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Complex Carbohydrate Research Center, Department of Biochemistry & Molecular Biology

GlycoAnalytics. Complex Carbohydrate Research Center, Department of Biochemistry & Molecular Biology University of Georgia. Compositional Analysis Using Derivitized Glycans often done by GLC Separation —Azadi Summer Course. HPAE-PAD Detection of Monosaccharides. GLC/GC Analysis

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Complex Carbohydrate Research Center, Department of Biochemistry & Molecular Biology

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  1. GlycoAnalytics Complex Carbohydrate Research Center, Department of Biochemistry & Molecular Biology University of Georgia

  2. Compositional Analysis Using Derivitized Glycans often done by GLC Separation —Azadi Summer Course

  3. HPAE-PAD Detection of Monosaccharides

  4. GLC/GC Analysis --Also taught in Azadi’s Summer Courses

  5. HPAEC GLC Derivatives No Yes Sensitivity >10pmol >lpmol Reproducibility Fair Good Interface to MS Not easily Yes Column life 2 – 3 years 2 – 3 years Column cost $800 $350 Machine cost $20-35,000 $7-10,000 Maintenance easy easy Miscellaneous eluants need He

  6. NCRR Biomedical Glycomics Tandem Mass Spectrometry-Based Approaches For the Characterization of Glycopeptides Lance Wells, Complex Carbohydrate Research Center, Department of Biochemistry & Molecular Biology, and Chemistry University of Georgia

  7. Site Mapping and Characterization-Glycans, N-linked Sites, O-linked Sites P M Q N G S W E K A F S D Y P R S T P G L C N A I H T C I G R E S S Q L N S V K M Current Efforts: Glycopeptides

  8. All MS do one thing: Measure m/z

  9. 729.5 2 5 M a r 3 1 M # 2 1 8 3 R T : 3 5 . 9 4 A V : 1 N L : 1 . 5 6 E 7 T : + c N S I F u l l m s [ 3 5 0 . 0 0 - 1 7 5 0 . 0 0 ] 7 2 9 . 5 1 0 0 9 5 542.5 5 4 2 . 8 9 0 Peptide mixtures 8 5 HPLC 8 0 7 5 TurboSEQUEST Cross-Correlation Comparison Database 7 0 1082.7 6 5 1 0 8 2 . 7 6 0 Protein 5 5 Nucleotide ESTs 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1486.1 1 5 1 4 8 6 . 1 1 0 Capillary Column Buffer A 5 Buffer B 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 1 7 0 0 LTQ ion trap Mass spectrometer Predicted MS/MS Nsi Peptides sequenced, Proteins Identified 1. Enolase EEALDLIVDAIK 2. P yruvate kinase NPTVEVELTTEK 3. Hexokinase IEDDPFVFLEDTDDIFQK 4. Hypusine APEGELGDSLQTAFDEGK 5. BMH1 QAFDDAIAELDTLSEESYK MS LC-nSI/MS/MS---Protein Identification Automated MS/MS CID MS/MS

  10. The Glycan Problem??: Neutral Loss Fragmentation of GlcNAc-CTD by CID-MS/MS Parent ion 535 ([M+2H+] + GlcNAc) NL 7.62e6 y8 866.3 b ions - 164 251 348 449 536 633 720 848 100 Y--S--P--T--S--P--S--K 95 866 703 616 519 418 331 234 147 - y ions 90 Poor Fragmentation No Site Information 85 O-GlcNAc (204) 80 75 70 Relative Abundance 65 60 55 50 45 40 35 GlcNAc 30 y6 410.3 819.2 25 203.8 616.2 b2 20 b8 433.8 15 250.9 10 848.4 476.8 5 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z

  11. Fragmentation of GlcNAc-CTD by CID-MS/MS/MS Parent ions 535/866 NL 2.88e6 b ions - 164 251 348 449 536 633 720 848 y6 b8 616.2 848.2 Y--S--P--T--S--P--S--K 50 866 703 616 519 418 331 234 147 - y ions Ion-Trap MS Allows MSn 45 Once sugar is off, fragmentation is good. MS/MS/MS works well for Sequencing, not site mapping 40 35 30 Relative Abundance 25 y3 331.0 20 830.2 15 598.2 y5 y4 b5 b7 10 518.0 804.3 y7 536.1 418.1 738.1 b6 720.1 499.9 y8 488.0 5 313.0 703.1 580.2 400.0 633.1 684.4 866.4 0 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z

  12. Site Mapping and Characterization-How Unsatisfying for Complex Glycans! P M Q N G S W E K A F S D Y P R S T P G L C N A I H T C I G R E S S Q L N S V K M Lance Wells CCRC, UGA

  13. Glycan Release/Permethylation of Glycans • Trypsin digestion of protein • Enzymatic release of N-glycans • b-elimination for O-linked- additon of NaOH • resulting in release of glycan structure from hydroxyl of Ser or Thr • Reduction with NaBH4 prevents re-attaching of glycan • Permethylation of glycan- OH→OMe • Addition of MeI • Analyzed permethylated glycans by applying MSn fragmentation as needed to completely determine the structure J. Am. Chem. Soc. (2003) 125(52): 16213-9.

  14. PNGase F Treatment and N-linked Glycosylation Site-mapping N-X-S/T Secreted Proteins from Adipocytes (insulin responsive vs insulin resistant) Sulfated glycoprotein 1 precursor (SGP-1) gi|3914939 (K)TVVTEAGNLLKDN#ATQEEILHYLEK(K)FSELIVNN#ATEELLVK (K)LVLYLEHNLEKN#STKEEILAALEK lipoprotein lipase gi|12832783 (R)TPEDTAEDTCHLIPGLADSVSNCHFN#HSSK vimentin gi|2078001 (R)QVQSLTCEVDALKGTN#ESLER Follistatin-related protein 1 precursor gi|2498391 (K)GSN#YSEILDK Haptoglobin gi|8850219 (K)VVLHPN#HSVVDIGLIK (K)NLFLN#HSETASAK (K)CVVHYEN#STVPEKK Adipsin gi|673431 (K)LSQN#ASLGPHVRPLPLQYEDK Decorin gi|6681143 (R)ISDTN#ITAIPQGLPTSLTEVHLDGNK Hemopexin gi|1881768 (R)SWSTVGN#CTAALR 56 proteins with 83 N-linked sites

  15. N-linked Site Mapping from ConA-enriched glycopeptides from Drosophila heads--272 sites mapped from197 Proteins Collaboration with Vlad Pannin, UTAM Pileup of the 272 N-linked Sites to Determine Consensus beyond N-X-S/T Concerns: PNGaseF/A, Deamidation, C-terminal O-18

  16. Characterizing Plant Protein PGIP • N-linked glycoprotein • 7 Putative Sites of Glycosylation • Collaboration with Carl Bergmann’s and Mike Tiemeyer’s group at the CCRC

  17. LC-MS/MS Analysis of O-18 labeled PGIP

  18. N-linked Site-Mapping on PGIP with PNGaseF Xcorr=4.82 gi|169684(K)IYGSIPVEFTQLNFQFLN@VSYNR@ = +3

  19. PGIP N-linked Site Mapping with PNGase F/A PNGase F (red = coverage) 3 sites identified DLCNPDDKKV LLQIKKAFGD PYVLASWKSD TDCCDWYCVT CDSTTNRINS LTIFAGQVSG QIPALVGDLP YLETLEFHKQ PNLTGPIQPAIAKLKGLKSL RLSWTNLSGS VPDFLSQLKN LTFLDLSFNN LTGAIPSSLS ELPNLGALRL DRNKLTGHIP ISFGQFIGNV PDLYLSHNQLSGNIPTSFAQ MDFTSIDLSR NKLEGDASVI FGLNKTTQIV DLSRNLLEFN LSKVEFPTSL TSLDINHNKI YGSIPVEFTQ LNFQFLNVSYNRLCGQIPVG GKLQSFDEYS YFHNRCLCGA PLPSCK PNGase A (red=coverage) 7 sites identified DLCNPDDKKV LLQIKKAFGD PYVLASWKSD TDCCDWYCVT CDSTTNRINS LTIFAGQVSG QIPALVGDLP YLETLEFHK Q PNLTGPIQPAIAKLKGLKSL RLSWTNLSGS VPDFLSQLKN LTFLDLSFNN LTGAIPSSLS ELPNLGALRL DRNKLTGHIP ISFGQFIGNV PDLYLSHNQLSGNIPTSFAQ MDFTSIDLSR NKLEGDASVI FGLNKTTQIV DLSRNLLEFN LSKVEFPTSL TSLDINHNKI YGSIPVEFTQ LNFQFLNVSYNRLCGQIPVG GKLQSFDEYS YFHNRCLCGA PLPSCK High Stringency Filter

  20. PNGF PNGA LTQ Permethylated 1505.0 1331.7 MALDI

  21. PNGaseF

  22. 1331 PNGaseF M3N2X GlcNAc-OMe -Xyl, -H2O GlcNAc-OMe -GlcNAc- - MeOH

  23. 1505 PNGaseA

  24. 1054.55 M3N2XF PNGaseA GlcNAc-OMe - MeOH -Fuc or -Xyl-H2O -Fuc or -Xyl-H2O -GlcNAc- - MeOH

  25. Michael Addition DTT (d0 or d6) H CH2 C N H C O Differential isotopic tagging of both cysteine and post-translationally modified ser/thr through b-elimination/Michael addition with light (d0) and heavy (d6) DTT. O ICH2 C NH2 S b-Elimination H CH2 C N H C CH2 O C Alkylated Cysteine N H C Light DTT (d0) or Heavy DTT (d6) O OH OH Dehydroalanine (or (GlcNAc or phosphate) HSCd2CdCdCd2SH HSCH2CHCHCH2SH O OH OH b-Elimination H CH2 C N H C O O-GlcNAc or O-phosphate Modified Serine (or threonine) Concerns: Specificity, efficiency, recovery

  26. Quantifying Peptides with D0- and D6-DTT

  27. Other Promising Approaches: ECD/ETD

  28. N-Glycans in Drosophila Embryos • Collaboration with Mike Tiemeyer, CCRC • Evidence for Complex, Hybrid Structures • Changes During Embryogenesis

  29. Glycan Analysis

  30. Permethylated Glycans from wildtype Drosophila embryos

  31. Hypoglycosylation and Congenital Muscular Dystrophy Current Opin. Strct. Bio. (2003) 13:621-30 Glycoconjugate J. (2004) 21:3-7

  32. Alpha-Dystroglycan/Dystophin Complex

  33. Comparison of relative abundance

  34. POMGnT1 appears to be essential for O-Man Extension

  35. Site Mapping and Characterization-Where we want to be! P M Q N G S W E K A F S D Y P R S T P G L C N A I H T C I G R E S S Q L N S V K M (75%) (25%)

  36. Alpha-Dystroglycan

  37. Figure 2. Released O-Man and O-GalNAc linked glycans

  38. Identification of Glycans using TIM-MS

  39. MS Survey Scan MS survey scan MS survey scan MS survey scan MS/MS scan Neutral loss? No Yes Yes Top N peaks? No SEQUEST ID Yes MS/MS/MS scan Pseudo Neutral Loss Activated Data Dependant MS3 for Glycopeptide Mapping If we have purified protein and glycomics data http://www.thermo.com

  40. Figure 3.Fragmentation of O-GalNAc a-DG glycopeptide

  41. Assignment of Glycan Structure by Residue

  42. C9 Z10 (Z10) C7 Z3 C3* Z9 C2 C5^ (C10) Z7* Z5^ C6^ Z8* C8 C4* Z4 Z6^ C10 Simultaneous O-Man and O-GalNAc Analysis by ETD MS/MS +3, 620.6 m/z +1, 1857.9 m/z Ac-IRT*T*T^S^GVPR-NH2 * Man^ GalNAc

  43. Where we are going next….. • NL triggered MSn • “non-NL” triggered MSn • Further exploring ECD/ETD capabilities • Enrichment Chromatography and HILIC • Quantification strategies

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