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HighGlycan Meeting Split 2013

HighGlycan Meeting Split 2013. LUMC Center for Proteomics and Metabolomics. HTP linkage-specific sialic acid stabilization for glycan analysis by MALDI-TOF-MS. Karli Reiding (PhD student). Linkage-specific sialic acid reactivity. α2 ,6 +28 Da. EDC/HOBt Ethanol. 1h 37°C. α2 ,3 -18 Da.

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HighGlycan Meeting Split 2013

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  1. HighGlycan MeetingSplit 2013 LUMC Center for Proteomics and Metabolomics

  2. HTP linkage-specific sialic acid stabilization for glycan analysis by MALDI-TOF-MS Karli Reiding (PhD student)

  3. Linkage-specific sialic acid reactivity α2,6 +28 Da EDC/HOBt Ethanol 1h 37°C α2,3 -18 Da EDC/HOBt Ethanol 1h 37°C

  4. Reaction comparison Direct plasma modification 1h 60°C methanol 2273.807 2273.823 No TFA 0.2% TFA 1968.714 1968.693 1647.605 2912.034 1647.587 2114.764 1809.661 1485.545 3058.082 2419.876 2114.749 1485.535 1809.641 2912.018 3058.072 2419.859 2638.952 2638.931 3550.226 3550.218 2273.800 DMT-MM 2273.798 DMT-MM 1968.693 1647.586 1485.535 1485.533 1968.693 1647.588 2114.746 2114.746 2419.845 2419.842 1809.638 1809.640 2638.906 2911.969 2638.893 2911.930 3058.017 3550.125 3057.948 3549.947 DCC + HOBt 2273.802 2273.805 DCC + HOBt 1485.533 1647.588 1968.695 1968.693 1647.587 1485.534 1809.640 1809.638 2114.748 2114.748 2419.861 2419.861 2912.065 2638.945 3058.155 3550.529 2638.932 2912.015 DCC + Oxy 2273.804 2273.800 DCC + Oxy 1968.693 1968.692 1647.585 1647.586 2114.749 2114.749 1809.638 1809.637 2419.847 1485.533 1485.533 2419.858 2911.937 3057.959 2638.898 2638.930 2911.996 3058.049 3549.935 3550.149 EDC + HOBt 2273.805 2273.800 EDC + HOBt 1647.586 1968.692 1968.695 1485.534 1647.587 1809.639 2114.750 1809.641 2114.747 2419.842 1485.534 2419.860 2638.873 2911.856 2638.934 3057.842 2912.012 m/z m/z 1000 1500 2000 2500 3000 3500 1000 1500 2000 2500 3000 3500 EDC + Oxy EDC + Oxy

  5. Linkage specificity methanol 100 100 1h 37°C 638.189 Intensity (%) Intensity (%) 670.217 0 0 620 640 660 680 700 720 m/z 670.217 1h 37°C 638.190 620 640 660 680 700 720 m/z

  6. Linkage specificity ethanol 100 100 1h 37°C 638.190 Intensity (%) Intensity (%) 684.232 0 0 620 640 660 680 700 720 m/z 684.232 1h 37°C 638.190 620 640 660 680 700 720 m/z

  7. Method comparison 100 100 Released fibrinogen N-glycans Carboxyethylation MALDI-TOF-MS 2AA-labeled HILIC HPLC Intensity (%) Intensity (%) 1982.708 0 0 2301.835 m/z 1800 2000 2200 2400 2600 14 16 18 20 22 24 26 min

  8. Plasma N-glycome profile 100 Intensity (%) 0 3824.345 100 3870.376 2301.842 3943.396 3970.407 3897.357 Intensity (%) 3989.429 4089.451 4043.408 4308.552 4581.669 4189.527 4235.608 4727.831 1982.710 m/z 1000 1500 2500 3000 3500 4000 4500 2000 3800 4000 4200 4400 4600 4800 m/z 2255.786 2940.050 1647.587 2447.895 2128.762 1809.640 3086.112 1485.533 2650.971 3532.227 1257.423 3259.139 3405.190 3678.283 3824.345 4189.527 4308.552 4581.669 4727.831 3943.396 4089.451 0

  9. Major peak repeatability

  10. Minor peak repeatability

  11. Workflow • Unpurified PNGase-F release mixture • 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 -20⁰C • HILIC recovery from 50% EtOH 50% ACN • Stable sialylated glycans in RP mode • Ethanol recrystallization removes shot to shot variability • Glycans detected up to 4800 Da

  12. 96-well format HILIC SPE Stephanie Holst (PhD Student) Emanuela Lonardi

  13. HILIC-SPE in 96-well format • 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 CAREFUL pipetting with multichannel pipette Total time required: ~ 40 min

  14. HILIC-SPE in 96-well format • 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 vacuum manifold AcroPrep 96 Filter Plates with 0.45 μm GHP membrane, 350 μL well volume (PALL) Centrifugation (@) Total time required: ~ 20min

  15. HILIC-SPE in 96-well format Person 1 • 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 Person 2 Samples: carboxyethylated N-glycans released from pooled control plasma

  16. HILIC-SPE in 96-well format • 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

  17. Automated workflow for the analysis of complex glycopeptide samples Bas Jansen (PhD student)

  18. Sample preparation Reduction, Alkylation & Digestion Plasma Cotton HILIC C18 SPE HPLC-MS(/MS) Deglycosylated peptide data Glycopeptide data

  19. Automated workflow conversion {raw}.mzML LC-MS/MS compounds. mzML {raw}.mzML Compound Spectra Compounding output.mgf output.mgf (deglyc.) Deglyc. peptide identification PeptMoiety peptides.csv human.fasta Uniprot peptides.csv Glycopeptide identification find_ glycopeps Glycopeptides results (pdf) compounds. mzML (glyco.)

  20. MS2(645.17), 1.1min #67 Compounding 366.11* 1.2 1.0 1.0 0.9 0.8 0.8 Intensity (104) 0.7 Scan: 67 Cumulative Intensity 0.6 528.22* Scan: 84 0.6 657.30* 0.5 911.31 893.32 775.28 749.32 0.4 784.26 204.00 690.18 0.4 0.3 1005.38 0.2 1276.51 1421.48 0.2 0.1 0.0 200 400 600 800 1000 1200 1400 m/z 0.0 200 400 600 800 1000 1200 1400 m/z MS2(645.44), 1.4min #84 MS2(645.44), 1.1min Combined spectrum 2.5 366.11* 366.11* 3.5 2.0 3.0 2.5 1.5 Intensity (104) Intensity (104) 2.0 657.19* 657.25* 1.0 1.5 775.25 784.28 784.25 775.21 528.21* 911.25 893.38 749.28 1.0 749.38 274.05* 274.00* 893.34 911.33 204.00 528.20* 0.5 1276.48 1276.51 1055.36 1421.48 0.5 0.0 0.0 400 600 800 1000 1200 1400 200 200 400 600 800 1000 1200 1400 m/z m/z

  21. Identification 8 MS2(995.51), 30.68min 657.23* 1333.52 6 Intensity (105) 4 366.14* 1107.80 1252.03 1516.04 1053.40 1011.76 504.26 1661.11 1171.61 2 882.63 1434.04 886.73 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z 3 MS2(887.26), 27.0min Alpha-2-HS-glycoprotein VCQDCPLLAPLN*DTR y6 (716.39 y7 (787.44) 2 Intensity (108) 1057.50 1 y10 (1110.66) 986.47 y8 (900.49) y2 (276.16) y4 (503.21) y3 (391.19) 969.44 769.43 1039.52 808.45 663.29 0 300 400 500 600 700 800 900 1000 1100 m/z

  22. Results

  23. Total plasma N-glycome genome-wide association study Karli Reiding (PhD Student)

  24. Genetics of glycosylation Plasma Lauc et al., PLoS Genet., 2010 IgG Lauc et al., PLoS Genet., 2013

  25. Measurement LLS 2AA-labeled plasma N-glycan GWAS (N=2342) (N=2299) HPLC MALDI-FTICR-MS 24 traits 87 derived 59 traits Discovery HPLC N loci - P<5 x 10 8 HPLC vs. FT CROATIA-Vis (N=1008) Replication CROATIA-Korcula (N=969) ORCADES (N=889) NSPHS (N=700) ML_1 - 37 MH_1 - 25 MALDI-FTICR-MS

  26. Trait calculation HPLC • Peak area relative to highest peak 24 traits MALDI-FTICR-MS • Traits • Peak intensity relative to set peak (H5N4S1) • Overlap allows for comparison low and high spectra • Glycan composition from mass • Derivedtraits • Summing of traits matching composition features (e.g. fucosylation, sialylation) 59 traits 87 traits

  27. Results • 1000 Genomes: 2322 hits found with p<1.0e-7 • Previous results replicated • Switch to 1000 genomes?

  28. High-throughput analysis of IgG Fab glycosylation Albert Bondt (PhD student)

  29. IgG Fc/Fab glycosylation analysis FT Eluate • contains Fc • and some undigested IgG containsFab2

  30. IgG Fc/Fab glycosylation analysis A Fab B Fc N-acetylglucosamine Galactose Mannose Fucose Sialic acid m/z 1400 1600 1800 2000 2200 Bondt et al., manuscript in preparation 2400 2600

  31. IgG Fc/Fab glycosylation analysis

  32. IgG Fc/Fab glycosylation analysis

  33. High-throughput analysis of IgA N- and O-glycopeptides Albert Bondt (PhD student)

  34. IgA glycosylation analysis Zauner et al., Mol Cell Prot (2013) 12(9): p.856-865c

  35. IgA glycosylation analysis

  36. IgA N-glycosylation assignment LAGKPTHVNVSVVMAEVDGTC(Y) LAG LAG-Y LAG-Y / LAG 4536.912 LAG-Y / LAG 4739.992 LAG-Y LAG LAG-Y LAG-Y LAG 4552.893 * 4756.067 4699.979 4903.059 4611.896 * 4448.904 * 4245.823 * * 4408.877 * Bondt et al., manuscript in preparation * oxidation products 4200 4300 4400 4500 4600 4700 4800 4900 m/z

  37. IgA N-glycosylation certainty LAG-Y peptide mass 2183.0709 Glycan H5N4F1S2 mass 2350.8303 + H+ = 4534.9091 Calculated mass Error -0.88 ppm • C-terminal Y truncated • Klapoetkeet al., 2011 Intens. 8 x10 4536.9098 1.0 4537.9119 4535.9075 0.8 4538.9144 0.6 0.4 4534.9051 4539.9175 0.2 4540.9206 4541.9231 4542.9282 0.0 4534 4536 4538 4540 4542 4544 m/z

  38. IgA O-glycosylation assignment HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR 6677.805 6967.886 6385.694 6764.809 6473.714 6223.658 6514.762 6182.642 6094.598 7130.978 6020.561 6699.786 6311.660 5932.544 6839.871 6990.870 6880.888 6407.682 7422.037 7260.009 6547.778 5891.530 6589.808 6787.809 5729.471 7172.004 7011.855 7055.925 6245.626 7152.967 6860.860 5641.469 6117.595 5803.518 6901.856 6042.544 5954.531 6334.642 7335.050 6609.771 5438.372 5770.494 7713.169 7445.044 5858.530 7282.001 5364.351 5567.417 5599.439 7551.098 5276.319 7466.024 5525.381 7625.177 5479.399 7595.087 7801.021 5000 5500 6000 6500 7000 7500 m/z Bondt et al., manuscript in preparation

  39. IgA O-glycosylation assignment certainty HYT O-glycopeptide mass 4135.8821 Glycan H4N5F0S4 mass 2827.9898 + H+ = 6964.8797 Calculated mass Error +0.46 ppm Intens. 8 x10 6968.8847 1.0 6967.8831 0.8 6969.8872 6966.8790 0.6 6970.8923 0.4 6965.8804 6971.8956 6972.8946 0.2 6964.8829 6973.9015 6974.9132 0.0 6964 6966 6968 6970 6972 6974 6976 m/z

  40. IgA O-glycosylation assignment • According to literature only 5 O-glycosylation sites are occupied • Mattu 1998: HYTNPSQDVTVPCPVPST225PPT228PS230PS232TPPT236PSPSCCHPR • Deshpande 2010:HYTNPSQDVTVPCPVPS224TPPTPS230PS232TPPTPS238PS240CCHPR • Takahashi 2012 JPR: 6 O-glycosylation sites HYTNPSQDVTVPCPVPST225PPT228PS230PS232T233PPT236PSPSCCHPR Intens. 203.0816 x107 7422.0187 6 162.0766 291.0765 4 2 7714.0929 7551.0392 7625.1003 7462.0144 7496.0327 7787.1769 0 7450 7500 7550 7600 7650 7700 7750 7800 m/z

  41. IgA O-glycosylation certainty? Can we be sure?????

  42. IgA O-glycosylation IgA- released with hydrazine GU 2.30 67% GU 1.75 GU 2.98 GU 4.58 21% 21% 2% IgA- washed with 0.1%TFA and released with hydrazine GU 1.75 GU 2.98 23% 51% GU 2.30 GU 4.58 21% 5% IgA- released with 50mM Ethylenediaminetetraacetic acid disodium salt dehydrate /hydrazine mix GU 1.75 GU 2.98 30% 53% GU 2.30 GU 4.58 3% 15% IgA- released with 20mM Ethylenediaminetetraacetic acid, anhydrous /hydrazine mix IgA-OHy Exp 6 IgA-TFA-OHy-B Exp 6 GU 1.75 IgA-MIX-1-A Exp 6 GU 2.98 31% IgA-MIX-4-B Exp 6 55% peeling GU 2.30 GU 4.58 3% 12% Rad Kozak

  43. Site-specific N-glycosylation analysis of human IgE Rosina Plomp (PhD student)

  44. Site-specific N-Glycosylation Analysis of Human IgE Polyclonal IgE (derived from human serum) Digestion with proteolyticenzymes (proteinase K, chymotrypsin and trypsin) Lab work LC-MS/MS glycan release with PNGaseF LC-MS/MS mass of peptide moiety glycan structure Sequence of peptide moiety Interpretation of LC-MS spectra TINIT 561.3 Da Identification of glycoprotein

  45. Site-specific N-Glycosylation Analysis of Human IgE LC-ESI-MS/MS spectrum of IgE glycopeptide at site Asn146 • 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 Stephanie Holst

  46. Site-specific N-Glycosylation Analysis of Human IgE Fragmentation of the peptide TINIT (deamidated)

  47. Site-specific N-Glycosylation Analysis of Human IgE Of the 7 potential N-glycosylation sites on IgE: • Asn21, 49, 99, 146 and 252 contain complex-type glycan species • Asn275 contains exclusively oligo-mannosidicglycans • Asn264 is unoccupied

  48. Site-specific N-Glycosylation Analysis of Human IgE • Similar N-glycosylation of IgEbetween single hyperimmune donor and healthy donors • IgEfrom myeloma patients differs • Myeloma IgE features: ↑ tri- and tetra-antennary glycan species ↓ glycans with a bisecting GlcNAc

  49. Glycosylation analysis of CRC cell lines Stephanie Holst (PhD student)

  50. Glycosylation analysis of CRC cell lines HT29 Screening of CRC cell lines: • HT29 • HCT116 • CaCo2 • Colo320 • RKO • SW480 • Co115 • CLL218 • SW480 • T84 • LOVO • DLD-1 • LS-180 • SW1463 • C10 • LS411N • SW620 • HCT81 F. Comes, et al. (2006). Cell Death & Differentiation. doi:10.1038/sj.cdd.4402076 → do they provide a similar glycosylation as observed in tissues?

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