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 --Also taught in Azadi’s Summer Courses

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

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 NCRR Biomedical Glycomics

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

All MS do one thing: Measure m/z

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

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

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 10 y4 b5 b7 518.0 804.3 418.1 536.1 y7 b6 720.1 738.1 499.9 y8 5 313.0 488.0 580.2 703.1 400.0 633.1 684.4 866.4 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z

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

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.

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

N-linked Site Mapping from ConA-enriched glycopeptides from Drosophila heads--272 sites mapped from 197 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

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

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

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

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

PNGF PNGA LTQ Permethylated 1505.0 1331.7 MALDI

PNGaseF

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

1505 PNGaseA

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

Concerns: Specificity, efficiency, recovery 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 H CH2 b-Elimination C N H C C N H O CH2 DTT (d0 or d6) CH2 Michael Addition O C Alkylated Cysteine N H C Light DTT (d0) or Heavy DTT (d6) O OH OH (GlcNAc or phosphate) Dehydroalanine (or HSCH2CHCHCH2SH HSCd2CdCdCd2SH 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

Quantifying Peptides with D0- and D6-DTT

Other Promising Approaches: ECD/ETD

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

Glycan Analysis

Permethylated Glycans from wildtype Drosophila embryos

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

Alpha-Dystroglycan/Dystophin Complex

Comparison of relative abundance

POMGnT1 appears to be essential for O-Man Extension

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

Alpha-Dystroglycan

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

Identification of Glycans using TIM-MS

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

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

Assignment of Glycan Structure by Residue

Simultaneous O-Man and O-GalNAc Analysis by ETD MS/MS Z3 Z4 Z5^ Z7* Z6^ Z8* Z9 Z10 (Z10) C8 +3, 620.6 m/z +1, 1857.9 m/z Ac-IRT*T*T^S^GVPR-NH2 * Man ^ GalNAc

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

Glycan Analysis

N- and O-glycan structures determined by “Total-ion monitoring” Full MS and TIM spectra obtained Spectra analyzed, glycans identified, and quantitated Identification based on accurate mass and fragmentation Expressed as “% of total” or “Fold change from standard sample” Method described in: J Biol Chem (2007) 282, 9127-9142

Relative quantification between 2 samples of released and permethylated N-glycans via isotope labeling with light/heavy iodomethane

+1 Da *

O-linked Glycan Mixture N-linked Glycan Mixture Amide-15N L-glutamine “GLN-15” Amide-14N L-glutamine “Gln-14” Light Medium Heavy Medium Harvest & Combine Mixed Protein Powder Homogenization and delipidation Peptide Mixture O-linked Glycan Mixture N-linked Glycan Mixture Tryptic digestion C18 reverse phase PNGase F digestion -elimination Permethylated Glycans Permethylation Deionization Characterization by MS/MS Quantification by Full MS Mass Spectrometry Heavy GLN-15 Light Gln-14

A. [M+Na]+: 1099.562 m/z (mono) Relative Abundance m/z 724.37 398.18 847.97 Dalton-MES-O # 401 RT: 9.68 AV: 1 NL: 5.62E3 T: ITMS + c NSI Full ms2 1100.00@cid25.00 [150.00-2000.00] 350 400 450 500 550 600 650 700 750 800 850 900 950 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 724.5 847.4 472.4 398.4

B. [M+Na]+: 1101.556 m/z (mono) Relative Abundance m/z 725.37 399.18 849.97 * Dalton-MES-HND3-O # 402 RT: 9.74 AV: 1 NL: 5.34E3 T: ITMS + c NSI Full ms2 1102.00@cid25.00 [150.00-2000.00] 350 400 450 500 550 600 650 700 750 800 850 900 950 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 725.6 473.3 849.5

G. [M+Na]+: 1256.636 + 1259.627 m/z (mono) Amide-15N-GLN = 0.83 1256.637 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1259.626 Amide-14N-Gln Amide-15N-GLN = 0.83 94% incorporation 1257.639 1260.628 1258.637 1261.631 1262.633

Glycoanalytics -Glycan Characterization and Site Mapping --robust methods -- sensitivity & quantification in progress -Intact glycopeptide analysis --very difficult, only pure samples, in infancy