Glycosylation analysis of immunoglobulin G

Glycosylation analysis of immunoglobulin G Maurice Selman Leiden University Medical Center Department of Parasitology Biomolecular Mass Spectrometry U...
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Glycosylation analysis of immunoglobulin G Maurice Selman Leiden University Medical Center Department of Parasitology Biomolecular Mass Spectrometry Unit Leiden – The Netherlands

Structure IgG

Structure of both N-glycans determine Fc conformation

R. Jefferis, Expert Opin. Biol. Ther. (2007) 7(9), 1401-13

Lack of core-fucosylation enhances IgG1 binding to Fcγ-receptors N-acetylglucosamine galactose mannose fucose sialic acid

antibody-dependent cellular cytotoxicity (ADCC)

macrophage

Shields et al, 2002, J. Biol. Chem., Iida et al, 2006, Clin. Cancer Res., Ferrara et al, 2011, PNAS

Research goal



Set up robust and fast analysis methods for IgG (subclass specific) Fc Nglycopeptide profiling – Sample preparation (miniaturized) – Analysis – Data processing



Study various IgG Fc N-glycosylation features such as fucosylation, sialylation, galactosylation and the incidence of bisecting GlcNAc in clinical samples

Tryptic peptides of the 4 human IgG subclasses

G0F E293EQYNSTYR301

IgG1

2634.0 Da

E293EQFNSTFR301

IgG2, IgG3

2602.1 Da

E293EQFNSTYR301

IgG4

2618.1 Da pep

IgG glycosylation profiling: sample work-up

sample work-up in the 96-well-plate format: up to 4 plates a day (384 samples)

purify IgG’s from plasma with Protein A or G affinity chromatography

overnight digestion with trypsin

nanoLC-ESI-MS

Desalt and purify digest (RP or HILIC)

(detailed picture, 110 injections a day)

MALDI-MS less detailed picture, 384 samples a day

Wada et al, 2007, Glycobiology

NanoLC sheath-flow ESI-MS analysis tryptic IgG glycopeptides BPC nanoLC capillary

sheath liquid

EIC IgG1

G0F G1F G2F G2FS

EIC IgG4

EIC IgG2

7

8

9

10

11

Time [min]

Selman et al, 2012, J. of Proteomics

dry gas (nitrogen)

Robustness nanoLC sheath-flow ESI-MS IgG1 100 Average preparation 1 90 Average preparation 2 80 Average preparation 3 70 Average all preparations 60 50 40 30 20 10 0

IgG2 100 90 80 70 60 50 40 30 20 10 0

G0F

G1F

G2F

G0FN

G1FN

pep

pep

pep

pep

pep

G2FN pep

G1FS pep

G2FS pep

G1FNS pep

G2FNS pep

G0 pep

G1 pep

G2 pep

G0N pep

G1N pep

G2N pep

G1S pep

G2S pep

Pregnancy related IgG Fc N-glycosylation changes

IgG2

Relative abundance (%)

IgG1

Relative abundance (%)

Galactosylation

Sialylation

SA/Gal

35

90 80

25 70 60

15

50

Bisecting GlcNAc

23

20

21

18

19

16

17

14

15

12

13

10

11

8

40

5

9

6

76

38

27

15

23

13

19

11

15

9

66 28 56 18 46 36

8

1

2

3

4

5

6

Pregnancy time point

11

1

2

3

4

5

6

Pregnancy time point

7

1

2

3

4

5

6

Pregnancy time point

1

2

3

4

5

6

Pregnancy time point

nanoLC sheath-flow ESI-MS conclusions



Sheath-flow ESI sprayer allows robust zero dead volume nanoLC-MS interfacing



In-spray mixing of sheath-liquid with LC eluent allows the use of TFA containing mobile phases



Fast and repeatable IgG subclass specific Fc N-glycopeptide profiles can be obtained (total analysis time = 13 minutes, 110 injection a day)



NanoLC sheath-flow ESI-MS is highly suitable for the analysis of large clinical and biopharmaceutical sample cohorts

IgG glycosylation profiling: sample work-up sample work-up in the 96-well-plate format: up to 4 plates a day (384 samples)

purify IgG’s from plasma with Protein A or G affinity chromatography

overnight digestion with trypsin

nano-HPLC-ESI-MS

Desalt and purify digest (RP or HILIC)

(detailed picture, 110 injections a day)

MALDI-MS less detailed picture, 384 samples a day

High throughput IgG glycopeptide desalting and purification

RP and HILIC desalting in the 96-well-plate format: up to 4 plates a day (384 samples)

reversed phase purification (C18): sample elution with acidified ACN (peptides and glycopeptides)

HILIC purification (Sepharose): Sample elution with water (glycopeptide specific)

RN-mode MALDI-TOF-MS with DHB pep

pep

pep

pep

RN-mode MALDI-TOF-MS with ClCCA

pep

pep

pep

pep

pep

pep

Pep Peptide moiety IgG1 glycopeptide

0.6

2762.10 pep

0.4 pep

3215.24 3247.24

0.2

*

0.0

pep

3215.24 3247.23

2956.14

2632.01

pep

pep

0.2

*

2396.96

0.4

2924.14

pep

2924.15 2956.16

0.6

2632.04

0.8

2794.09

0.8

2794.10

1.0

2396.96

Relative intensity

1.0

2600.04

2600.05

IgG 2 glycopeptide

0.0 2400

2500

2600

2700

2800

m/z

2900

3000

3100

3200

2400

2500

2600

2700

2800

m/z

2900

3000

3100

3200

40000

*

2762.55

40000

2761.98

* 30000

30000 pep Intensity [a.u.]

pep

20000

20000

10000 10000

0

2761

2762

2763

2764 2765 m/z

2766

2767

2768

2769

0

2761

2762

2763

2764 2765 m/z

2766

2767

2768

2769

Theoretical mass IgG2 G1F: 2762.09 Da Matrix

Measured mass (Da)

Error (ppm)

95% confidence interval (Da)

S/N ratio

Cl-CCA (A)

2761.98*

40*

0.05

175*

11083*

DHB (B)

2762.55*

167*

0.16

85*

6490*

THAP/diammonium citrate

2762.25¥

68 ¥

0.09

40¥

7710 ¥

*Values represent average of 10 replicates ¥Values represent average of 5 replicates

Selman et al, 2012, Proteomics

Resolution

Conclusions MALDI-MS



Entire workflow (IgG capturing, digestion and glycopeptide desalting) is suitable for robotized platforms



High throughput sample preparation and MALDI-MS analysis is possible (384 samples in less than 36 hours)



Cold matrixes allow the registration of intact sialylated species by negative ion mode MALDI-TOF-MS



ClCCA has a high potential for high throughput glycopeptide profiling



Data evaluation (automated peak picking and integration)

Acknowledgments

LUMC, Biomolecular Mass Spectrometry Unit • Rico Derks • Carolien Koeleman • Bart Schoenmaker • Liam Mcdonnell • Magnus Palmblad • Gerhild Zauner • André Deelder • Manfred Wuhrer

Roche • Dietmar Reusch • Niklas Engler • Markus Haberger

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