HighGlycan Meeting Split 2013
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HighGlycan MeetingSplit 2013
LUMCCenter for Proteomics and Metabolomics
HTP linkage-specific sialic acid stabilization for glycan analysis by MALDI-TOF-MS
Karli Reiding (PhD student)
20-04-232
Linkage-specific sialic acid reactivity
20-04-233
1h 37°C
1h 37°C
EDC/HOBtEthanol
EDC/HOBtEthanol
α2,6+28 Da
α2,3-18 Da
Reaction comparison
20-04-2342
27
3.8
07
19
68
.69
3
16
47
.58
7
21
14
.74
9
14
85
.53
5
18
09
.64
1
29
12
.01
8
24
19
.85
9
30
58
.07
2
26
38
.93
1
35
50
.21
8
22
73
.79
8
14
85
.53
5
19
68
.69
3
16
47
.58
8
21
14
.74
6
24
19
.84
5
18
09
.64
0
29
11
.96
9
30
58
.01
7
26
38
.90
6
35
50
.12
5
22
73
.80
5
19
68
.69
3
16
47
.58
7
14
85
.53
4
18
09
.63
8
21
14
.74
8
24
19
.86
1
29
12
.06
5
26
38
.94
5
30
58
.15
5
35
50
.52
9
22
73
.80
0
19
68
.69
3
16
47
.58
6
21
14
.74
9
18
09
.63
8
24
19
.84
7
14
85
.53
3
29
11
.93
7
30
57
.95
9
26
38
.89
8
35
49
.93
5
22
73
.80
0
19
68
.69
5
16
47
.58
7
21
14
.75
0
18
09
.64
1
24
19
.84
2
14
85
.53
4
26
38
.87
3
29
11
.85
6
30
57
.84
2
1000 1500 2000 2500 3000 3500 m/z
22
73
.82
3
19
68
.71
4
16
47
.60
5
29
12
.03
4
21
14
.76
4
18
09
.66
1
14
85
.54
5
24
19
.87
6
30
58
.08
2
35
50
.22
6
26
38
.95
2
22
73
.80
0
19
68
.69
3
16
47
.58
6
14
85
.53
3
21
14
.74
6
24
19
.84
2
18
09
.63
8
29
11
.93
0
30
57
.94
8
26
38
.89
3
35
49
.94
7
22
73
.80
2
14
85
.53
3
16
47
.58
8
19
68
.69
5
18
09
.64
0
21
14
.74
8
24
19
.86
1
26
38
.93
2
29
12
.01
5
22
73
.80
4
19
68
.69
2
16
47
.58
5
21
14
.74
9
18
09
.63
7
14
85
.53
3
24
19
.85
8
29
11
.99
6
30
58
.04
9
26
38
.93
0
35
50
.14
9
22
73
.80
5
16
47
.58
6
19
68
.69
2
14
85
.53
4
18
09
.63
9
21
14
.74
7
24
19
.86
0
26
38
.93
4
29
12
.01
2
1000 1500 2000 2500 3000 3500 m/z
DMT-MM DMT-MM
DCC + HOBt DCC + HOBt
DCC + Oxy DCC + Oxy
EDC + HOBt EDC + HOBt
EDC + Oxy EDC + Oxy
No TFA 0.2% TFA
Direct plasma modification 1h 60°C methanol
Linkage specificity methanol
63
8.1
89
67
0.2
17
620 640 660 680 700 720
Inte
nsi
ty (
%)
0
100
m/z
Inte
nsi
ty (
%)
0
100
67
0.2
17
63
8.1
90
620 640 660 680 700 720m/z
1h 37°C
1h 37°C
20-04-235
Linkage specificity ethanol
63
8.1
90
68
4.2
32
620 640 660 680 700 720m/z
Inte
nsi
ty (
%)
0
100
68
4.2
32
63
8.1
90
620 640 660 680 700 720
Inte
nsi
ty (
%)
0
100
m/z
1h 37°C
1h 37°C
20-04-236
Method comparison
20-04-237
19
82
.70
8
23
01
.83
5
1800 2000 2200 2400 2600m/z
Inte
nsi
ty (
%)
0
100
Carboxyethylation MALDI-TOF-MS 2AA-labeled HILIC HPLC
Released fibrinogen N-glycans
Inte
nsi
ty (
%)
0
100
14 16 18 20 22 24 26 min
Plasma N-glycome profile
20-04-238
23
01
.84
2
19
82
.71
0
22
55
.78
6
16
47
.58
7
29
40
.05
0
21
28
.76
2
24
47
.89
5
18
09
.64
0
14
85
.53
3
30
86
.11
2
26
50
.97
1
12
57
.42
3
35
32
.22
7
32
59
.13
9
36
78
.28
3
34
05
.19
0
38
24
.34
5
39
43
.39
6
40
89
.45
1
43
08
.55
2
45
81
.66
9
41
89
.52
7
47
27
.83
1
1000 15002000
2500 3000 3500 4000 4500 m/z
Inte
nsit
y (
%)
0
100
38
24
.34
5
38
70
.37
6
39
43
.39
6
39
70
.40
7
38
97
.35
7
39
89
.42
9
40
89
.45
1
43
08
.55
2
40
43
.40
8
45
81
.66
9
41
89
.52
7
42
35
.60
8
47
27
.83
1
3800 4000 4200 4400 4600 4800 m/z
Inte
nsit
y (
%)
0
100
Major peak repeatability
20-04-239
Minor peak repeatability
20-04-2310
Workflow
20-04-2311
- Add and react 30 min at 37°C - Full sialic acid linkage specificity- Reaction mixture stable at -20 for > 1 month
- 15 min precipitation at -20 C⁰- HILIC recovery from 50% EtOH 50% ACN
- Unpurified PNGase-F release mixture
- Stable sialylated glycans in RP mode- Ethanol recrystallization removes shot to shot variability- Glycans detected up to 4800 Da
96-well format HILIC SPE
Stephanie Holst (PhD Student)Emanuela Lonardi
20-04-2312
HILIC-SPE in 96-well format
20-04-2313
Cotton HILIC protocol
•Equilibration • 3x mQ• 3x 85% ACN
•Sample loading • 20x, do not pipet precipitate
•Wash• 3x 85% ACN 1% TFA • 3x 85% CAN
•Elution• 5x in 10µl mQ
CARE
FUL
pipe
tting
with
m
ultic
hann
el p
ipett
e
Total time required: ~ 40 min
HILIC-SPE in 96-well format
20-04-2314
Plate HILIC protocol (optimized for HP from Burkina et al.)
•Equilibration • 200µl 70% EtOH• 200µl mQ• 200µl ACN 100%
•Sample loading • Samples in 50µl ACN (after precipitation @-20°C),
loaded onto filter plate• @50xg 1min
•Wash• 3x 200µl 96% ACN • @500xg 1 min• 1x @1000xg 1 min (to remove residual solvent)
•Elution• 10µl mQ • @1000xg 1 min
The samples are applicable for MS and UPLC
vacu
um
man
ifold
Cent
rifug
ation
(@)
Total time required: ~ 20min
AcroPrep 96 Filter Plates with 0.45 μm GHP membrane, 350 μL well volume (PALL)
HILIC-SPE in 96-well format
20-04-2315
23
02
.02
9
19
82
.87
7
16
47
.74
2
14
85
.68
4
22
55
.98
0
24
43
.16
3
18
09
.79
8
21
28
.94
2
14
19
.62
5
12
57
.56
5
29
40
.29
2
16
88
.76
7
19
05
.79
8
23
32
.03
82
35
8.0
73
26
51
.18
9
30
86
.35
7
25
84
.30
6
15
81
.68
2
21
98
.97
3
13
39
.61
8
11
93
.04
0
09092013SH86_ManuA1-12_Manu 0:L11 MS Raw
0
1
2
3
5x10
Inte
ns.
[a.u
.]
23
02
.49
5
16
48
.08
2
19
83
.29
0
14
85
.98
5
22
56
.44
0
24
43
.65
1
18
10
.17
6
21
29
.38
2
14
19
.90
8
12
57
.80
5
29
40
.85
0
16
89
.11
8
19
06
.19
7
23
32
.51
22
35
8.5
44
25
84
.81
5
30
86
.93
7
26
51
.70
7
15
82
.00
5
11
92
.25
6
13
39
.88
3
21
86
.41
0
10
30
.18
8
09092013SH86_ManuA1-12_Stephi 0:M11 MS Raw
0.0
0.5
1.0
1.5
2.0
2.5
5x10
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500 4000m/z
GHP-plate purification:
•High-throughput
•Better reproducible & standardized
•No/small inter-person variation
•Relative standard deviation at ~ 1.5% between 12 samples based on highest peak
•Re-use of plates is possible
Samples: carboxyethylated N-glycans released from pooled control plasma
Person 1
Person 2
HILIC-SPE in 96-well format
20-04-2316
13
05
.95
3
13
93
.99
9
12
17
.89
7 TEST1_75lp 0:B12 MS Raw
0
2000
4000
6000
Inte
ns.
[a.u
.]
13
05
.94
1
13
93
.98
7
12
33
.88
4
23
01
.68
3
TEST2_75lp 0:B13 MS Raw
0
1000
2000
3000
4000
Inte
ns.
[a.u
.]
TEST3_75lp 0:B14 MS Raw
0
1000
2000
3000
Inte
ns.
[a.u
.]
TEST4_75lp 0:B15 MS Raw
0
1000
2000
3000
Inte
ns.
[a.u
.]
23
01
.87
3
TEST5_75lp 0:B16 MS Raw
0
1000
2000
3000
4000
5000
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500 4000 4500m/z
Spectra after cleaning GHP with :
•3x200µL 10%ACN and 1x200µL mQ (vacuum
manifold)
•remove residual solvent (1 min @1000xg)
•elute in 10µL mQ (1 min @1000xg);
•Samples: ethylated Visicon plasma
→ Negligible intensities of Polymer (PEG from Tape)
and retaining G2S2
Automated workflow for the analysis of complex glycopeptide samples
Bas Jansen (PhD student)
20-04-2317
Sample preparation
20-04-2318
Plasma
Reduction, Alkylation & Digestion
C18 SPECotton HILIC
HPLC-MS(/MS)
Glycopeptide dataDeglycosylated peptide data
Automated workflow
20-04-2319
conversion
{raw}.mzML
output.mgf
compounds.mzML
CompoundSpectra
human.fasta
Glycopeptides results (pdf)
peptides.csv
LC-MS/MS {raw}.mzML
PeptMoiety
output.mgf (deglyc.)
find_glycopeps
peptides.csv
compounds.mzML (glyco.)
Com
pou
nd
ing
Un
ipro
t
Deg
lyc.
pep
tid
e
iden
tifi
cati
on
Gly
cop
ep
tid
e
iden
tifi
cati
on
Compounding
20-04-2320
0.5
1.0
1.5
2.0
2.5
3.0
3.5
200 400 600 800 1000 1200 1400
Inte
nsi
ty (
10
4)
m/z
14
21
.48
12
76
.51
36
6.1
1*
65
7.2
5*
52
8.2
1*
77
5.2
5
78
4.2
5
91
1.2
5
27
4.0
0*
89
3.3
8
74
9.3
8
20
4.0
0
MS2(645.44), 1.1min Combined spectrum
0.0
36
6.1
1*
52
8.2
2*
65
7.3
0*
77
5.2
8
89
3.3
2
10
05
.38
12
76
.51
14
21
.48
MS2(645.17), 1.1min #67
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Inte
nsi
ty (
104)
200 400 600 800 1000 1200 1400
91
1.3
1
20
4.0
0
74
9.3
26
90
.18
78
4.2
6
m/z
27
4.0
5*
36
6.1
1*
52
8.2
0*
65
7.1
9*
78
4.2
8
91
1.3
3
10
55
.36
12
76
.48
MS2(645.44), 1.4min #84
0.0
0.5
1.0
1.5
2.0
Inte
nsi
ty (
10
4)
200 400 600 800 1000 1200 1400m/z
77
5.2
17
49
.28
89
3.3
4
2.5
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
200 400 600 800 1000 1200 1400
Scan: 67Scan: 84
0.0m/z
Cum
ula
tive
Inte
nsi
ty
Identification
20-04-2321
MS2(995.51), 30.68miny2
(2
76
.16
)
y3
(3
91
.19
)
y4
(5
03
.21
)
y6
(7
16
.39
y7
(7
87
.44
)
y8
(9
00
.49
) 10
57
.50
0
1
2
Inte
nsi
ty (
10
8)
300 400 500 600 700 800 900 1000 1100m/z
y1
0 (
11
10
.66
)
Alpha-2-HS-glycoproteinVCQDCPLLAPLN*DTR
MS2(887.26), 27.0min 3
36
6.1
4*
65
7.2
3*
0
2
4
6
Inte
nsi
ty (
10
5)
400 600 800 1000 1200 1400 1600 1800 2000m/z
16
61
.11
15
16
.04
14
34
.04
12
52
.03
13
33
.52
8
98
6.4
7
10
39
.52
96
9.4
4
80
8.4
5
76
9.4
3
66
3.2
9
11
07
.80
11
71
.61
10
53
.40
10
11
.76
50
4.2
6
88
2.6
3
88
6.7
3
Results
20-04-2322
Antithrombin-III LGACN128DTLQQLMEVFK 0 0
Beta-2-glycoprotein 1 VYKPSAGN162NSLYR 1 5
Ceruloplasmin EHEGAIYPDN138TTDFQR 1 1
Clusterin LAN374LTQGEDQYYLR 2 2
Complement component C3 TVLTPATNHMGN85VTFTIPANR 0 0
Complement component C4B GLN1328VTLSSTGR 1 1
Complement factor B SPYYN122VSDEISFH 3 6
Complement factor H SQPPQIEHGTIN882SSR 1 2
Complement factor H ISEEN911ETTCYMGK 0 0
Complement factor H MDGASN1029VTCINSR 0 0
Complement factor I light chain FLNN103GTCTAEGK 5 5
Complement factor I light chain LFQPN464DTCIVSGWGR 1 5
Haptoglobin VVLHPN241YSQVDIGLIK 0 0
Haptoglobin HN184LTTGATLINEQWLLTTAK 0 0
Haptoglobin NLFLN211HSENATAK 1 3
Hemopexin ALPQPQN453VTSLLGCTH 0 0
Hemopexin SWPAVGN187CSSALR 1 2
Ig Alpha-2 SEAN131LTCTLTGLR 1 1
Ig Alpha-2 SVTWSESGQNVTAR 0 0
Ig Gamma-1 EEQYN180STYR 3 2
Ig Gamma-2 EEQFN180STFR 4 10
Ig Gamma-4 EEQFN180STYR 0 0
Ig Mu GLTFQQN209ASSMCVPDQDTAIR 0 0
Ig Mu YKN46NSDISSTR 7 15
Kalikrein heavy chain IYSGILN453LSDITK 0 0
Protein Peptide MS1 MS2
Total plasma N-glycome genome-wide association study
Karli Reiding (PhD Student)
20-04-2323
Genetics of glycosylation
20-04-2324
Plasma
IgG
Lauc et al., PLoS Genet., 2010
Lauc et al., PLoS Genet., 2013
Measurement
20-04-2325
HPLC
N loci-P<5 x 108
HPLC vs. FT
CROATIA-Vis (N=1008)
ORCADES (N=889)
NSPHS (N=700)
Dis
cove
ryR
ep
lica
tion
LLS 2AA-labeled plasma N-glycan GWAS
MALDI-FTICR-MS
24 traits 59 traits 87 derived
CROATIA-Korcula (N=969)
(N=2299)(N=2342)
HPLC
MALDI-FTICR-MSML_1 - 37 MH_1 - 25
Trait calculation
20-04-2326
HPLC
MALDI-FTICR-MS
- Peak area relative to highest peak
- Traits- Peak intensity relative to set peak
(H5N4S1)- Overlap allows for comparison low and
high spectra- Glycan composition from mass
- Derived traits- Summing of traits matching
composition features (e.g. fucosylation, sialylation)
24 traits
59 traits
87 traits
Results
20-04-2327
Chr. location P val Effect size Effect size SE Lowest P trait Glycogenes in interval Hits Traits
1 161674108 2.11E-09 -0.21422 0.03577 LC_4 RPL31P11(dist=19066),FCRLA(dist=2654) 42 3
1 173066780 2.77E-11 0.46521 0.06987 G2qb TNFSF18(dist=46677),TNFSF4(dist=86090) 1 8
5 10928594 1.01E-10 0.46135 0.07135 G4S4qb DAP(dist=167207),CTNND2(dist=43358) 1 4
5 79525260 4.23E-11 0.42744 0.0648 G2qb SERINC5 1 6
5 175046210 1.75E-09 0.59219 0.09839 G1S0qb SFXN1(dist=90589),HRH2(dist=38830) 2 19
7 73087652 9.94E-11 0.77977 0.12056 ML_5qb VPS37D(dist=1212),DNAJC30(dist=7596) 5 9
7 79923948 1.94E-09 0.75782 0.12624 S0Fqb GNAI1(dist=75223),CD36(dist=74943) 13 2
8 23276844 2.58E-11 0.63019 0.0945 ML_20qb ENTPD4 3 7
9 75160111 1.83E-11 0.80224 0.1194 ML_11qb TMC1 2 21
11 109912508 7.16E-10 0.52985 0.08598 G1qb C11orf87(dist=612615),ZC3H12C(dist=51418) 6 11
11 126250680 2.37E-19 0.32058 0.03564 G4Sqb ST3GAL4 69 9
12 121423659 1.81E-11 0.20986 0.03123 G3Fqb HNF1A 52 9
13 27449712 1.65E-09 0.55616 0.09225 G4S4perG4qb GPR12(dist=114790),USP12(dist=190575) 1 1
14 65884030 3.26E-19 0.2355 0.02628 MH_10qb FUT8 437 6
16 72102813 8.20E-11 0.21131 0.03253 LC_11 HPR 101 15
19 5.83E+06 2.22E-40 -1.12188 0.08433 S3Fqb FUT6, FUT3, FUT5 28 26
19 50443846 1.60E-09 0.33108 0.05486 Gqb ATF5(dist=6653),SIGLEC11(dist=8404) 2 14
- 1000 Genomes: 2322 hits found with p<1.0e-7
- Previous results replicated- Switch to 1000 genomes?
LC FT
1 0
0 1
0 1
0 1
0 1
0 1
0 1
0 1
0 1
0 1
1 1
1 1
0 1
1 1
1 1
1 1
0 1
High-throughput analysis of IgG Fab glycosylation
Albert Bondt (PhD student)
20-04-2328
IgG Fc/Fab glycosylation analysis
20-04-2329
Eluatecontains Fcand some undigested IgG
IgG Fc/Fab glycosylation analysis
20-04-2330
N-acetylglucosamineGalactose
Mannose
Fucose
Sialic acid
1400 1600 1800 2000 2200 2400 2600m/z
A
B
Fab
Fc
Bondt et al., manuscript in preparation
IgG Fc/Fab glycosylation analysis
20-04-2331
IgG Fc/Fab glycosylation analysis
20-04-2332
High-throughput analysis of IgA N- and O-glycopeptides
Albert Bondt (PhD student)
20-04-2333
IgA glycosylation analysis
April 20, 202334
Zauner et al., Mol Cell Prot (2013) 12(9): p.856-865c
IgA glycosylation analysis
April 20, 202335
IgA N-glycosylation assignment
April 20, 202336
4245.823
4408.877
4448.904
4536.912
4552.893
4611.896 4699.979
4739.992
4756.067
4903.059
4200 4300 4400 4500 4600 4700 4800 4900 m/z
LAG-Y
*
*
**
* *
LAGKPTHVNVSVVMAEVDGTC(Y)
Bondt et al., manuscript in preparation* oxidation products
IgA N-glycosylation certainty
LAG-Y peptide mass 2183.0709Glycan H5N4F1S2 mass 2350.8303+ H+= 4534.9091
Calculated mass
Error -0.88 ppm•C-terminal Y truncated•Klapoetke et al., 2011
April 20, 202337
4534.9051
4535.9075
4536.9098
4537.9119
4538.9144
4539.9175
4540.9206
4541.9231
4542.9282
0.0
0.2
0.4
0.6
0.8
1.0
8x10Intens.
4534 4536 4538 4540 4542 4544 m/z
IgA O-glycosylation assignment
April 20, 202338
5276
.319
5364
.351
5438
.372
5479
.399
5525
.381
5567
.417
5599
.439 56
41.4
69 5729
.471
5770
.494
5803
.518
5858
.530
5891
.530
5932
.544
5954
.531
6020
.561
6042
.544
6094
.598
6117
.595
6182
.642
6223
.658
6245
.626
6311
.660
6334
.642
6385
.694
6407
.682
6473
.714
6514
.762
6547
.778
6589
.808
6609
.771
6677
.805
6699
.786
6764
.809
6787
.809
6839
.871
6860
.860
6880
.888
6901
.856
6967
.886
6990
.870
7011
.855
7055
.925
7130
.978
7152
.967
7172
.004 72
60.0
0972
82.0
01
7335
.050
7422
.037
7445
.044
7466
.024
7551
.098
7595
.087
7625
.177
7713
.169
7801
.021
5000 5500 6000 6500 7000 7500 m/z
HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR
Bondt et al., manuscript in preparation
IgA O-glycosylation assignment certainty
HYT O-glycopeptide mass 4135.8821Glycan H4N5F0S4 mass 2827.9898+ H+= 6964.8797
Calculated mass
Error +0.46 ppm
April 20, 202339
6964.8829
6965.8804
6966.8790
6967.8831
6968.8847
6969.8872
6970.8923
6971.8956
6972.8946
6973.9015
6974.9132
0.0
0.2
0.4
0.6
0.8
1.0
8x10
Intens.
6964 6966 6968 6970 6972 6974 6976 m/z
IgA O-glycosylation assignment
April 20, 202340
• According to literature only 5 O-glycosylation sites are occupied• Mattu 1998: HYTNPSQDVTVPCPVPST225PPT228PS230PS232TPPT236PSPSCCHPR • Deshpande 2010:
HYTNPSQDVTVPCPVPS224TPPTPS230PS232TPPTPS238PS240CCHPR
• Takahashi 2012 JPR: 6 O-glycosylation sites HYTNPSQDVTVPCPVPST225PPT228PS230PS232T233PPT236PSPSCCHPR
7422.0187
7462.0144 7496.03277551.0392 7625.1003
7714.0929
7787.1769
162.0766
203.0816
291.0765
0
2
4
6
x107
Intens.
7450 7500 7550 7600 7650 7700 7750 7800 m/z
IgA O-glycosylation certainty?
Can we be sure?????
April 20, 202341
IgA O-glycosylation
April 20, 202342
IgA-OHy Exp 6 IgA-TFA-OHy-B Exp 6 IgA-MIX-1-A Exp 6 IgA-MIX-4-B Exp 6
GU 2.30
GU 2.98 GU 4.58GU 1.75
GU 2.30
GU 2.98
GU 4.58
GU 1.75
GU 2.30
GU 2.98
GU 4.58
GU 1.75
GU 2.30
GU 2.98
GU 4.58
GU 1.75
21%
67%
21% 2%
23%
21%
51%
5%
30%
15%
53%
3%
31%
12%
55%
3%
peeling
IgA- released with hydrazine
IgA- washed with 0.1%TFA and released with hydrazine
IgA- released with 50mM Ethylenediaminetetraacetic acid disodium salt dehydrate /hydrazine mix
IgA- released with 20mM Ethylenediaminetetraacetic acid, anhydrous /hydrazine mix
Rad Kozak
Site-specific N-glycosylation analysis of human IgE
Rosina Plomp (PhD student)
20-04-2343
Site-specific N-Glycosylation Analysis of Human IgE
04/20/2344
Digestion with proteolytic enzymes(proteinase K, chymotrypsin and trypsin)
Digestion with proteolytic enzymes(proteinase K, chymotrypsin and trypsin)
glycan release with PNGaseF glycan release with PNGaseF
Polyclonal IgE (derived from human serum)
LC-MS/MS LC-MS/MS
LC-MS/MS LC-MS/MS
Lab work
Interpretation of LC-MS spectra
glycan structure
mass of peptide moiety Sequence
of peptide moiety
TINIT561.3 Da
Identification of glycoprotein
Site-specific N-Glycosylation Analysis of Human IgE
04/20/2345Stephanie Holst
97
1.3
63+ 9
71
.683
+
97
2.0
13+
15
99
.67+
19
64
.79+
21
10
.79+
14
53
.60+
18
18
.71+
36
6.1
2+
65
7.2
2+
76
4.4
0+
91
0.4
5+
80
3.2
9+
81
9.2
8+ 10
55
.872
+
92
2.6
0++
10
47
.402
+
12
38
.002
+
12
91
.59+
11
28
.582
+
13
46
.46+
13
83
.582
+
14
37
.63+
pep
pep
pep
peppep peppep
pep
peppep
pep
13
10
.972
+
pep
pep
A
m/z972971 973
144TINIT148
Calculation of peptide mass
PNGase F digest on same sample → confirmation of the peptide moiety mass
LC-MS/MS of peptide → sequence of the peptide
LC-ESI-MS/MS spectrum of IgE glycopeptide at site Asn146
Site-specific N-Glycosylation Analysis of Human IgE
04/20/2346
Fragmentation of the peptide TINIT (deamidated)
201.02
330.13
443.20
544.25
D + 0.1
I/L
D
I/L - 0.1T + 0.44
T
+MS2(562.6), 9.7-10.4min #(756-815)
0.00
0.25
0.50
0.75
1.00
1.25
7x10Intens.
100 200 300 400 500 600 700 m/z
Site-specific N-Glycosylation Analysis of Human IgE
04/20/2347
Of the 7 potential N-glycosylation sites on IgE:
•Asn21, 49, 99, 146 and 252 contain complex-type glycan species
•Asn275 contains exclusively oligo-mannosidic glycans
•Asn264 is unoccupied
Site-specific N-Glycosylation Analysis of Human IgE
• Similar N-glycosylation of IgE between single hyperimmune donor and healthy donors
• IgE from myeloma patients differs
• Myeloma IgE features:
↑ tri- and tetra-antennary glycan species
↓ glycans with a bisecting GlcNAc
04/20/2348
Glycosylation analysis of CRC cell lines
Stephanie Holst (PhD student)
20-04-2349
Glycosylation analysis of CRC cell lines
20-04-2350
→ do they provide a similar glycosylation as observed in
tissues?
F. Comes, et al. (2006). Cell Death & Differentiation. doi:10.1038/sj.cdd.4402076
HT29
Glycosylation analysis of CRC cell lines
20-04-2351
↔ in solution
Glycosylation analysis of CRC cell lines
20-04-2352
0.5x106 cells
1x106 cells
2x106 cells
PaTu-S PVDF protocol
17
43
.77
0
15
81
.70
2
19
05
.83
2
12
57
.56
4
20
12
.91
9
10
79
.49
8
21
00
.98
8
14
85
.69
5
21
58
.98
8
23
05
.05
2
18
09
.82
6
16
88
.79
0
19
55
.89
3
14
44
.66
6
15
51
.84
4
18
66
.84
9
23
78
.07
3
25
24
.14
0
11
47
.78
0
13
39
.62
0
26
51
.18
9
16
22
.72
6
24
32
.09
2
22
16
.01
9
27
43
.22
6
30
16
.35
0
32
89
.46
5
30
86
.37
5
35
62
.60
4
29
24
.30
6
28
15
.26
6
38
35
.75
4
33
59
.49
1
36
32
.62
9
31
62
.42
5
28
72
.29
5
34
35
.55
9
32
32
.44
6
35
05
.58
5
37
08
.68
7
162.07 162.07 162.06162.06 162.07162.07
146.07 146.07 146.07 146.07 146.06 146.07 146.08 146.09 146.08
203.10 162.07 162.06203.10
146.08
273.11
162.06 162.07203.09
273.12 273.12273.12
203.08
273.15
146.07162.06
203.09
365.16203.10 203.10 162.05
146.06 162.06 365.16
273.13 273.13
273.14
273.13
RP_HT29_ethyl_75lp 0:F16 MS, BaselineSubtracted
0
1
2
3
4
5
6
5x10
Inte
ns. [a
.u.]
1000 1500 2000 2500 3000 3500m/z
Glycosylation analysis of CRC cell lines
20-04-2353
→ high-mannose structures are high abundant→ whole cell lysate: intra-&extra cellular→ complex type glycans also found in tissue
1x106 cells, in-solution
Glycosylation analysis of CRC cell lines
20-04-2354
17
43
.76
0
14
19
.62
5
15
81
.69
4
20
12
.91
9
19
05
.82
6
12
57
.55
5
10
79
.48
8
23
32
.06
7
21
58
.98
7
26
51
.21
3
14
85
.68
9
18
09
.82
7
25
24
.13
9
16
63
.75
0
22
63
.04
2
13
78
.59
0
28
43
.28
8
24
51
.11
8
27
43
.22
7
30
16
.37
7
31
62
.45
2
29
40
.32
3
33
08
.52
3
RP_CaCo2_ethyl_75lp 0:F15 MS Raw
0
1
2
3
4
5
5x10
Inte
ns.
[a.u
.]
14
19
.64
3
17
43
.78
4
15
81
.71
4
12
57
.57
3
10
54
.46
9
19
05
.85
0
28
94
.34
8
21
84
.05
2
22
56
.06
5
21
01
.02
3
19
96
.95
5
24
78
.17
7
32
13
.50
9
36
32
.70
9
38
35
.85
3
RP_CLL218_ethyl_75lp 0:F12 MS Raw
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Inte
ns.
[a.u
.]
14
19
.64
0
17
43
.77
6
15
81
.70
9
24
02
.10
9
19
05
.84
3
12
57
.56
9
11
25
.77
6
21
28
.99
8
22
56
.04
0
28
94
.31
3
13
24
.92
7
30
40
.38
8
19
82
.92
6
10
54
.46
7
27
67
.26
9
36
32
.66
9
26
21
.19
7
34
05
.57
4
32
13
.47
3
31
32
.43
2
39
97
.89
9
RP_Co115_ethyl_75lp 0:F10 MS Raw
0.0
0.5
1.0
1.5
2.0
5x10
Inte
ns.
[a.u
.]
12
57
.51
4
14
19
.58
1
17
43
.71
5
15
81
.64
7
19
05
.77
1
10
79
.45
7
21
28
.92
2
18
09
.77
1
24
94
.06
7
19
82
.85
0
28
59
.21
7
14
85
.64
4
16
63
.70
7
13
78
.54
8
12
16
.48
7
22
62
.99
8
23
48
.00
3
12
98
.54
2
27
13
.14
8
32
24
.37
8
26
21
.09
9
29
40
.24
5
31
32
.32
6
35
89
.55
1
RP_colo320_ethyl_75lp 0:F17 MS Raw
0
1
2
3
4
5x10
Inte
ns.
[a.u
.]
14
19
.63
5
17
43
.77
1
12
57
.56
5
15
81
.70
5
10
54
.46
4
19
05
.83
9
11
22
.14
2
13
25
.23
2
14
85
.70
2
28
94
.30
6
21
01
.98
7
18
09
.83
7
16
88
.79
9
20
12
.93
6
22
56
.03
9
21
84
.03
6
24
67
.13
8
26
70
.23
1
23
56
.06
6
32
13
.48
1
27
97
.27
7
29
94
.35
3RP_HCT81_ethyl_75lp 0:F13 MS Raw
0.0
0.5
1.0
1.5
2.0
5x10
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500 4000 4500m/z
Glycosylation analysis of CRC cell lines
20-04-2355
14
19
.62
8
17
43
.77
0
15
81
.70
2
19
05
.83
2
12
57
.56
4
10
79
.49
8
20
12
.91
9
14
85
.69
5
21
00
.98
8
21
84
.01
8
23
05
.05
2
18
09
.82
6
16
88
.79
0
11
47
.78
0
13
39
.62
0
23
78
.07
3
25
24
.14
0
26
51
.18
9
24
50
.11
5
27
43
.22
6
30
16
.35
0
30
86
.37
5
28
15
.26
6
29
24
.30
6
32
89
.46
5
35
62
.60
4
33
59
.49
1
38
35
.75
4
31
62
.42
5
36
32
.62
9
34
35
.55
9
37
08
.68
7
RP_HT29_ethyl_75lp 0:F16 MS Raw
0.0
0.5
1.0
1.5
2.0
2.5
5x10
Inte
ns.
[a.u
.]
17
43
.77
4
14
19
.63
6
12
57
.56
6
15
81
.70
5
19
82
.92
2
19
05
.84
0
16
63
.76
6
23
48
.07
3
10
95
.49
2
27
13
.23
1
17
79
.81
9
14
60
.66
1
21
01
.01
4
13
78
.59
9
22
63
.08
3
26
21
.18
8
21
86
.01
3
30
78
.40
6
29
86
.35
7
25
51
.16
7
34
43
.58
9
33
51
.53
9
RP_HTC116_ethyl_75lp 0:F14 MS Raw
0
1
2
3
4
5
5x10
Inte
ns.
[a.u
.]
19
05
.76
4
17
43
.70
4
15
81
.64
3
14
19
.57
8
12
57
.51
1
10
54
.41
5
14
85
.63
9
20
67
.82
3
16
88
.73
7
22
62
.98
5
13
78
.55
4
18
09
.76
7
19
55
.83
0
24
94
.05
8
21
83
.95
1
28
59
.20
8
31
32
.32
2
24
02
.01
3
27
67
.16
3
26
40
.11
8
30
05
.27
4
32
78
.39
4
34
05
.44
7
RP_RKO_ethyl_75lp 0:F18 MS Raw
0.0
0.5
1.0
1.5
2.0
2.55x10
Inte
ns.
[a.u
.]
17
43
.69
6
14
19
.57
0
19
05
.75
5
23
01
.96
9
12
57
.50
6
10
79
.44
8
15
81
.63
3
28
94
.17
8
18
09
.75
5
21
74
.90
0
16
47
.69
6
14
85
.63
1
13
24
.85
2
19
82
.83
0
25
40
.04
2
20
67
.81
5
24
48
.01
1
26
86
.10
3
32
13
.32
5
23
77
.98
4
28
13
.14
5
30
86
.26
6
29
86
.25
0
RP_SW48_ethyl_75lp 0:F21 MS Raw
0.0
0.5
1.0
1.5
5x10
Inte
ns.
[a.u
.]
17
43
.68
9
14
19
.56
4
19
05
.74
9
15
81
.62
8
12
57
.50
0
10
79
.44
4
21
00
.90
4
14
85
.62
7
20
12
.83
8
22
62
.96
4
21
83
.93
6
18
09
.75
6
16
83
.60
4
23
31
.98
5
24
78
.04
2
26
70
.11
2
28
94
.18
3
30
35
.26
6RP_SW480_ethyl_75lp 0:F19 MS Raw
0.0
0.5
1.0
1.5
2.0
2.55x10
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500 4000 4500m/z
Glycosylation analysis of CRC cell lines
20-04-2356
14
19
.64
3
17
43
.78
4
15
81
.71
4
12
57
.57
3
10
54
.46
9
19
05
.85
0
28
94
.34
8
21
84
.05
2
22
56
.06
5
21
01
.02
3
19
96
.95
5
24
78
.17
7
32
13
.50
9
36
32
.70
9
38
35
.85
3
RP_T84_ethyl_75lp 0:F12 MS Raw
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Inte
ns.
[a.u
.]
14
19
.43
6
17
43
.51
6
19
05
.55
1
15
81
.47
7
21
28
.65
4
12
57
.39
6
24
93
.74
0
19
82
.60
6
16
83
.41
7
24
01
.70
1
18
09
.56
2
27
66
.79
6
22
55
.66
8
28
93
.81
7
RP_C10_1_ethyl_APACI_75lp 0:D7 MS Raw
0
1
2
3
4
5
4x10
Inte
ns.
[a.u
.]
17
43
.47
0
19
05
.50
0
14
19
.41
3
15
81
.43
9
12
57
.38
1
29
39
.91
7
16
63
.47
9
22
09
.59
3
20
28
.56
3
23
01
.64
5
23
93
.67
2
28
47
.83
1
25
74
.72
5
32
13
.05
1
26
66
.78
1
34
86
.20
8
RP_DLD-1_2_ethyl_APACI_75lp 0:C16 MS Raw
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Inte
ns.
[a.u
.]
17
43
.53
6
19
05
.57
3
15
81
.49
6
14
19
.46
7
12
57
.43
4
RP_HCT81_3.2_ethyl_APACI_75lp 0:C3 MS Raw
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns.
[a.u
.]
19
05
.52
1
17
43
.48
9
14
19
.42
8
15
81
.45
9
10
63
.99
8
12
57
.39
2
13
37
.37
9
11
75
.35
2
14
99
.41
3
28
93
.89
8
25
39
.75
9
RP_LOVO_3_ethyl_APACI_75lp 0:C12 MS Raw
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500 4000 4500m/z
Glycosylation analysis of CRC cell lines
20-04-2357
19
05
.52
1
17
43
.48
9
14
19
.42
8
15
81
.45
9
10
63
.99
8
12
57
.39
2
13
37
.37
9
11
75
.35
2
14
99
.41
3
28
93
.89
8
25
39
.75
9
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns.
[a.u
.]
19
05
.58
3
17
43
.54
5
14
19
.47
2
15
81
.50
9
12
57
.42
8
22
74
.74
9
21
28
.70
6
RP_LS180_1_ethyl_APACI_75lp 0:D1 MS Raw
0
1
2
3
4x10
Inte
ns.
[a.u
.]
17
43
.47
3
14
19
.40
2
19
05
.50
8
15
81
.43
6
12
57
.36
5
10
79
.34
4
21
28
.60
3
16
88
.50
5
22
74
.63
5
28
93
.73
9
24
93
.68
1
RP_LS411N_1_ethyl_APACI_75lp 0:D13 MS Raw
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns.
[a.u
.]
17
43
.42
1
15
81
.39
6
19
05
.44
7
14
19
.37
3
11
75
.30
7
13
37
.32
9
18
23
.40
3
16
61
.37
5
14
99
.35
4
19
85
.43
0
21
47
.45
9
12
57
.34
4
24
01
.61
4
23
09
.50
6
24
71
.54
8
26
33
.61
0
RP_SW1463_1_ethyl_APACI_75lp 0:C20 MS Raw
0.0
0.5
1.0
1.5
4x10
Inte
ns.
[a.u
.]
17
43
.41
1
19
05
.44
0
14
19
.34
9
15
81
.38
0
10
63
.93
9
13
40
.92
3
11
37
.88
3
17
02
.39
1
12
57
.31
9
20
12
.50
2
28
93
.64
4
RP_SW620_3_ethyl_APACI_75lp 0:D23 MS Raw
0.0
0.5
1.0
1.5
2.0
2.54x10
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500 4000 4500m/z
Glycosylation analysis of CRC cell lines
Future plans:
•Cell line glycans as source for a new generation microarray (screening for auto-antibodies)
•Down-scaling (2x106 cells → 1000 cells?)
•Sequential analysis of GSL-, N- and O-glycans
•Isolation of plasma membrane → specific analysis of cell surface glycans
•Cell culture experiments: role of glycans and how can we influence the glycosylation?
20-04-2358
Acknowledgements
04/20/2359
• IgG, IgA, IgE Glycosylation
• Glycan microarrays
• HTP Glycosylation profiling
• Cancer glycomics
• Inflammatory bowel disease
• Glycosylation storage diseases
• Bioinformatics, Databases
• O-Glycan analysis Center for Proteomics and Metabolomics – Glycomics group
Thank you!
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