Isomer Separation of Positively Labeled -glycans by CE-ESI … · Isomer Separation of Positively...

Isomer Separation of Positively Labeled N-glycans by CE-ESI-MS

G.S.M. Kammeijer

Center for Proteomics and MetabolomicsCE IN THE BIOTECHNOLOGY & PHARMACEUTICAL INDUSTRIES

19TH SYMPOSIUM ON THE PRACTICAL APPLICATIONS FOR THE ANALYSIS OF PROTEINS, NUCLEOTIDES AND SMALL MOLECULES (CE PHARM 2017)

LATE BREAKING SESSION – 18TH OF SEPTEMBER 2017

GLYCOSYLATION

Highly prevalent co- and post-translational protein modification (PTM)

Thought to be the most complex PTM

More than 50% of human proteins are glycosylated

Large influence on protein function

Associated with physiological conditions and the effect of biopharmaceuticals

IgG1 [P01857]

2 10/1/2017G.S.M. Kammeijer - [email protected]

GLYCOPROTEOMICS

Fucose146.0579 Da

N-Acetyl-D-galactosamine

203.0794 Da

Glucose162.0528 Da

Mannose162.0528 Da

N-Acetyl-D-glucosamine

203.0794 Da

N-Acetylneuraminicacid

291.0954 Da

Galactose162.0528 Da

The most abundant monosaccharides in humans are:


• Analysis of complex samples

• Not site-specific• Not protein-specific

• Site-specific• Protein specific

• Fast increase of complexity of the data

ANALYSIS APPROACHES

4

INTACT GLYCOPROTEINS GLYCOPEPTIDES RELEASED GLYCANS

• Different isoforms• Protein specific

• Not site-specific• Requires isolated proteins


GLYCAN CHARACTERIZATION

OUTLINE

5

IMPROVING SENSITIVITY

10/1/2017G.S.M. Kammeijer - [email protected]

IMPROVING SEPARATION?

IN-HOUSE METHODS

GLYCAN ANALYSIS

CE-ESI-MS

Sample PNGase F release

PNGase F releaseSample CE-ESI-MS

Di-methylamidation

Labeling


TPNG PROFILE WITH CE-ESI-MSBARE-FUSED SILICA CAPILLARY

Injection: 5 psi 60sec40 nL

0.0

2.0

4.0

6.0

x105Intens.

30 31 32 33 34 35 36 37 38 39 Time [min]

10/1/20177 Kammeijer, G.S.M. et al. Manuscript in preparation

0.0

2.0

4.0

6.0

x105Intens.

0 5 10 15 20 25 30 35 40 45 Time [min]

BFS capillary – 90 cm, 30µm id, BGE composition: 10 % AA, capillary coolant: 20°C, voltage: 20 kV, injection sample: 5 psi, 60 sec (40 nL)

TPNG PROFILE WITH CE-ESI-MSBARE-FUSED SILICA CAPILLARY

Injection: 5 psi 60sec40 nL

0.0

2.0

4.0

6.0

x105Intens.

30 31 32 33 34 35 36 37 38 39 Time [min]


VS VS

BFS capillary – 90 cm, 30µm id, BGE composition: 10 % AA, capillary coolant: 20°C, voltage: 20 kV, injection sample: 5 psi, 60 sec (40 nL)

TPNG PROFILE WITH CE-ESI-MSBFS VS DYNAMIC COATED NEUTRAL CAPILLARY

Bare-Fused Silica CapillaryInjection: 5 psi 60sec (40 nL)

0.0

0.2

0.4

0.6

0.8

x106

Intens.

30 31 32 33 34 35 36 37 38 39 Time [min]

0.0

0.5

1.0

1.5

2.0

2.5

x107

Intens.

37 38 39 40 41 42 43 44 45 46 Time [min]

Dynamic Coated Neutral CapillaryInjection: 5 psi 60sec (40 nL)


Dynamic neutral coated BFS capillary 90 cm, 30µm id, , BGE: 10 % AA, capillary coolant: 20°C, voltage: 20 kV, injection sample: 5 psi, 60 sec (40 nL)

BFS capillary 90 cm, 30µm id, , BGE: 10 % AA, capillary coolant: 20°C, voltage: 20 kV, injection sample: 5 psi, 60 sec (40 nL)

IN SOURCE DECAY

10/1/201710 G.S.M. Kammeijer - [email protected]

0

1

2

3

4

x105

Intens.

39.0 39.5 40.0 40.5 41.0 41.5 Time [min]

Advantage of using CE for glycan characterizationIn-source decay can be easily detected and corrected

IN-SOURCE DECAY


Advantage of using CE for glycan characterizationIn-source decay can be easily detected and corrected

Derivatization method 1 Derivatization method 2

Courtesy of Sander Wagt

DATA AUTOMATIONLACYTOOLS

Jansen, B.C. et al. J. Proteome Res., 2016, 15 (7), pp 2198-2210


DATA AUTOMATIONALIGNMENT


DATA AUTOMATIONALIGNMENT

Alignment of reference file(Run#1)

Alignment of analysis(Run #2)

Alignment of analysis(Run #3)


DATA AUTOMATION DATA INTEGRATION

Aligned mzXML file will be used for further data integration

Reference file –

Provide composition listPEP

Expected migration time PEP

Migration windowPEP

Min. charge state PEP

# Peak RT Mass Window Time Window Min Charge Max Calibrant

protonLoss1gip1H5N2 3104 50

protonLoss1gip1H3N4F1 3207 45

protonLoss1gip1H4N4 3233 55




protonLoss1gip1H5N4E1 3395 55 2

protonLoss1gip1H5N4F1E1 3426 55 2

protonLoss1gip1H5N4E2 3475 55 2

protonLoss1gip1H5N4Am1E1 3482 70 2







OUTLINE

16




INCREASING SENSITIVITY OF GLYCAN ANALYSIS WITH CE-ESI-MS

Significant increase in sensitivity with nano-LC-MS in combination with acetonitrile enriched nitrogen (DEN)- gas

`

Improvement factor of approximately one order of magnitudeApplication Note # LCMS-93 amaZon speed ETD: Exploring glycopeptides in protein mixtures using Fragment Triggered ETD and CaptiveSpray nanoBooster-2014

Nitrogen gas coming in

Acetonitrile in bottle

Nitrogen gas passes the

headspace of the acetonitrile bottle

Dopant enriched nitrogen gas is

introduced in the ESI source

The solvent vapor acts as a dopant for enrichment. The

charge state of multiple charged ions are modified

optimizing the signal intensity.

17

10/1/201717 Kammeijer, G.S.M. et al. Anal. Chem., 2016, 88 (11), pp 5849-5856


Applicable on CE-ESI-MS?

10/1/201718 Kammeijer, G.S.M. et al. Anal. Chem., 2016, 88 (11), pp 5849-5856

279.157

327.006

391.282

430.241

536.162

922.000

311.973

430.242622.030

0.0

0.2

0.4

0.6

0.8

x104

Intens.

0.0

0.2

0.4

0.6

0.8

x104

200 400 600 800 1000 1200 1400 1600 1800 m/z


19

Background MS signal

A – CE-ESI-MS

C – CE-ESI-MS with DEN-gas D

B

An overall lower background observed

especially in the higher mass region


0

1

2

3

4

5x105

Intens.

0 10 20 30 40 50 60 Time [min]

0

1

2

3

4

5x105

Intens.

0 10 20 30 40 50 60 Time [min]


20

Improvement (total plasma N-glycome)



21




22


3x


GLYCOMICS LOD DETERMINATION

23

PNGase F release

Derivatization

Labeling - Addition of label with cationic charge

CE-ESI-MS analysis

α2,3 N-Acetylneuraminic acid α2,6 N-Acetylneuraminic acidVS

Sample

Purification and enrichment - Cotton HILIC

10/1/201723 Haan, de N., et al., Anal. Chem., 2015, 87 (16), pp 8284–8291, DOI: 10.1021/acs.analchem.5b02426cc

LOD – EXPERIMENTAL SET-UP 2 GLYCAN STANDARDS

Dilution series

Dilution factor

MALDI-TOF-MS Volume 1 µL

CE-ESI-MS Injection volume 44 nL

Concentration Amount used Concentration (addition of LE, 9:1)

Injected amount

1 1000 fmol/µL 1000 fmol 900 fmol/µL 39.6 fmol




200 5 fmol/µL 5 fmol 4.5 fmol/µL 0.198 fmol


2000 0.5 fmol/µL 0.5 fmol 0.45 fmol/µL 0.020 fmol


24 10/1/201724 Kammeijer, G.S.M. et al. Manuscript in preparation


00%

10%

20%

30%

40%

50%

60%

70%

Re

lati

ve p

eak

are

a

1x –

MA

LDI-

TOF-

MS

10

x –

MA

LDI-

TOF-

MS

20

x –

MA

LDI-

TOF-

MS

20

x –

CE-

ESI-

MS

10

x –

CE-

ESI-

MS

1x –

CE-

ESI-

MS

10

00

x –

CE-

ESI-

MS

20

0x –

CE-

ESI-

MS

10

0x –

CE-

ESI-

MS

20

00

x –

CE-

ESI-

MS

1x –

MA

LDI-

TOF-

MS

10

x –

MA

LDI-

TOF-

MS

20

x –

MA

LDI-

TOF-

MS

20

x –

CE-

ESI-

MS

10

x –

CE-

ESI-

MS

1x –

CE-

ESI-

MS

10

00

x –

CE-

ESI-

MS

20

0x –

CE-

ESI-

MS

10

0x –

CE-

ESI-

MS

20

00

x –

CE-

ESI-

MS


Dilution series

Dilution factor



Concentration Amount used Concentration Injected amount

0 6.39 pmol/µL 12.8 pmol 3.83 pmol/µL 169 fmol

2 3.20 pmol/µL 6.39 pmol 1.92 pmol/µL 84.3 fmol







Dilution series

Dilution factor



Concentration Amount used Concentration (addition of LE; 9:1)

Injected amount







2000 0.5 fmol/µL 1 fmol 0.45 fmol/µL 0.020 fmol


CE-ESI-MS with DEN-gas shows a ~100x higher sensitivity compared to MALDI-TOF-MS



OUTLINE

27




OPTIMIZATIONDYNAMIC NEUTRALLY COATED CAPILLARY

• 10 % AA • 20 % AA • 20 % AA 10 % MeOH

BGE COMPOSITION

• 15˚C• 20˚C• 25˚C

TEMPERATURE COOLANT

VOLTAGE

• 20 kV• 25 kV• 30 kV

28

Flow

• 0 psi• 0.3 psi• 0.5 psi

TPNG PROFILE WITH CE-ESI-MS– NEUTRALS

25 30 35 40 45 50 Time [min]0.0

0.5

1.0

1.5

2.0

x107

Intens.Dynamic Coated Neutral Capillary

Injection: 5 psi 60sec (40 nL)

0

1

2

3x106

Intens.

45 50 55 60 65 70 Time [min]

Static Coated Neutral CapillaryInjection: 1 psi 60sec (9 nL)


Static neutrally coated BFS capillary, 90 cm 30µm id, BGE: 10 % AA, capillary coolant: 25°C, voltage: 20 kV, injection sample: 1 psi, 60 sec (9 nL)

Dynamic neutrally coated BFS capillary, 90 cm 30µm id, BGE: 10 % AA, capillary coolant: 25°C, voltage: 20 kV, injection sample: 5 psi, 60 sec (40 nL)

0.0

0.5

1.0

1.5

2.0

x107

Intens.

25 30 35 40 45 50 Time [min]

0

1

2

3x106

Intens.

45 50 55 60 65 70 Time [min]

TPNG PROFILE WITH CE-ESI-MS– NEUTRALS

Dynamic Coated Neutral CapillaryInjection: 5 psi 60sec (40 nL)

Static Coated Neutral CapillaryInjection: 1 psi 60sec (9 nL)


ZERO-FLOW PRINCIPLE

10 min

20 min

30 min

50 min


0

1

2

3x106

Intens.

0

2

4

6

8

012345

0

2

4

6

01

2

34

5

0 20 40 60 80 100 Time [min]

x106

x106

x106

x106

Time [min]

0

2

4

60.0 61.0 62.0 Time [min]

1

2

3

65.0 66.0 67.0 Time [min]

2

4

71.0 72.0 73.0

1

2

3

80.0 81.0 82.0 Time [min]

1

53.0 54.0 55.0 Time [min]

HIG

H M

AN

NO

SE

0 min

10 min

20 min

30 min

50 min

Time [min]

Time [min]

Time [min]

Time [min]

0.20

0.40

55.0 56.0 57.0

0.00

0.50

1.00

63.5 64.5

0.00

0.25

0.50

0.75

67.5 68.5 69.5 Time [min]

0.00

0.50

1.00

74.0 75.0 76.0

0.00

0.25

0.50

0.75

82.5 83.5 84.5

Inte

nsx

105

Inte

nsx

105

Inte

nsx

105

Inte

nsx

105

Inte

nsx

105

GA

LAC

TOSY

LATI

ON

Time [min]

Time [min]

Time [min]

Time [min]

Time [min]

0.25

0.50

0.75

54.5 55.5 56.5

0.00

1.00

2.00

62.0 63.0 64.0

0.00

0.50

1.00

1.50

67.0 68.0 69.0

0.00

0.50

1.00

1.50

73.5 74.5 75.5

0.00

0.50

1.00

82.0 83.0 84.0

0 min

10 min

20 min

30 min

50 min

Inte

nsx

105

Inte

nsx

105

Inte

nsx

105

Inte

nsx

105

Inte

nsx

105

0 min

10 min

20 min

30 min

50 min

Inte

nsx

104

Inte

nsx

104

Inte

nsx

104

Inte

nsx

104

Inte

nsx

104

VSVS

VS

VS

EFFECT OF “ZERO-FLOW” ON THE SEPARATION OF ISOMERIC N-GLYCANS

EFFECT OF “ZERO-FLOW” ON THE SEPARATION OF ISOMERIC N-GLYCANS

SIA

LYLA

TIO

N

Time [min]

Time [min]

Time [min]

Time [min]

Time [min]

0.0

0.5

1.0

1.5

2.0

62.0 63.0

0.0

2.0

4.0

71.5 72.5

0.0

1.0

2.0

3.0

77.5 78.5

0.0

2.0

4.0

84.5 85.5 86.5

0.0

1.0

2.0

3.0

94.5 95.5 96.5

0 min

10 min

20 min

30 min

50 min

Inte

nsx

106

Inte

nsx

106

Inte

nsx

106

Inte

nsx

106

Inte

nsx

106

Time [min]

Time [min]

Time [min]

Time [min]

Time [min]

0 min

10 min

20 min

30 min

50 min

Inte

nsx

106

Inte

nsx

106

Inte

nsx

106

Inte

nsx

106

Inte

nsx

104

0.00

0.20

0.40

61.5 62.5 63.5

0.00

0.50

1.00

71.0 72.0 73.0

0.00

0.25

0.50

0.75

77.0 78.0 79.0

0.00

0.25

0.50

0.75

84.5 85.5 86.5

0.00

0.20

0.40

94.5 95.5 96.5

VS

BIS

ECTI

ON

/GA

LAC

TOSY

LATI

ON

Time [min]

Time [min]

Time [min]

Time [min]

Time [min]

1

2

3

54.5 55.5 56.5

0

1

2

3

4

5

6

67.5 68.5 69.5

0

1

2

3

4

5

6

67.5 68.5 69.5

0

2

4

6

73.5 74.5 75.5

1

2

3

4

82.5 83.5 84.5

0 min

10 min

20 min

30 min

50 min

Inte

nsx

104

Inte

nsx

104

Inte

nsx

104

Inte

nsx

104

Inte

nsx

104

VS

VS

o Glycan analysis is possible after derivatization and labeling

o CE-ESI-MS found to be ~100 times more sensitive than MALDI-TOF-MS

o CE-ESI-MS shows great potential for characterization of N-linked

glycosylation in complex mixtures

o Usage of static coated neutral capillary shows high potential

o Isomeric separation visible for several N-glycan species

GLYCAN ANALYSIS WITH CESI-MS IN POSITIVE IONIZATION MODE

34

Center for Proteomics and Metabolomics June 15, 2016

Characterization of various analytes

o Investigation usage for biopharmaceutical characterization

o Investigate the usage for limited sample amounts

o Further explore the potential of isomeric separation using zero-flow

o Comparison between positive and negative ionization mode

o Investigate different labels

o Identification of the different isomers

PERSPECTIVES

35

ACKNOWLEDGEMENTS

Manfred Wuhrer

Noortje de Haan

Sander Wagt

Pablo Mohaupt

Jan Nouta

Karli Reiding

Gerda Vreeker

Bas Jansen

Ludger Ltd.

Rad Kozak

Daniel Spencer

Richard Gardner

Darryl Fernandes

Center for Proteomics and Metabolomics Department of Chemistry & Chemical Biology

Alexander Ivanov

Isomer Separation of Positively Labeled -glycans by CE-ESI … · Isomer Separation of Positively...

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