MALDI-MS capillary LC fractionation MS/MS sequencing nanoESI

MALDI-MS capillary LC fractionation MS/MS sequencing nanoESI

MALDI-MS
capillary LC
fractionation
nanoESI-MS/MS
sequencing
low info, high throughput
MALDI- MS/MS
sequencing
LC-MALDI-MS/MS
LC-ESI-MS/MS
high info content, lower throughput
Purified
protein or
2D gel
protein spot
Combination
of PMF and
MS/MS search
Single protein ID
MALDI-MS
Protein Mix
(sub)-Proteomes
MALDIMS/MS
sequencing
Probable identity of
unassigned peaks /
confirm matched
PMF peaks
When do we need LC-MS/MS ?
capillary LC
fractionation
nanoESI-MS/MS
sequencing
MALDI-MS/MS
sequencing
LC-MALDI-MS/MS
LC-ESI-MS/MS
Which LC-MS/MS Workflow ?
• Offline LC-MALDI-MS/MS removes
temporal constraints for targeted analysis
• LC-ESI-MS/MS allows specific online
detection of modified peptides
• LC-MS/MS ensures detection
and MS/MS of more peptides;
• Better quality CID MS/MS on
doubly and triply charged
peptides better ion score
• ESI and MALDI affords
complementary sets of peptides
alleviates problems of ion
suppression – increase
coverage and/or protein hits
Workflow Design
Purified
protein or
2D gel
protein spot
Combination
of PMF and
MS/MS search
MALDI MS +
auto MS/MS
targeted
MALDI MS/MS
Protein
Mix
Probable identity of
unassigned peaks /
confirm matched
PMF peaks
LC-separation
Choice of 1D vs 2D;
time + gradient
offline
online
MALDI MS +
(auto MS/MS)
targeted
MALDI MS/MS
Confident
Positive ID
LC-ESI-MS/MS
Targeted and
PID mode ESIMS/MS
Manual
inspection / de
novo sequencing
increase coverage;
identify modifications
and/or additional
proteins
Tandem Mass Spectrometry (MS/MS)
Sample Mixtures
Ionized
Molecular Ion
MS 1
Select Sample for MS/MS
Fragment Ions
Fragment Ions
MS 2
CID
Fragment Ions
Molecular Ion
Molecular Ion
m/z
m/z
m/z
Molecular Ions
Individual MS/MS sequentially
m/z
GC/LC-MS and Tandem Mass Spectrometry (MS/MS)
Sample Mixtures
chromatogram
time
GC/LC
separation
Ionized
Online
Molecular Ion
MS 1
online DDA of MS/MS
Fragment Ions
Fragment Ions
MS 2
CID
Fragment Ions
Molecular Ion
Molecular Ion
m/z
m/z
m/z
Molecular Ions
Molecular Ions
Individual MS/MS sequentially
Molecular Ions
m/z
m/z
m/z
Individual MS sequentially
M1
M3
M2
MS scan
M4
Product ion scan
M4
Set at
precursor m/z
m/z
MS/MS
M4
Precursor ion scan
M4
M1
Scan for
parent ions
Product ion scan
M3
M4
M2
M4
Neutral loss scan
for M -
Set at
product m/z
Neutral loss scan
M4
Precursor ion
scan
for
Scan
product ions
M4
Set at parent
m/z = m
Set at product
m/z = m- x
GC/LC-MS and Tandem Mass Spectrometry (MS/MS)
Abs
Sample Mixtures
Other detector
M1
M3
GC/LC
separation
M2
M4
time
chromatogram
TIC
MS detector
M2
M1
M4
M3
Direct MS
time
m/z
m/z
TIC
m/z
SIM / SRM / MRM
m/z
Precursor ion scan
for
Neutral loss scan
for M LC-MS
time
m/z
m/z
MS survey, possible to trigger MS/MS
Use of LC-MS/MS for selective
detection
LC-separation
nanoLC
Choice of 1D vs 2D;
time + gradient
offline
online
MALDI MS +
(auto MS/MS)
targeted
MALDI MS/MS
LC-ESI-MS/MS
Alternate Low /High
CID energy MS survey
Targeted and
PID mode ESIMS/MS
Manual
inspection / de
novo sequencing
No
Desired
fragment ion
present ?
or
Desired
neutral loss
detected ?
Yes
from low energy MS survey, create
list of candidate parent ion masses
trigger MS/MS
Specific online LC-MS detection of
modified peptides
• True precursor ion and neutral loss
scans on triple quads, linear Q-Trap
• Pseudo-precursor ion / neutral loss
scan modes (reconstructed
chromatogram) on ion traps or Q/TOF
• Precursor Ion Discovery modes on
Q/TOF
acquire 1 verification scan of MS/MS
spectra for each of the shortlisted
candidate precursor ions
No
Pass the precursor ion
or neutral loss criteria ?
Yes
acquire full duration MS/MS spectra
for each the verified parent ions
Strategy for Characterizing a Glycoprotein
(glyco)-Peptide
Digests
± PNGase F
± endo F
RP nanoLC
Released total
glycan pools
Lectin
columns
MALDI-MS/MS
nanoESI-MS/MS
Enriched
subsets of
Glycopeptides
Peptide Map
What glycans ?
On which sites ?
Detection of de-Nglycosylated peptides
Site Occupancy
MALDI-MS Mapping +
MALDI-MS/MS Sequencing
Glyco-peptides
MS/MS sequencing
Specific MS
Detection of
Glycopeptides
Glyco-proteomics - Concerted proteomic analysis of glycosylated and
deglycosylated sub-proteomes, and glycomics analysis of the released glycans
(glyco)-Peptides
± PNGase F
/endo F
Proteome
Glycomics
Total proteome extracts
SCX + RP
2D nanoLC
nonretained
Targeted
from Cell, tissues,
Glycoproteome
fluids etc
Lectin/mAb
columns
MALDI-MS/MS
nanoESI-MS/MS
Glycome
Enriched
subsets of
Glycopeptides
Proteomic Map
Precursor Ion Discovery
mode analysis
Specific online LC-MS
Detection of Glycopeptides
Released total
glycan pools
MALDI-MS Mapping +
MALDI-MS/MS Sequencing
What glycans ?
On which glycoproteins ?
Glycopeptides
MS/MS sequencing
Experimental Approach
Glycoprotein
extracts from
Biological
source
Protease
Glycopeptides
PNGase F
fractionation
±
± permethylated
N- glycans
( with -3,6 or -3
core fucosylation)
Glycans
±
Exo-glycosidases
O- glycans
Mild periodate oxidation
MALDIMS/MS
O
O
O
A1
A2
Mucin
Acidic Glycans
Methyl Esterified
OH
Sialylated
B2 C 2 A3
RP SPE
Neutral Glycans
O
O
PNGase A
Ion Exchange
X1 Y1 Z1
HO
Branching Pattern
& Sequence
Reductive
elimination
C2 / C4
halves
MALDI-MS
Molecular
Composition
Glycopeptides
N- glycans
(with/without -6
core fucosylation)
Chemical derivatization
±
RP SPE
Ion Exchange
Sulfated
Periodate Oxidation and Smith Degradation
NaIO 4
H H
HO OH periodate
oxidation
adjacent OH
O
O
reduction
Periodic acidSchifff (PAS)
staining
OH
OH
O
O
O
OH
4
O
3
OH
RO
OH
O
HO
OH
3
OH
NHAc
O
RO
OH
O
NHAc HO
HO
O
OH
HO
OH
HO
O
oxidation
OH
O
HO
mild periodate
O H Ser/Thr
3
NHAc
HO
OR
O
HO
OH
OOH
NHAc
4
O
OH
O
O
OH
6
6
HO NHAc
HO NHAc
HO
OH
3
O
RO
O
RO
RO
cleaved
HO
HO
H H
HO
OH
Glycanspecific
labeling for
detection
OH
O
OH
O
HO
OH
OH
OH
O
O
O
O
3
OH
OH
O
HO
NHAc HO
3
O
NHAc
HO
OH
O
HO
O
HO
OH
OR
HO
O
4
4
OH
OR
Smith Degradation
OH
OH
O
O
O
3
HO
NHAc
OH
OH
OH
4
4
O
HO
NHAc HO
Chemical Derivatization for structural analysis
Partially methylated
alditol acetates
6
5
HO
6
OH
O
1
4
3
HO
MeO
1
4
MeO
O
MeO
2
O
Hydrolysis
& Reduction
PerMethyl Derivatives
MeO
OAc
Acetylation
OH
6
C H2OMe
1
C DHOAc
2
OAc
MeO
MeO
OAc
OH
6
OH H
N
O
OAc
MeO
OH
Linkage Analysis
FAB-, ESI-, MALDI- MS
and CID MS/MS Analysis
HO
MeO
2
C DHOAc
2
GC -EI-MS
Reducing end tagging
for HPLC
HO
1
4
3
3
perMe
2
O
1
OMe
OH
5
O
5
O
6
OMe
C H 2OMe
2-AB
O
H2N
2-aminobenzamide
PA
H2N
N
2-aminopyridine
Reducing end derivatization allows distinction
between reducing end and non-reducing end
fragment ions
ESI-MS/MS vs MALDI MS/MS on QTOF for biantennary structues
H +/N a +
897
1851
1035.5
O
464
O
H +/N a +
1143
O
O
662
866
Na+
1402
HO
1606
Na+
486
O
O
HO
1143
939
1329
1793
O
O
486
O
O
1606
O
545
1402
1793
Permethylation facilitates CID MS/MS Analysis
O
O
O
935
486
O
O
1157
545
953
Major N-glycans from
Pigeon Egg White Ovalbumin
Occurrence of Gal1-4Gal (P1 epitope)
JBC (2001) 276, 23221-23229; JBC (2001) 276, 23230-23239
Major N-glycans from Pigeon Egg White Ovalbumin
Monosialylated,
Monosialylated, Gal
Gal1-4Gal containing PA-glycans
Penta-antennary
668
6
ms-7
ms-7
4
2
PA
4
2
825
Tetra-antennary
4300
t-Gal, 4-Gal, 6-Gal, 2,4-Man,
2,4,6-Man, 3,6-Man, 4-GlcNAc
Tri-antennary
After neuraminidase, -galactosidase and -GlcNAcase
6-Gal and 2,4-Man disappeared, no 2-Man
Major N-glycans from Pigeon Serum IgG
Sialylated,
Sialylated, Fucosylated,
Gal
Gal1-4Gal containing
glycans
Major N-glycans from Pigeon Serum IgG
±
±
±
±
±
±
±
±
±
Major N-glycans from Pigeon Serum IgG
±
±
±
Hex6HexNAc4CF
Hex8HexNAc4CF
Sialylated,
Sialylated, Fucosylated,
Gal
Gal1-4Gal containing
glycans
2884
2216
2625
1956
Hex6HexNAc4CF
3292
Me
2216
2625
2884
3088
Hex7HexNAc4CF
2420
1956
2829
2160
3496
Me
2216
2625
Me
2884
Hex8HexNAc4CF
Me
690
3292
2420
1548
O
O
O
2216
719
O
1289
3088
1289
HO
MS/MS sequencing revealed
unusual terminal sequences
HO
HO
648
O
HO
3700
3033
2160
O
O
O
838
O
O
894
Me
1956
O
2829
HO
1956
474
Major N-glycans from Pigeon Serum IgG
Sialylated,
Sialylated, Fucosylated, Gal
Gal1-4Gal
containing glycans
±
±
±
Hex6HexNAc4CF
Hex8HexNAc4CF
Linkage Analysis
±
±
±
±
±
±
±
Major N-glycans from Pigeon Serum IgG
±
±
±
±
±
±
±
±
3292
690
2420
1548
O
O
O
894
O
O
O
How to interprete and assign
MS/MS spectra of
permethylated N-glycans
based on characteristic
fragmentation pattern
719
474
1289
2216
3088
HO
HO
3033
2160
3700
HO
O
O
O
HO
O
HO
838
HO
838
1097
486
HO
690
HO
O
648
HO
1956
2829
852
1111
PA
894
566
HO
Mapping of non-reducing termini,
number of antenna, and core
fucosylation
847
HO
HO
866
Structures Common to Different Types of Glycans
Variation on common themes
Biosynthetic regulation : Fucosyltransferases; Sialyltransferases; Sulfotransferases
Sda
4
HNK-1
N-Glycans, O-Glycans
and Glycosphingolipids
"C ore" Synthesis
Terminal
Glycosylation
SO 4-3 GlcA1-3
Elongation and Branching
6', 6 bis-sulfo-sialy l
6 sulfo-sialy l
6' sulfo-sialy l
±SO 4
±SO 4
Lewis X
6 6
SO 4
PO 4
2 Fuc
3/ 4 Fuc
LacdiNAc
Type 1
3
1-4 GalT
Type 2
4
3 Gal
6 Fuc
SO 4 4
1-3 GalT
1-3 GlcNAcT
n
4
Poly-N -acetyllactosamine
±
3 N euAc
n
6 N euAc
4 GalNAc
1-6 GlcNAcT
n
4
±
MeO
MeO
What are the characteristic
fragmentation
?
..
O
O
N-glycans
O
MeO1143
HO
MeO
O
486
OMe
X2 Y2
OR
OO,4A
RO
NMeAc
MeO
O
OMe
Z2
O
O
O
O
oxonium
O
OH
O
1303
HO
O
O
474
HO
660
MeO
852
HO
2,4A
O
MeO
O-glycans
1129
HO
MeOMeNAc
MeO
852
HO
847
O
H+
Fuc- OHO
HO
690
3
866
OMe
OMe
O
B2
2
O
C2
O,2A
O 6 NMeAc
284
HO
O
O
MeO
C4
O
620
O
AO,2
OMe
694
868
486
660
1055
1331
O
O
881
O
418
C4
OR
OMeO 6
mild
3
periodate
O
Elimination
of
3-substituent
cleavage
MeO
298O
3
O +
O
O
O
MeO
546
O AO,4
O
O
4
3
MeO
3
MeO
Core 1
3,5A
HO
3
3
Fuc- O
O
Core 2
OMe
+
MeO
449
708
O
O
636
OMe
C4
486
C4
NMeAc
708
O
O
432
245
What are the characteristic fragmentation ?
N-glycans
HO
RO
866
O
O
486
O
O
O
OH
O
1303
HO
MeNAc
852
HO
847
O
O
HO
HO
690
X 2 Y 2 Z2
O,4A
1143
2,4A
O
474
HO
660
O-glycans
1129
HO
Core 2
546
O AO,4
O
C4
O
620
O
AO,2
OMe
694
868
486
660
1055
1331
O
O
881
O
418
O
O
C4
449
708
3
O
3
3
Core 1
4
3
mild
periodate
cleavage
284
O
HO
3,5A
O6
3
O
MeO
C2
O 6
852
HO
B2
2
O,2A
O
298
C4
O
O
636
O
C4
486
708
O
O
432
245
Concerted Glycomics and Glycoproteomics Strategies
Molecular
Composition
MALDI-MS
MALDI-TO F
MALDIMS/MS
Branching Pattern
/ Sequence /
Terminal Epitope
MALDI-TO FTO F
MALDI
t arge t plat e
MALDI-Q -TO F
GAGs
Mucin
Glycomics
Glycome
Cell, tissue,
organism
C18 Capillary
nanoLC column
Sequential release of
Glycan Pools / Fractionation
Membrane
glycoproteins
/ glycolipids
Secreted
glycoproteins
/ tissue fluids
Direct offline
nanoESI
Glycopeptides
nanoESIQ -TO F
Glycopeptide Detection
and MS/MS Sequencing
GlycoProteomics
Glycosylation Site
and Protein ID
Profiling of N-glycans from mouse serum
Normal
1
2
1
Higher abundance in
NeuAc relative to NeuGc
3
2
CT-26
NeuGc/NeuAc content
similar to normal mice
Higher abundance in
disialylated antenna
TSA
biantennary core
+
HO
HOHO-
HOHO-
+
Differential Liver Glycomic Profiles from Normal and
Schistosoma mansoni-Infected Mice
From infected mouse
NeuGc
1
M7
+
2
M9
M6
NeuAc
1
M8
M5
+
2
+
+
+
Normal, uninfected
+ neuraminidase
C MP-NeuAc
CMP-NeuAc hydroxylase
(CMPH)
C MP-NeuGc
normal
[
]3
[
1 3 5
]4
[
infected
7 9 12 1 3 5
7 9 12 week
CMPH
]5 [
]6
Beta-actin
Strategy for Characterizing a Glycoprotein
(glyco)-Peptide
Digests
± PNGase F
± endo F
RP nanoLC
MALDI-MS/MS
Released total
glycan pools
MALDI-MS Mapping +
MALDI-MS/MS Sequencing
nanoESI-MS/MS
What glycans ?
Peptide Map
Detection of de-Nglycosylated peptides
Site Occupancy
MALDI-QTOF MS and MS/MS of the tryptic digests of a glycoprotein
Peptide Mass Fingerprinting
followed by
Unusually high, dominant peak,
not mapped to any predicted
peptide
MALDI MS/MS Sequencing
Glycopeptide not observed
y”2
322
pyroGlutamate-D-STQNLI-PA-PSLLTV-PLQ-PD-FR
y7
y21
y13
y2
y15
x20
MALDI MS and MS/MS of de-N
de-N-glycosylated proteins for mapping of
glycosylation site occupancy.
Protein 1, 2 potential N-glycosylation sites
on two separate tryptic peptides
[M+H]+ = 1627.83
118
131
NSSNFHLNQLQGLR
D
+ 0.984
482
[M+H]+ = 1742.9
497
TAGWNIPMGLLANQTR
Mox (+16 u)
D
+ 0.984
Protein 2, 2 potential sites on a single tryptic glycopeptide
x4
TEGSFTMYSTIYELQE-N-N-SYD-VTSILVR
y12
x3
y10
y7
Total mass increment = 1
only one site
is occupied
Parent
Strategy for Characterizing a Glycoprotein
(glyco)-Peptide
Digests
± PNGase F
± endo F
RP nanoLC
Released total
glycan pools
Lectin
columns
MALDI-MS/MS
nanoESI-MS/MS
Enriched
subsets of
Glycopeptides
Peptide Map
What glycans ?
On which sites ?
Detection of de-Nglycosylated peptides
Site Occupancy
MALDI-MS Mapping +
MALDI-MS/MS Sequencing
Glyco-peptides
MS/MS sequencing
Specific MS
Detection of
Glycopeptides
Amino acid sequences of the
secreted (S-EGFR) and the
extracellular domain (EC-EGFR) of
the full-length EGF receptor with
the potential N-glycosylation sites
highlighted. The difference between
the two sequences lies in the Ctermini, as indicated by the split.
N615 is a potential site of
glycosylation in S-EGFR but not in
EC-EGFR.
from Zhen et al., (2003) Biochemistry 42,
5478-5492. Characterization of glycosylation
sites of the epidermal growth factor receptor.
when the Lys-C digested S-EGFR was analyzed using
MALDI-MS, 16 peptides were observed, which include
almost all the peptides with no potential glycosylation sites,
except the first two N-terminal peptides.
Observation of four
potential glycopeptides as
nonglycosylated peptides
suggests that these four
glycosylation sites are
either not glycosylated or
are only partially
glycosylated in A431 cells
MALDI-MS analysis of the fraction containing peak 1 (Figure 3) revealed a series of peaks (from
m/z 5500 to 8000) centered at m/z 6800 (Figure 4). The mass differences between these peaks
were 365, 291, and 162 Da (±1 Da), characteristic of glycopeptides.
After the treatment with PNGase F, a new peptide with a monoisotopic mass of m/z
4448.3 was observed in the sample (Figure 5), attributable to the peptide spanning
residues 477-514, which has a theoretical monoisotopic mass of 4447.0 Da for the
nonglycosylated peptide with an Asn at position 504 (Figure 5). The observed 1-Da
increase in the mass of the deglycosylated peptide corresponds to the conversion of
the Asn504 to Asp by PNGase F.
The peptide was further digested with Glu-C, and the digestion products were analyzed by MALDIMS. Two new peptides were observed with monoisotopic masses of 1429.6 and 2535.1 Da (data
not shown), which correspond to peptides spanning residues 477-489 and 490-510, respectively.
The peptides from the Glu-C digestion were further analyzed using nano-ESI MS/MS.
GlycoMod :
GlycoMod is a program designed to find all possible compositions of a glycan structure from its
experimentally determined mass. This is done by comparing the mass of the glycan to a list of precomputed masses of glycan compositions.
The program can be used with free or derivatised glycans and for glycopeptides where the peptide
mass or protein is known. Compositional constraints can be applied to the output.
Structural analysis using GlycoMod suggests
that the ion at m/z 5691.5 most likely has the
following glycan structure:
When analyzing the m/z 7532.8 glycoform
using GlycoMod, six possible sugar
compositions were suggested. However,
based on the observation (Figure 4) that this
glycoform contains at least four Hex, three
HexNAc, one NeuNAc, and one fucose residue,
only two of the six possible compositions
meet these criteria:
Database search using the software GlycoSuiteDB
suggests that the glycan with m/z 7532.8 is most
likely to have the following structure:
Since the mass of a sialic acid residue (291 Da)
is about twice the mass of a fucose residue (146
Da), the m/z 7532.8 glycoform mathematically
fits both compositions.
Carr SA, Huddleston MJ and Bean MF (1993) Protein Sci, 2, 183-186
nanoESI-MS/MS of a glycopeptide
366
204
657
MS/MS on 4+ parent
N
N
MS/MS on 5+ parent
Selective Detection of Glycopeptides during LC-MS
orifice voltage ramped
120V at m/z 150
65V at m/z 500
Glycopeptides
65V held constant tp m/z 2000
Glycopeptides
Parent Ion Scan, TIC
A number of parent ions are present
indicates that one or more of the
parents contain glycan
parent ion scan specifically detect
only the glycopeptide
Roberts et al
+ ve
Parent ion scan
for m/z 204
m/z 86
for I/L immonium ion
- ve
m/z 147
for y1=Lysine
Parent ion scan
for m/z 168
parent ion scan for m/z 79
m/z 175
for y1=Arginine
Detected Glycopeptide
Precursor Ion Discovery
Precursor Ion
Discovery
nanoLC
peptide and
glycopeptide
mixture
LC-MS peptide map
Alternate Low
/High CID energy
MS survey
[low energy, no CID]
+
Selected product
ion monitoring
profile [high energy]
No Desired fragment
glycopeptides
selected for
MS/MS
ion present ?
Yes
from low energy MS survey,
create list of candidate
Precursor ion masses
trigger MS/MS
acquire 1 verification
scan of MS/MS spectra
for each of the shortlisted
candidate precursor ions
analysis time
not spent on
false positives
No
Desired
fragment ion
present in
MS/MS
spectrum of the
candidate
precursor
?
Yes
acquire full duration
MS/MS spectra for each
the verified precursor ions
examples of false positive
SIM profile of high
enery MS survey
product ions
monitored
Low energy MS survey
region where
glycopeptides eluted
204
366
Glycan
specific
fragment ions
N
2+
657
Peptide core ion,
specific to each set
of glycopeptides
Glycan sequence
informative y fragment ions
1+
N
N
Glycopeptides differ in the
number of sialylated
antenna share majority of
fragment ions
N
204
366
Glycan
specific
fragment ions
LCPDCPLLAPLNDSR
y13
N
2
+
y2
y10
y6
y7
y6
657
y7
MS/MS on core
peptide to
derive peptide
sequence
y10
y8
y9
N
y2
y11 y13
N
N
N
N
N
N
N
N
Glycopeptides differ in the
number of sialylated antenna
share majority of fragment ions
N
N
Glycan
specific
fragment ions
2+
y7
y9
y5
2+
y6
SNPCLNGGSCK
y5
y8 D
y6
y7
D
Cam
1620
1502
1634
P
MaxE3 transformed
Peptide core
SNPCLNGGSCK
y9
y8
L
1766
1340
2131
SNPCLNGGSCK
1867
1766
1999
Parent ion
Lactoferrin Glycopeptide heterogeneity
131
118
NSSNFHLNQLQGLR
m/z for M+2H+ = 915.9
3+
for 2x
m/z for M+3H+ = 1327.57
m/z for M+3H+ = 1327.23
+
482
497
TAGWNIPMGLLANQTR
for 2x
m/z for
M+2H+ = 973.5
m/z for M+3H+
= 1365.93
3+
m/z for
M+3H+ =
1365.59
+
nanoESI-MS/MS sequencing of glycopeptides from lactoferrin
482
204
657
366
497
TAGWNIPMGLLANQTR
1574.09
N
+
N
131
118
NSSNFHLNQLQGLR
512
N
N
658
989.43
1516.6
+
CID MS/MS of permethylated N-glycans with Lewis X/Y epitopes
Eliminate
Fuc
Eliminate
Fuc
1113
HOHOHOEliminate
Fuc
HO-
Eliminate
Fuc
Only Lewis X,
no Lewis Y
Fuc3-biantennary structures
from seminal vesicle fluid
HOHOEliminate
Fuc
Fuc3-biantennary structures
from uterine luminal fluid
Glyco-proteomics - Concerted proteomic analysis of glycosylated and
deglycosylated sub-proteomes, and glycomics analysis of the released glycans
(glyco)-Peptides
± PNGase F
/endo F
Proteome
Glycomics
Total proteome extracts
SCX + RP
2D nanoLC
nonretained
Targeted
from Cell, tissues,
Glycoproteome
fluids etc
Lectin/mAb
columns
MALDI-MS/MS
nanoESI-MS/MS
Glycome
Enriched
subsets of
Glycopeptides
Proteomic Map
Precursor Ion Discovery
mode analysis
Specific online LC-MS
Detection of Glycopeptides
Released total
glycan pools
MALDI-MS Mapping +
MALDI-MS/MS Sequencing
What glycans ?
On which glycoproteins ?
Glycopeptides
MS/MS sequencing