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

Isomer Separation of Positively Labeled N-glycans by CE-ESI-MS G.S.M. Kammeijer Center for Proteomics and Metabolomics CE IN THE BIOTECHNOLOGY & PHARMACEUTICAL INDUSTRIES 19 TH SYMPOSIUM ON THE PRACTICAL APPLICATIONS FOR THE ANALYSIS OF PROTEINS, NUCLEOTIDES AND SMALL MOLECULES (CE PHARM 017) LATE BREAKING SESSION 18 TH OF SEPTEMBER 017

GLYCOSYLATION Highly prevalent co- and posttranslational 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] G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

GLYCOPROTEOMICS The most abundant monosaccharides in humans are: Glucose 16.058 Da Galactose 16.058 Da Mannose 16.058 Da Fucose 146.0579 Da N-Acetyl-Dglucosamine 03.0794 Da N-Acetyl-Dgalactosamine 03.0794 Da N-Acetylneuraminic acid 91.0954 Da 3 G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

ANALYSIS APPROACHES INTACT GLYCOPROTEINS GLYCOPEPTIDES RELEASED GLYCANS Different isoforms Protein specific Site-specific Protein specific Analysis of complex samples Not site-specific Requires isolated proteins Fast increase of complexity of the data Not site-specific Not protein-specific 4 G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

OUTLINE GLYCAN CHARACTERIZATION IMPROVING SENSITIVITY? IMPROVING SEPARATION 5 G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

IN-HOUSE METHODS GLYCAN ANALYSIS CE-ESI-MS Sample PNGase F release Sample PNGase F release Di-methyl amidation Labeling CE-ESI-MS 6 G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

TPNG PROFILE WITH CE-ESI-MS BARE-FUSED SILICA CAPILLARY Intens. x10 5 6.0 4.0.0 0.0 0 5 10 15 0 5 30 35 40 45 Time [min] Intens. x10 5 Injection: 5 psi 60sec 40 nl 6.0 4.0.0 0.0 30 31 3 33 34 35 36 37 38 39 Time [min] BFS capillary 90 cm, 30µm id, BGE composition: 10 % AA, capillary coolant: 0 C, voltage: 0 kv, injection sample: 5 psi, 60 sec (40 nl) 7 Kammeijer, G.S.M. et al. Manuscript in preparation

TPNG PROFILE WITH CE-ESI-MS BARE-FUSED SILICA CAPILLARY VS VS Intens. x10 5 Injection: 5 psi 60sec 40 nl 6.0 4.0.0 0.0 30 31 3 33 34 35 36 37 38 39 Time [min] BFS capillary 90 cm, 30µm id, BGE composition: 10 % AA, capillary coolant: 0 C, voltage: 0 kv, injection sample: 5 psi, 60 sec (40 nl) 8 Kammeijer, G.S.M. et al. Manuscript in preparation

TPNG PROFILE WITH CE-ESI-MS BFS VS DYNAMIC COATED NEUTRAL CAPILLARY Intens. x10 6 0.8 Bare-Fused Silica Capillary Injection: 5 psi 60sec (40 nl) 0.6 0.4 0. 0.0 30 31 3 33 34 35 36 37 38 39 Time [min] BFS capillary 90 cm, 30µm id,, BGE: 10 % AA, capillary coolant: 0 C, voltage: 0 kv, injection sample: 5 psi, 60 sec (40 nl) Intens. x10 7.5.0 1.5 1.0 0.5 Dynamic Coated Neutral Capillary Injection: 5 psi 60sec (40 nl) 0.0 37 38 39 40 41 4 43 44 45 46 Time [min] Dynamic neutral coated BFS capillary 90 cm, 30µm id,, BGE: 10 % AA, capillary coolant: 0 C, voltage: 0 kv, injection sample: 5 psi, 60 sec (40 nl) 9 Kammeijer, G.S.M. et al. Manuscript in preparation

IN SOURCE DECAY Intens. x10 5 Advantage of using CE for glycan characterization In-source decay can be easily detected and corrected 4 3 1 0 39.0 39.5 40.0 40.5 41.0 41.5 Time [min] 10 G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

IN-SOURCE DECAY Advantage of using CE for glycan characterization In-source decay can be easily detected and corrected Derivatization method 1 Derivatization method Courtesy of Sander Wagt 11 Kammeijer, G.S.M. et al. Manuscript in preparation

DATA AUTOMATION LACYTOOLS Jansen, B.C. et al. J. Proteome Res., 016, 15 (7), pp 198-10 1 Kammeijer, G.S.M. et al. Manuscript in preparation

DATA AUTOMATION ALIGNMENT 13 Kammeijer, G.S.M. et al. Manuscript in preparation

DATA AUTOMATION ALIGNMENT Alignment of reference file (Run#1) Alignment of analysis (Run #) Alignment of analysis (Run #3) 14 Kammeijer, G.S.M. et al. Manuscript in preparation

DATA AUTOMATION DATA INTEGRATION PEP PEP PEP PEP Aligned mzxml file will be used for further data integration Reference file Provide composition list Expected migration time Migration window Min. charge state # Peak RT Mass Window Time Window Min Charge Max Calibrant protonloss1gip1h5n 3104 50 protonloss1gip1h3n4f1 307 45 protonloss1gip1h4n4 333 55 protonloss1gip1h4n4f1 374 60 protonloss1gip1h4n5f1 38 50 protonloss1gip1h5n4f1 3338 50 protonloss1gip1h5n4e1 3395 55 protonloss1gip1h5n4f1e1 346 55 protonloss1gip1h5n4e 3475 55 protonloss1gip1h5n4am1e1 348 70 protonloss1gip1h5n4f1e 3498 55 protonloss1gip1h5n5f1e 3504 55 protonloss1gip1h6n5am1e 3617 60 protonloss1gip1h7n6am3e1 374 70 15 Kammeijer, G.S.M. et al. Manuscript in preparation

OUTLINE GLYCAN CHARACTERIZATION IMPROVING SENSITIVITY? IMPROVING SEPARATION 16 G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

INCREASING SENSITIVITY OF GLYCAN ANALYSIS WITH CE-ESI-MS 17 Significant increase in sensitivity with nano-lc-ms in combination with acetonitrile enriched nitrogen (DEN)- gas ` Nitrogen gas coming in Nitrogen gas passes the headspace of the acetonitrile bottle Dopant enriched nitrogen gas is introduced in the ESI source Acetonitrile in bottle The solvent vapor acts as a dopant for enrichment. The charge state of multiple charged ions are modified optimizing the signal intensity. Improvement factor of approximately one order of magnitude Application Note # LCMS-93 amazon speed ETD: Exploring glycopeptides in protein mixtures using Fragment Triggered ETD and CaptiveSpray nanobooster-014 17 Kammeijer, G.S.M. et al. Anal. Chem., 016, 88 (11), pp 5849-5856

INCREASING SENSITIVITY OF GLYCAN ANALYSIS WITH CE-ESI-MS Applicable on CE-ESI-MS? 18 Kammeijer, G.S.M. et al. Anal. Chem., 016, 88 (11), pp 5849-5856

INCREASING SENSITIVITY OF GLYCAN ANALYSIS WITH CE-ESI-MS Background MS signal Intens. x10 4 0.8 0.6 0.4 0. A CE-ESI-MS 536.16 79.157 391.8 37.006 430.41 9.000 Intens. x10 5 5 4 3 1 B 0 0 10 0 30 40 50 60 Time [min] 0.0 x10 4 0.8 0.6 0.4 C CE-ESI-MS with DEN-gas 311.973 An overall lower background observed especially in the higher mass region Intens. x10 5 5 4 3 1 D 0 0 10 0 30 40 50 60 Time [min] 0. 0.0 430.4 6.030 00 400 600 800 1000 100 1400 1600 1800 m/z 19 Kammeijer, G.S.M. et al. Manuscript in preparation

INCREASING SENSITIVITY OF GLYCAN ANALYSIS WITH CE-ESI-MS Improvement (total plasma N-glycome) 0 Kammeijer, G.S.M. et al. Manuscript in preparation

INCREASING SENSITIVITY OF GLYCAN ANALYSIS WITH CE-ESI-MS Improvement (total plasma N-glycome) 1 Kammeijer, G.S.M. et al. Manuscript in preparation

INCREASING SENSITIVITY OF GLYCAN ANALYSIS WITH CE-ESI-MS Improvement (total plasma N-glycome) 3x Kammeijer, G.S.M. et al. Manuscript in preparation

GLYCOMICS LOD DETERMINATION Sample PNGase F release Derivatization α,3 N-Acetylneuraminic acid α,6 N-Acetylneuraminic acid VS Purification and enrichment - Cotton HILIC Labeling - Addition of label with cationic charge CE-ESI-MS analysis 3 Haan, de N., et al., Anal. Chem., 015, 87 (16), pp 884 891, DOI: 10.101/acs.analchem.5b046cc

LOD EXPERIMENTAL SET-UP GLYCAN STANDARDS Dilution factor MALDI-TOF-MS Volume 1 µl Dilution series 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 10 100 fmol/µl 100 fmol 90 fmol/µl 3.96 fmol 0 50 fmol/µl 50 fmol 45 fmol/µl 1.98 fmol 100 10 fmol/µl 10 fmol 9 fmol/µl 0.396 fmol 00 5 fmol/µl 5 fmol 4.5 fmol/µl 0.198 fmol 1000 1 fmol/µl 1 fmol 0.9 fmol/µl 0.040 fmol 000 0.5 fmol/µl 0.5 fmol 0.45 fmol/µl 0.00 fmol 10000 0.1 fmol/µl 0.1 fmol 0.09 fmol/µl 0.004 fmol 4 Kammeijer, G.S.M. et al. Manuscript in preparation

Relative peak area LOD EXPERIMENTAL SET-UP GLYCAN STANDARDS 000x CE-ESI-MS 1000x CE-ESI-MS 00x CE-ESI-MS 100x CE-ESI-MS 0x CE-ESI-MS 10x CE-ESI-MS 1x CE-ESI-MS 1x MALDI-TOF-MS 10x MALDI-TOF-MS 0x MALDI-TOF-MS 000x CE-ESI-MS 1000x CE-ESI-MS 00x CE-ESI-MS 100x CE-ESI-MS 0x CE-ESI-MS 10x CE-ESI-MS 1x CE-ESI-MS 1x MALDI-TOF-MS 10x MALDI-TOF-MS 0x MALDI-TOF-MS 70% 60% 50% 40% 30% 0% 10% 00% 5 Kammeijer, G.S.M. et al. Manuscript in preparation

LOD EXPERIMENTAL SET-UP GLYCAN STANDARDS MALDI-TOF-MS Dilution series Dilution series CE-ESI-MS MALDI-TOF-MS CE-ESI-MS Volume µl µl Injectionvolume volume 44 44 nl nl Dilution Dilution factor Concentration Concentration Amount used used Concentration Concentration Injected Injected amount factor (addition of LE; 9:1) amount 0 6.39 pmol/µl 1.8 pmol 3.83 pmol/µl 169 fmol 1 1000 fmol/µl 000 fmol 900 fmol/µl 39.6 fmol 10 3.0 pmol/µl 100 fmol/µl 6.39 pmol 00 fmol 1.9 pmol/µl 90 fmol/µl 84.3 fmol 3.96 fmol 5 0 1.8 pmol/µl 50 fmol/µl.56 pmol 100 fmol 0.77 pmol/µl 45 fmol/µl 33.7 fmol 1.98 fmol 10 100 0.64 pmol/µl 10 fmol/µl 1.8 pmol 0 fmol 0.38 pmol/µl 9 fmol/µl 16.9 fmol 0.396 fmol 0 0.3 pmol/µl 0.64 pmol 0.19 pmol/µl 8.4 fmol 00 5 fmol/µl 10 fmol 4.5 fmol/µl 0.198 fmol 50 0.13 pmol/µl 0.6 pmol 0.07 pmol/µl 3. fmol 1000 1 fmol/µl fmol 0.9 fmol/µl 0.040 fmol 100 000 0.06 pmol/µl 0.5 fmol/µl 0.13 pmol 1 fmol 0.04 pmol/µl 0.45 fmol/µl 1.6 fmol 0.00 fmol 10000 0.1 fmol/µl 0. fmol 0.09 fmol/µl 0.004 fmol CE-ESI-MS with DEN-gas shows a ~100x higher sensitivity compared to MALDI-TOF-MS 6 Kammeijer, G.S.M. et al. Manuscript in preparation

7 OUTLINE GLYCAN CHARACTERIZATION IMPROVING SENSITIVITY? IMPROVING SEPARATION 7 G.S.M. Kammeijer - g.s.m.kammeijer@lumc.nl

OPTIMIZATION DYNAMIC NEUTRALLY COATED CAPILLARY 8 BGE COMPOSITION 10 % AA 0 % AA 0 % AA 10 % MeOH TEMPERATURE COOLANT 15 C 0 C 5 C VOLTAGE 0 kv 5 kv 30 kv Flow 0 psi 0.3 psi 0.5 psi

TPNG PROFILE WITH CE-ESI-MS NEUTRALS Intens. x10 7.0 Dynamic Coated Neutral Capillary Injection: 5 psi 60sec (40 nl) 1.5 1.0 0.5 0.0 5 30 35 40 45 50 Time [min] Dynamic neutrally coated BFS capillary, 90 cm 30µm id, BGE: 10 % AA, capillary coolant: 5 C, voltage: 0 kv, injection sample: 5 psi, 60 sec (40 nl) Intens. x10 6 3 Static Coated Neutral Capillary Injection: 1 psi 60sec (9 nl) 1 0 45 50 55 60 65 70 Time [min] Static neutrally coated BFS capillary, 90 cm 30µm id, BGE: 10 % AA, capillary coolant: 5 C, voltage: 0 kv, injection sample: 1 psi, 60 sec (9 nl) 9 Kammeijer, G.S.M. et al. Manuscript in preparation

TPNG PROFILE WITH CE-ESI-MS NEUTRALS Intens. x10 7.0 Dynamic Coated Neutral Capillary Injection: 5 psi 60sec (40 nl) 1.5 1.0 0.5 0.0 5 30 35 40 45 50 Time [min] Intens. x10 6 3 Static Coated Neutral Capillary Injection: 1 psi 60sec (9 nl) 1 0 45 50 55 60 65 70 Time [min] 30 Kammeijer, G.S.M. et al. Manuscript in preparation

Intens. x10 6 3 x10 6 x10 6 x10 6 1 0 8 6 4 0 5 4 3 1 0 6 4 ZERO-FLOW PRINCIPLE 10 min 0 min 30 min 0 x10 6 5 4 3 50 min 1 0 0 0 40 60 80 100 Time [min] 31 Kammeijer, G.S.M. et al. Manuscript in preparation

HIGH MANNOSE EFFECT OF ZERO-FLOW ON THE SEPARATION OF ISOMERIC N- GLYCANS GALACTOSYLATION Intensx10 5 0.40 0 min Intensx10 5 0.75 0 min Intensx10 4 0 min 0.0 0.50 1 0.5 Intensx10 5 55.0 56.0 57.0 10 min Time [min] Intensx10 5 54.5 55.5 56.5 10 min Time [min] Intensx10 4 53.0 54.0 55.0 Time [min] 10 min 1.00 VS.00 4 0.50 1.00 0.00 63.5 64.5 Time [min] 0.00 6.0 63.0 64.0 Time [min] 0 60.0 61.0 6.0 Time [min] Intensx10 5 0.75 0.50 0 min Intensx10 5 1.50 1.00 0 min VS Intensx10 4 3 0 min VS 0.5 0.50 1 0.00 67.5 68.5 69.5 Time [min] 0.00 67.0 68.0 69.0 Time [min] 65.0 66.0 67.0 Time [min] Intensx10 5 1.00 0.50 30 min VS Intensx10 5 1.50 1.00 0.50 30 min Intensx10 4 4 30 min 0.00 Intensx10 5 74.0 75.0 76.0 50 min Time [min] 0.00 Intensx10 5 73.5 74.5 75.5 50 min Time [min] Intensx10 4 71.0 7.0 73.0 Time [min] 50 min 0.75 0.50 0.5 1.00 0.50 3 1 0.00 8.5 83.5 84.5 Time [min] 0.00 8.0 83.0 84.0 Time [min] 80.0 81.0 8.0 Time [min]

EFFECT OF ZERO-FLOW ON THE SEPARATION OF ISOMERIC N- GLYCANS BISECTION/GALACTOSYLATION SIALYLATION Intensx10 4 Intensx10 4 Intensx10 4 Intensx10 4 Intensx10 4 3 1 6 5 4 3 1 0 6 5 4 3 1 0 6 4 0 4 3 1 0 min 54.5 55.5 56.5 10 min 67.5 68.5 69.5 0 min 67.5 68.5 69.5 30 min 73.5 74.5 75.5 50 min Time [min] Time [min] Time [min] Time [min] 8.5 83.5 84.5 Time [min] VS VS Intensx10 6.0 1.5 1.0 0.5 0.0 Intensx10 6 4.0.0 0.0 Intensx10 6 3.0.0 1.0 0.0 Intensx10 6 4.0.0 0.0 Intensx10 6 3.0.0 1.0 0.0 0 min 6.0 63.0 Time [min] 10 min 71.5 7.5 Time [min] 0 min 77.5 78.5 Time [min] 30 min 84.5 85.5 86.5 Time [min] 50 min 94.5 95.5 96.5 Time [min] Intensx10 6 0.40 0.0 0.00 Intensx10 6 1.00 0.50 0.00 Intensx10 6 0.75 0.50 0.5 0.00 Intensx10 6 0.75 0.50 0.5 0.00 Intensx10 4 0.40 0.0 0.00 0 min 61.5 6.5 63.5 Time [min] 10 min 71.0 7.0 73.0 Time [min] 0 min 77.0 78.0 79.0 Time [min] 30 min 84.5 85.5 86.5 Time [min] 50 min 94.5 95.5 96.5 Time [min] VS

34 GLYCAN ANALYSIS WITH CESI-MS IN POSITIVE IONIZATION MODE 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 Center for Proteomics and Metabolomics June 15, 016

35 PERSPECTIVES 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

ACKNOWLEDGEMENTS Center for Proteomics and Metabolomics 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 Department of Chemistry & Chemical Biology Alexander Ivanov