ion eluted at the moment t

CE buffer: 40mM ammonium acetate/ammonia pH 11.8. CE separation voltage 30kV directpolarity, 6 s injection by pressure. CE column length 100 cm. Nanosprayer potential 600V,sampling cone potential 12V. MS signal acquisition: 15 min after injection.

.m/z

575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975 10001025 1050 10750

100

%

933.12

727.84

933.32

933.52

933.72

933.92

[M-5H]5-

0

100

%

933.12

933.32

933.52

933.72

933.92

934.13934.33

4,5 DIdoA-GalNAc {GlcA-GalNAc}9(11S)

calculated monoisotopic: m/z 933.126

ProteoglycansSpectrum derived from the 7th peak

D 4,5[ HexA -HexNAc (S)]11m/z (1400-1700) u.

1450 1500 1550 1600 1650 1700m/z0

100

%

1502.23

1434.58 1469.66

1434.90 1470.1

1528.88

1502.5

1502.9

1529.20

1529.55

1605.38

1534.20

1535.25 1565.40

1596.37

1605.87

1693.351653.22

1693.84

C19 (9S)

3-

Y19 (10S)

3-

[M-3H] 3-

2-

C14 (6S)

Z14 (7S)

2-

Y14 (7S)

2-

2- 2-

Y15 (7S)Y15 (6S)

[M-SO 3 -3H] 3-

1495.15

Y19(11S)

1489.28 Z19(11S)

on-line CE/nanoESI MS/MS of the eicosasaccharide bearing 11 sulfate groups

4,5 DIdoA-GalNAc {GlcA-GalNAc}9(11S)

B19(9S)

Proteoglycans

4,5D-IdoA-O-GalNAc- GlcA-GalNAc -GlcA-O-GalNAc-O

S S S

B1

Y19

C1

Z19

B19C19

Y1Z1

8

S

Fragmentation scheme of the eicosasaccharide and

localization of the sulfate groups from MS/MS data

Proteoglycans

Polysaccharides

HO O

HO

OOH

OH

O

HOOH

OH

O O

HOOH

OH

OHn-2

HO O

HO

OOH

OH

O

HOOH

OH

O OH

HO

OH

OH

n-2

HN

NH23

HOO

HO

OOH

OH

HO O

HO

OOH

HOO

HOOH

OH

n-2

HOO

HO

OOH

OH

HO O

HO

OOH

HOOH

HOOH

n-2HN

NH23

H2N CH2 NH26

80oC, 2 days

NaBH3CN

DMSODMFAcOH

I. Perdivara, E. Sisu, N. Dinca, K. Tomer, M. Przybylski, A. D. Zamfir, Rapid Commun Mass Spectrom. 2008

500 600 700 800 900 1000 1100 1200 m/z0

Glc6HMD

Glc7HMD Glc8HMD

Glc5HMD

*GlcnHMD – [M+2H]2+

Glc9HMD

Glc10HMD

Glc11HMD

Glc12HMDGlc14HMD

Glc13HMD

Glc2HMD

GlcnHMD – [M+H]+

Glc3HMD

Glc4HMD

Glc5HMD

Glc6HMD

Glc7HMD

# #

##

#

##

GlcnHMD – [M+2H]2+ (+14)

#

GlcnHMD – [M+H]+ (+28)

< 1 ppm

443.2603

471.2553

NHNH2

NHNH

O

HGlcn Glcn3 3

DMF

Side reaction

OH

OHO

OH

OH

O

OHO

OH

OH

O

OHOH

OH

OH

NH

NH23

OH

OHO

OH

OH

O

OHO

OH

OH

O

OHOH

OH

OH

NH

NH23

n#

*

*

* *

*

*

*

*

*

*

(+) ESI FTICR MS of long-chain polysaccharides(+) ESI FTICR MS of long-chain polysaccharides


1400 1600 1800 2000 2200 2400 m/z

GlcnHMD – [M+3H]3+

GlcnHMD – [M+2H+Na]3+

Glc42HMD

Glc41HMD

Glc40HMDGlc39HMD

Glc38HMD

Glc37HMD36

35

34

Glc33HMD

3231

30

29

28

27

26Glc25HMD

GlcnHMD – [M+2H]2+

Glc16HMD

17

18

Glc19HMD

Glc15HMD

OOH

OH

OH

OH

O

OOH

OH

OH

O

OHOH

OH

OH NH

NH23

n

OOH

OH

OH

OH

O

OOH

OH

OH

O

OHOH

OH

OH NH

NH23

n

*

*

*

*

*

*

§

§

§

§

§ §

§

§

§

§ §

§§

§

§

§

§


(+) ESI FTICR MS of long-chain polysaccharides(+) ESI FTICR MS of long-chain polysaccharides

525.25

687.28

849.32

1011.36

1173.42

1335.49

1497.55

1659.57

1000

2000

3000

Intens.

600 800 1000 1200 1400 1600 1800 2000 m/z

Glc4-AGL

Glc2-AGL

Glc8-AGL

Glc3-AGL

Glc5-AGL

Glc7-AGL

Glc6-AGL

Glc9-AGL

Glc10-AGL1821.55

Glc11-AGL1983.47

525.25

687.28

849.32

1011.36

1173.42

1335.49

1497.55

1659.57

1000

2000

3000

Intens.

600 800 1000 1200 1400 1600 1800 2000 m/z

Glc4-AGL

Glc2-AGL

Glc8-AGL

Glc3-AGL

Glc5-AGL

Glc7-AGL

Glc6-AGL

Glc9-AGL

Glc10-AGL1821.55

Glc11-AGL1983.47


of polydisperse maltodextrins derivatized with aromatic amines

Average MW:1800 Da

Conditions: Solvent: H2O; concentration 10 pmol/L; acquisition time 2 min; Chip ESI: -0.60 kV; capillary exit: -20 V.

Glc4-AGL849.43

1011.50

1173.56

1335.67

1497.74

1659.79

1821.84

1983.88

0

1

2

3

4

5

6

x105

Intens.

800 1000 1200 1400 1600 1800 2000 2200 m/z

Glc6-AGL

Glc5-AGL

Glc8-AGL

Glc7-AGL

Glc10-AGL

Glc9-AGL

Glc11-AGL

MS2

O

OHOH

OHO

O

OH

OHO

OH

OH

OHOH

NH

OHOH

n

CH2 NH2

Glc4-AGL849.43

1011.50

1173.56

1335.67

1497.74

1659.79

1821.84

1983.88

0

1

2

3

4

5

6

x105

Intens.

800 1000 1200 1400 1600 1800 2000 2200 m/z

Glc6-AGL

Glc5-AGL

Glc8-AGL

Glc7-AGL

Glc10-AGL

Glc9-AGL

Glc11-AGL

MS2

O

OHOH

OHO

O

OH

OHO

OH

OH

OHOH

NH

OHOH

n

CH2 NH2

AGL



Average MW:2800 Da

Area m/z: (800-2400)


0

2000

4000

6000

8000

2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z

2146.00

Glc13-AGL

Glc12-AGL

Glc15-AGL

Glc14-AGLGlc16-AGL

2307.00

2469.402632.00 2794.00

2956.20

Glc17-AGL

0

2000

4000

6000

8000

2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z

2146.00

Glc13-AGL

Glc12-AGL

Glc15-AGL

Glc14-AGLGlc16-AGL

2307.00

2469.402632.00 2794.00

2956.20

Glc17-AGL

O

OHOH

OHO

O

OH

OHO

OH

OH

OHOH

NH

OHOH

n

CH2 NH2

0

2000

4000

6000

8000

2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z

2146.00

Glc13-AGL

Glc12-AGL

Glc15-AGL

Glc14-AGLGlc16-AGL

2307.00

2469.402632.00 2794.00

2956.20

Glc17-AGL

0

2000

4000

6000

8000

2100 2200 2300 2400 2500 2600 2700 2800 2900 m/z

2146.00

Glc13-AGL

Glc12-AGL

Glc15-AGL

Glc14-AGLGlc16-AGL

2307.00

2469.402632.00 2794.00

2956.20

Glc17-AGL

O

OHOH

OHO

O

OH

OHO

OH

OH

OHOH

NH

OHOH

n

CH2 NH2

AGL

Area m/z: (2100-3000)





of the singly charged ion at 1173.46 corresponding to Glc6-AGL


Intens.

363.17

453.18

471.16

487.21

506.06

525.23

585.19

649.27

687.28

747.28

768.26

811.33

849.33

909.36

930.83

1011.371071.40

1173.46

0

1000

2000

3000

4000

5000

6000

300 400 500 600 700 800 900 1000 1100 1200 m/z

MS3 [M+H]+

[M+H]+-H2O1155.46

4

YY11

YY22 YY33

YY44

YY55

BB33

BB44

BB33

BB44

BB55


of the singly charged ion detected in MS2 at 363.20


0

1

2

3

4

5

Intens.

50 100 150 200 250 300 350 400 450 m/z

363.20

[M+H]+

345.00

274.00

256.00

238.20

201.00184.90

103.20 [M+H]+-H2O

201.00

274.00 256.00-H2O

238.20

-NH2

184.90

-H2O

103.20

Glyco(sphingo)lipids:

• ...contain one carbohydrate epitope per molecule

• ...of the same monosaccharide sequence show conformational diversity according to the specificity of glycosidic linkages

•...their carbohydrate chains conformational degrees of freedom are restricted according to the linkage sites (e.g.3- vs. 6-) and steric hindrance (e.g.3- vs. 4-)

Glycosphingolipids:

• ...are organized in cell surface lipid microdomains (rafts) to associate with specific signaling molecules

• ...show specificity of expression in different normal and pathological states according to their carbohydrate epitopes

Glycosphingolipids:

• ...are to be analyzed to characterize their composition regarding the carbohydrate and the lipid moiety, their way of attachment/sequence, patterns of branching and the anomericity of the linkages

• ...the task for analytical services is to identify already known and still unknown molecular species by mapping and de-novo sequencing

•...mass spectrometry is the most sensitive and accurate analytical method for this task(Methods Enzymol. 193, 1990; Mass Spectrom. Rev. 1994).

• ...are sialylated glycosphingolipids organized in cell surface lipid microdomains (rafts) to associate with specific signaling molecules

• ...show specificity of expression in different normal and pathological states according to their carbohydrate epitopes

•....have the highest abundance in central nervous system, being biomarkers of brain disorders, neurodegeneration and cancer

• ...are to be analyzed to characterize their composition regarding the carbohydrate and the lipid moiety, their way of attachment/sequence, patterns of branching and the anomericity of the linkages

• ...the task for analytical services is to identify already known and still unknown molecular species by mapping and de-novo sequencing

Gangliosides

Neu5Ac3Galß4GlcCer GM3 GalNAcß4(Neu5Ac 3)Galß4GlcCer GM2 Galß3GalNAcß4(Neu5Ac 3)Galß4GlcCer GM1a Neu5Ac 3Galß3GalNAcß4Galß4GlcCer GM1b Neu5Ac 8Neu5Ac 3Galß4GlcCer GD3 GalNAcß4(Neu5Ac 8Neu5Ac 3)Galß4GlcCer GD2 Neu5Ac 3Galß3GalNAcß4(Neu5Ac 3)Galß4GlcCer GD1a Galß3GalNAcß4(Neu5Ac 8Neu5Ac 3)Galß4GlcCer GD1b Neu5Ac 8Neu5Ac 3Galß3GalNAcß4(Neu5Ac 3)Galß4GlcCer GT1a Neu5Ac 3Galß3GalNAcß4(Neu5Ac 8Neu5Ac 3)Galß4GlcCer GT1b Galß3GalNAcß4(Neu5Ac 8Neu5Ac 8Neu5Ac 3)Galß4GlcCer GT1c Neu5Ac 8Neu5Ac 3Galß3GalNAcß4(Neu5Ac 8Neu5c 3)Galß4GlcCer GQ1b

GM1a

Gangliosides

8

O

OH

O

HO

OHNH

O

OH

OH

1

2

3

4

5

6

7

10 11

8

9

9

1110

7

6

5

4

3

2

1

6

54

3

21

6

5

4

32

1

O

OH

O

HO

OHNH

O

OH

O

O

OH OH

O

OHO

O

OHHO

OH

OHN

O

Ceramide

Sphingosine

Fatty acidGlcGal

Neu5Ac

Neu5Ac

OH

2-8, 2-3-Disialolactosylceramide (GD3)

Normal brain histology. Section of the human cerebellum

Neurohistopathological features of Glioblastoma multiforme/gliosarcoma

nanoESI chip-QTOF MS of the gliosarcoma ganglioside mixture

Solvent: MeOH; ESI tip: 1.60 kV; sampling cone: 80 V; acquisition 2 minaverage sample consumption: 0.5 pmols

1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 20000

100

%

x3x2.5 x12x61470.971179.55

1264.44

1180.56

1263.44

1235.49

1181.53

1207.48

1382.151265.45

1354.201263.41

1383.16

1464.10

1462.08

1552.92

1540.88

1472.01

1526.92

1512.89

1553.891628.80

1554.91

1756.55

1629.80

1673.64

1630.79

1674.65

1675.66

1729.58

1757.53

1835.35

1758.52

1759.51

1918.26

1914.20

1889.28

1919.24

1992.531920.22

GD1(d18:1/18:0)

GD1(d18:1/24:1)

GD1(d18:1/24:0)

GD3(d18:1/18:0)

GD3(d18:1/20:0)

O-Ac-GD3(d18:1/18:0)

O-Ac-GD3(d18:1/20:0)

GD3(d18:1/24:0)

GD3(d18:1/24:1)

GM3(d18:1/24:0)

GM3(d18:1/16:0)

GM3(d18:1/18:0)

GM3(d18:1/22:0)

GM3(d18:1/20:0)

GM2(d18:1/16:0)

GM2(d18:1/18:0)

GM2(d18:1/20:0)

GD2(d18:1/18:0)

GD2(d18:1/22:0)

GD2(d18:1/24:1)

GD2(d18:1/24:0)

GM1(d18:1/20:0)

GM1(d18:1/24:0)

Could a GM1 (d18:1/18:0) species be present? Calc.M-H= m/z1544.86

?

1539 1540 1541 1542 1543 1544 1545 1546 1547 15480

100

%

1540.88

1539.04

1541.85

1542.87

1543.84

O-Ac-GD3(d18:1/20:0)

m/z 1544.86 GM1?

?

Solvent: MeOH; ESI tip: 1.60 kV; collision energy: 45-85 eV;signal acquisition 50 min. Average sample consumption 15 pmols

nanoESI chip-QTOFMS/MS of the singly charged ion at m/z 1544.86

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 15000

100

%

x10290.12

179.16

202.20

564.79

364.25

291.15

332.22

424.38

562.81466.39

888.77

726.69

565.77

708.68833.52727.70

728.71

889.77

890.76

1544.89995.52916.81

B1

1091.84

[M-H] -

Y0

Y1

Z1

Y2

Y3

1253.79

Y4

C4

Hex -

GalNAc -

GalGalNAc -

C52,4A3/B1

603.01

Y0 (GD3 18/20)

Y2 (GD3 18/20)

GD3 (18/20)NeuAc-NeuAc-Gal-Glc-Cer

GM1 (18/18)

364

NeuAc-Gal-GalNAc-Gal-Glc-Cer

290

888

833

726/708

10911253 564

995B1

290 916 603

*

B2 (Na)(GD3 18/20)

592

603 (Na)

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 15000

100

%

x10x4x6x16

281.24

255.22

179.04

202.06

581.20

419.25

290.35

364.13

537.20437.29

465.32

1253.84

701.54

582.19

623.22

1249.84

702.53888.68

729.54 851.321073.71

1207.741091.67

1540.96

1544.93290.09

161.03

220.08

m/z

MS/MS of the singly charged ion at m/z 1540.88

Hex-

-H2O

GalNAc-

B1

B1

GalGalNAc-

B2

-CO2

***

Y2

B2(Na)

Y4

Y3-H2O

-Ac

B2 (Ac)**

Y3 (Ac)-H2O

[M-H]-

[M-H]-

M= GM1 (18/18)M= O-Ac GD3 (18/20)

735.53

836.68

917.60

1063.72

1207.01

1471.031572.02

0.0

0.5

1.0

1.5

2.0

4x10

Intens.

800 1000 1200 1400 1600 1800 2000 m/z

931.72

1049.26

1077.73

1139.01

1179.90

1237.90 1249.95 1279.88

1259.92

1353.031375.03

1519.06

1544.16

1553.07

1653.21

1671.11

1756.01

1757.51

1858.32

1885.08

1918.11

MS2

Fully automated (-) nanoESI chip HCT MS1 of An28 native ganglioside mixture from glial islands of anencephalic fetus

Conditions: Solvent: MeOH; sample concentration 5 pmol/μL; acquisition time 10 min; Chip ESI: -0.8 kV; capillary exit: -50 V.

735.52 836.71

917.58

1544.20

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Intens.

800 1000 1200 1400 1600 1800 m/z

1836.20-

0

2000

4000

6000

1700 1750 1800 1850 1900 1950 2000 2050

851.60

931.72

952.80

1037.60

1041.60

1049.18

1063.33

1065.63

1077.71

1104.78

1139.01

1151.71

1165.80

1167.82

1179.74

1181.75

1206.77

1221.33

1235.81

1383.21

1354.79

1301.82

1279.81

1259.79

1471.03

1444.80

1858.20

1990.50


of FL27 native ganglioside mixture from normal fetus frontal lobe

Conditions: Solvent: MeOH; sample concentration 5 pmol/μL; acquisition time 10 min; Chip ESI: -0.8 kV; capillary exit: -50 V.

(d18:1/18:0) + +

(d18:1/18:1) + -

(d18:1/24:1) + -

(d18:1/24:0) + -

GD2

(d18:1/20:0) - +

(d18:0/18:0) + +

(d18:1/16:0) - +

(d18:0/16:0) - +

(d18:1/18:1) - +

(d18:1/18:0) + +

GD3

(d18:1/24:1) + -

(18:1/16:0) + +

(d18:1/18:0) + +

(d18:0/20:0) + -

(d18:1/20:0) + +

GT1

(d18:1/24:0) - +

GT3 (d18:1/18:0) - +

(d18:1/24:0) - +

(d18:1/24:1) + -

GQ1 (d18:1/18:0) + -

HexNAcHex2Cer (d18:0/14:0) or (d16:0/16:0)

- +

HexNAcHex2Cer (d18:0/16:0) - +

HexNAcHex2Cer (t18:0/22:0) or (d18:0/h22:0) or (d18:2/24:4)

- +

Asialo-GG species

HexHexNAcHex2Cer(d18:1/18:0) - +

Comparative overview upon gangliosides and asialo-gangliosides detected in An28 and FL27 mixtures by NanoMate/HCT

-+(d18:0/18:0)

-+(d18:1/24:1)

+-(d18:1/23:0)

++(d18:1/20:0)

++(d18:1/18:0)GD1

+-(d18:1/20:2)GM4

+-O-Ac-GM3 (d18:1/24:2)

++O-Ac-GM3 (d18:0/22:0) (or GM3(20:0/23:0)

+-O-Ac-GM3 (d18:1/22:0) (or GM3(20:1/23:0)

+-O-Ac-GM3 (d18:1/22:1) or GM3(20:1/23:1)

+-O-Ac-GM3 (d18:1/20:0) or GM3(18:1/23:0)

+-(d18:1/24:0)

+-(d18:1/24:1)

++(d18:0/24:0)

++(d18:1/24:2)

++(d18:0/22:0)

+-(d18:1/22:0)

++(d18:1/20:0)

+-(d18:0/18:0)

++(d18:1/18:0)

+-(t18:1/16:0) or (d18:1/h16:0) or HexNAcHex2Cer(d18:1/24:4)

+-(d18:1/16:0)

++(d18:1/14:0) or (d18:1/h14:0) or HexNAcHex2Cer (d18:1/22:4)

GM3

+-(d18:1/22:0)

+-(d18:1/18:0)

++(d18:1/16:0)

-+nLM1 and/or LM1 (d18:0/20:0)

++nLM1 and/or LM1 (d18:1/18:0)

++nLM1 and/or LM1 (d18:0/16:0)GM1

FL27An28Proposed structureGG species

-+(d18:0/18:0)

-+(d18:1/24:1)

+-(d18:1/23:0)

++(d18:1/20:0)

++(d18:1/18:0)GD1

+-(d18:1/20:2)GM4

+-O-Ac-GM3 (d18:1/24:2)

++O-Ac-GM3 (d18:0/22:0) (or GM3(20:0/23:0)

+-O-Ac-GM3 (d18:1/22:0) (or GM3(20:1/23:0)

+-O-Ac-GM3 (d18:1/22:1) or GM3(20:1/23:1)

+-O-Ac-GM3 (d18:1/20:0) or GM3(18:1/23:0)

+-(d18:1/24:0)

+-(d18:1/24:1)

++(d18:0/24:0)

++(d18:1/24:2)

++(d18:0/22:0)

+-(d18:1/22:0)

++(d18:1/20:0)

+-(d18:0/18:0)

++(d18:1/18:0)

+-(t18:1/16:0) or (d18:1/h16:0) or HexNAcHex2Cer(d18:1/24:4)

+-(d18:1/16:0)

++(d18:1/14:0) or (d18:1/h14:0) or HexNAcHex2Cer (d18:1/22:4)

GM3

+-(d18:1/22:0)

+-(d18:1/18:0)

++(d18:1/16:0)

-+nLM1 and/or LM1 (d18:0/20:0)

++nLM1 and/or LM1 (d18:1/18:0)

++nLM1 and/or LM1 (d18:0/16:0)GM1

FL27An28Proposed structureGG species

B2βY4α

2-907.32

Y4β

Y4α

290.211252.83

Y4β/B1α

888.83

Y2α/B2β

537,32

B2β- CO2

[M-2H]2-

[M-2H]2-- CO2

1041.21

1054.21

[M-2H]2-- H2O

1526.811859.011818.01

Z4α

581.31

917.32

1063.41

1544.81

1837.01

0

1000

2000

3000

Intens.

400 600 800 1000 1200 1400 1600 1800 2000 m/z

B2βY4α

2-907.32

Y4β

Y4α

290.211252.83

Y4β/B1α

888.83

Y2α/B2β

537,32

B2β- CO2

[M-2H]2-

[M-2H]2-- CO2

1041.21

1054.21

[M-2H]2-- H2O

1526.811859.011818.01

Z4α

581.31

917.32

1063.41

1544.81

1837.01

0

1000

2000

3000

Intens.

400 600 800 1000 1200 1400 1600 1800 2000 m/z

Z4α2-

Z4β

Y4α+Na

MS3

Fig3a

B1α1253.89

537.32

581.31

1041.21

1526.82

1544.71

1054.21

1837.01

1859.01B2β

Y4α2-907.32

Y4β

Y4α

290.211252.83

Y4β/B1α

888.83

Y2α/B2β

537,32

B2β- CO2

[M-2H]2-

[M-2H]2-- CO2

1041.21

1054.21

[M-2H]2-- H2O

1526.811859.011818.01

Z4α

581.31

917.32

1063.41

1544.81

1837.01

0

1000

2000

3000

Intens.

400 600 800 1000 1200 1400 1600 1800 2000 m/z

B2βY4α

2-907.32

Y4β

Y4α

290.211252.83

Y4β/B1α

888.83

Y2α/B2β

537,32

B2β- CO2

[M-2H]2-

[M-2H]2-- CO2

1041.21

1054.21

[M-2H]2-- H2O

1526.811859.011818.01

Z4α

581.31

917.32

1063.41

1544.81

1837.01

0

1000

2000

3000

Intens.

400 600 800 1000 1200 1400 1600 1800 2000 m/z

Z4α2-

Z4β

Y4α+Na

MS3

Fig3a

B1α1253.89

537.32

581.31

1041.21

1526.82

1544.71

1054.21

1837.01

1859.01

Y4α/B2β

NanoESI chip HCT CID MS2 of the doubly charged ion at m/z 1063.34 corresponding to GT1 (d18:1/18:0) ganglioside species detected in An28 mixture


O

NeuAc

O

NeuAc

O

NeuAc

O

NeuAc

NeuAc – O – Gal – O – GalNAc – O – Gal – O – Glc – O – Cer

B1α C1α

Z4αY4α

Z4β

Y4βB2β

C2β

Y4β /B1α

Y2α /B2β

B1β

Fig3b

Y4α/B2β

MS2 fragmentation pathway of [M-2H]2- ion at m/z 1063.34

GT1b (d18:1/18:0)GT1b (d18:1/18:0)

290.21 581.32B2β

564.62 887.63

Z3β/C2α orZ2α/B2β

908.27

917.27

1254.02

1382.92

Y4β/B1αor

Y3α/B1β

1544.87

Y4β

[M-H2O-2H]2-

[M-2H]2-

0

20

40

60

80

Intens.

200 400 600 800 1000 1200 1400 m/z

982.941023.92

Z4β

[M-2H]2- -H2O

1161.50

Z4β/B2α orY2α/C1β

Y0 Y3β

MS4

B1β

1091.32

Z3α/C2β

Fig3c

1500.69

Y4β/CO2

888.63

Z3α/C2β

1161.50Y2α/B2β

Z2α/C1β

NanoESI chip HCT CID MS3 using as a precursor

Y4α2- ion detected at m/z 917.32 in MS2


Gal – O – GalNAc – O – Gal – O – Glc – O - Cer

O

NeuAc

O

NeuAc

Y0

Z3β

Y3β

Z4β

Y4β

B1β

C1β

B2β

C2βZ3β/C2α or Z2α/B2β

Z4β/B2α or Y2α/C1β

Y4β/B1α or Y3α/B1β

Z3α/C2β


O

NeuAc

O

NeuAc

O

NeuAc

O

NeuAc

Y0

Z3β

Y3β

Z4β

Y4β

B1β

C1β

B2β

C2βZ3β/C2α or Z2α/B2β

Z4β/B2α or Y2α/C1β

Y4β/B1α or Y3α/B1β

Z3α/C2β

Z2α/B2β

Z2α/C1β

Y3α/B1β

Z3α/C2β

Y2α/B2β

MS3 fragmentation pathway of the ion detected in MS2 at m/z 917.32

0

5

10

15

Intens.

600 700 800 900 1000 1100 1200 1300 1400 1500 m/z

564.62

888.42Y2α/B1β

870.42

Y2α/C1β

1253.81

Y3β

1544.87

[M-H]-

1526.87

[M-H]--H2O

980.32

707.63

1024.45

1212.61

1389.01

1375.83

Y0

Z1

Y1

726.22

B4

1179.48

Y2α

1346.50

1364.56

Z3α

Z3α-H2O

1501.82

[M-H]--CO2

1090.80

Y3α/B1β


NanoESI chip HCT CID MS4 using as a precursor

the Y4β- ion detected at m/z 1544.87 in MS3


O

NeuAc

O

NeuAc

B4

Z0Y0Y2α

Y3β

Y2α/B1β or

Y2α/C1β or

Fig3f

Z1Y1

C1β

B1β

Y3β /B2α

Z3β /B2αB2α

Z3αY3α

Y3α/B1β


O

NeuAc

O

NeuAc

B4

Z0Y0Y2α

Y3β

Y2α/B1β or

Y2α/C1β or

Fig3f

Z1Y1

C1β

B1β

Y3β /B2α

Z3β /B2αB2α

Z3αY3α

Y3α/B1β

Y2α/B1β

MS4 fragmentation pathway of the ion detected in MS3 at m/z 1544.87

MS2

931.72

1077.65

0

2

4

6

8

5x10

Intens.

600 800 1000 1200 1400 1600 m/z

1063.201085.20

1088.60

1049.25

1033.36

1020.16

939.60

926.63

918.11

717.80

918.11

1063.67

1077.65

1088.68

0

2

4

6

8

5x10

Intens.

900 925 950 975 1000 1025 1050 1075 1100 m/z

926.63

939.60

1020.16

1033.06

1049.25

1071.20

1074.66

1085.60

1098.20

918.11

931.72

1063.67

1077.65

1088.68

0

2

4

6

8

5x10

Intens.

900 925 950 975 1000 1025 1050 1075 1100 m/z

926.63

939.60

1020.16

1033.06

1049.25

1077.20

1071.20

1074.66

1085.20

1098.20

917.60

1063.20

1074.20

1088.20

TOP DOWN ANALYSIS OF GANGLIOSIDES BY MULTISTAGE CID

MSMS11

581.32

932.10

1068.28

1282.06

1573.21

1864.21

0

1

2

3

4

5

4 x10

Intens.

400 600 800 1000 1200 1400 1600 1800 2000 m/z

B2β

603.30

537.40

655.40

Y3α/B1β/Y1

B2β /CO2

1055.20

[M-2H]2-- H2O

[M-2H]2-- H2O

[M-2H]2-

1120.00Y3α/B2β

916.80

Y2α/B2β

1254.00

B5/B1β

Y4α/B2β

1514.60Y2α

1545.10

1555.20

Z2β (and Y4α /C1β)

Y2β (and Y4α /B1β)

754.80

736.80

Y3β (and Y4α)

1846.27Z3β - H2O

1077.20

Y1

Z1

1852.20

1820.20

Y3β /CO2

MS3

1836.13

B5 - H2O

Y3β2- (and Y4α)

n.a.

n.a.

n.a.

MSMS22

NeuAc – O – Gal – O – GalNAc – O – Gal – O – Glc – O – Cer

O

NeuAc

O

NeuAc

Z2β

Y2β

Z3β

Y3β

B2β

Y3α/B2β

Y3α/B1β

Y2α/B2β

Z1Y1Y2αY3α/B1β/Y1

Y4α

Y4α/B1β

B1β

B5

B5/B1β

C1β

FRAGMENTATION PATTERNFRAGMENTATION PATTERNIN IN MSMS22

916.80

1053.90

1282.06

1820.29

0

1000

2000

3000

Intens.

600 800 1000 1200 1400 1600 1800 m/z

1573.21

Y3β(and Y4α /B1β)

MS4

1864.21

[M-H]-

1846.27

B2β

581.32

562.40

B2β - H2O

736.80

754.80

Z1

Y1

Y2α/B2β

[M-H]- - H2O

1802.20

1555.20Z 3β(and Z4α/C1β)

1528.60

1514.60

1496.60

1481.20

1338.85

Y3β/CO2

1374.80

Y2β

1208.00

1025.00

1011.80994.66

Y2α

Z2α

2,4X3

Y2α/C1β

Z3/B2β/CH2O/H2O

Z3/B2β/CH3CO/CH2O/H2O

n.a.

n.a.

n.a.

n.a.

n.a.

MSMS33


O

NeuAc

O

NeuAc

Y2β

Z3β

Y3β

B2βY2α/C1β

Y2α/B1β

Z1Y1Z2αY2α

C1β

Z3α

B1β


592.66

916.80

1282.06

0

100

200

300

Intens.

200 400 600 800 1000 1200 1400 1600 1800m/z

1573.21[M-H]-

MS5

1555.20

1529.20

[M-H]-/CO2

[M-H]-- H2O

Y2β

1374.80

Z3α - H2O

1207.40

Y2α

1163.80

1120.00

Y3α/B1β

1102.00

Y3α/C1β

Y2α/B1β

754.80

Y1

736.80

898.00

Y2α/C1β

Z1

Y0

574.60

1025.60

1054.00

850.60

514.60

1512.20

2,4X3

1408.80

Y3α

655.08

Y3α/Y1

997.22

C4

Y2α /CO2

Z0

Z3α/B1β/CH2O/H2O

n.a.

n.a.n.a.

n.a.n.a.

n.a.

MSMS44


O

NeuAc

Z0Y0Z1Y1Y2αZ3α

Y3α

Y2β B1β

C1β

Y3α/B1β

Y2α/B1β

Y2α/C1βY3α/C1β

C4


Gal – O – GalNAc – O – Gal – O – Glc – O – Cer

Y0Z1Y1Y2Z3

0

5

10

15

20

25

Intens.

200 400 600 800 1000 1200 1400 1600 m/z

1282.06

[M-H]-

592.66

Y0

1053.80

1101.801011.60

916.60

754.80

736.80

Y1

Z1

Y2

Z3

MS6

Z3/CH2O/H2O

Z3/CH3CO/CH2O/H2O

MSMS55

0

2

4

6

8

10

Intens.

200 300 400 500 600 700 800 m/z

592.66

[M-H]-

562.60

544.60

339.20

327.60

308.80

283.40

265.80

[M-H]-/CH2O

[M-H]-/CH2O/H2O

P

V

T

MSMS66 OH OH

NH

CO

V

P

T

20:1/18:0 = Cer

OH OH

NH

CO

V

P

T

20:1/18:0 = Cer


curs carbohydrate

Documents

specific signaling

polydisperse

unknown molecular

singly charged

doubly charged

esi fticr

gag chain

proteoglycans