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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
GlycoproteinsTypes of O-Glycans
GalNAc
Man
Fuc
GlcNAc
NeuAc
Glycoproteins
GalNAc
Man
Fuc
GlcNAc
NeuAc
Glycoproteins
GalNAc
Man
Fuc
GlcNAc
NeuAc
Glycoproteins
GalNAc
Man
Fuc
GlcNAc
NeuAc
Glycoproteins
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
Fully automated (-) nanoESI chip HCT MS1
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
Fully automated (-) nanoESI chip HCT CID MS2
of the singly charged ion at m/z 538.11
Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.3-0.6 V
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
I. Perdivara, E. Sisu, N. Dinca, K. Tomer, M. Przybylski, A. D. Zamfir, Rapid Commun Mass Spectrom. 2008
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
*
*
*
*
*
*
§
§
§
§
§ §
§
§
§
§ §
§§
§
§
§
§
I. Perdivara, E. Sisu, N. Dinca, K. Tomer, M. Przybylski, A. D. Zamfir, Rapid Commun Mass Spectrom. 2008
(+) 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
Fully automated (-) nanoESI chip HCT MS1
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
Fully automated (-) nanoESI chip HCT MS1
of polydisperse maltodextrins derivatized with aromatic amines
Average MW:2800 Da
Area m/z: (800-2400)
Conditions: Solvent: H2O; concentration 10 pmol/L; acquisition time 2 min; Chip ESI: -0.60 kV; capillary exit: -20 V.
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)
Conditions: Solvent: H2O; concentration 10 pmol/L; acquisition time 2 min; Chip ESI: -0.60 kV; capillary exit: -20 V.
Fully automated (-) nanoESI chip HCT MS1
of polydisperse maltodextrins derivatized with aromatic amines
Fully automated (-) nanoESI chip HCT CID MS2
of the singly charged ion at 1173.46 corresponding to Glc6-AGL
Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.5-0.8 V
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
Fully automated (-) nanoESI chip HCT CID MS3
of the singly charged ion detected in MS2 at 363.20
Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.2-0.4 V
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
Fully automated (-) nanoESI chip HCT MS1
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
Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.6-1.0 V
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
Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.6-1.0 V
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
Gal – O – GalNAc – O – Gal – O – Glc – O - Cer
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
Conditions: isolation width: 2 u; variable RF signal amplitudes within 0.6-1.0 V
NanoESI chip HCT CID MS4 using as a precursor
the Y4β- ion detected at m/z 1544.87 in MS3
Gal – O – GalNAc – O – Gal – O – Glc – O - Cer
O
NeuAc
O
NeuAc
B4
Z0Y0Y2α
Y3β
Y2α/B1β or
Y2α/C1β or
Fig3f
Z1Y1
C1β
B1β
Y3β /B2α
Z3β /B2αB2α
Z3αY3α
Y3α/B1β
Gal – O – GalNAc – O – Gal – O – Glc – O - Cer
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
Gal – O – GalNAc – O – Gal – O – Glc – O - Cer
O
NeuAc
O
NeuAc
Y2β
Z3β
Y3β
B2βY2α/C1β
Y2α/B1β
Z1Y1Z2αY2α
C1β
Z3α
B1β
FRAGMENTATION PATTERNFRAGMENTATION PATTERNIN IN MSMS33
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
Gal – O – GalNAc – O – Gal – O – Glc – O - Cer
O
NeuAc
Z0Y0Z1Y1Y2αZ3α
Y3α
Y2β B1β
C1β
Y3α/B1β
Y2α/B1β
Y2α/C1βY3α/C1β
C4
FRAGMENTATION PATTERNFRAGMENTATION PATTERNIN IN MSMS44
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
FRAGMENTATION PATTERNFRAGMENTATION PATTERNIN IN MSMS66