HighGlycan Meeting Split 2013

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


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)


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


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

.]


0

1000

2000

3000

Inte

ns.

[a.u

.]


0

1000

2000

3000

Inte

ns.

[a.u

.]

23

01

.87

3


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


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


20-04-2331


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


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


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


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


• 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


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


20-04-2351

↔ in solution


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


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


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


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


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


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


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!

HighGlycan Meeting Split 2013

Documents

major peak repeatability

minor peak repeatability

mzcarboxyethylat malditof

reaction comparison

method comparison

glycan analysis

mskarli reiding phd

tfadirect plasma modification