Tandem Mass Spectrometry-Based Approaches For the Characterization of Glycans and Glycopeptides Lance Wells, Jae-Min Lim, Dan Sherling, Bryan Woosley, Dawei Lin, Ron Orlando, Enas El-Karim, Chin-Fin Teo, Olga Stuchlik, Michael Tiemeyer& Carl Bergman Complex Carbohydrate Research Center, Department of Biochemistry & Molecular Biology University of Georgia

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

What We Really Need? A method that allows for mapping of sites A method that allows for enrichment of modified peptides A method that can be used to do quantitative mass spectrometry (isotope labeling of PTM)

BEMAD Methodology for Sites of O-Glycosylation

b-elimination/Michael addition with light (d0) and heavy (d6) DTT. Differential isotopic tagging of post-translationally modified ser/thr through b-elimination/Michael addition with light (d0) and heavy (d6) DTT. O CH2 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)

Quantifying Peptides with D0- and D6-DTT

LTQ: BEMAD (Light/Heavy) Quantified by LC-MS (200 amol) Theoretical: 1:1, Experimental: 1:0.88

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 CH2 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)

Comparison of Quantitative ICAT vs BEMAD * * ** *= site of phosphorylation, ** = site of O-GlcNAc Modification Quantification for both ICAT and BEMAD differed by <25% Collaboration with Keith Vosseller in Alma Burlingame’s Laboratory

BEMAD and Neutral Loss Approaches for Other O-Glycans O-Man (not extended) on fungal proteins Complex O-Glycosylation

Neutral Loss and BEMAD Mapping of O-Man Sites on PGC

BEMAD for Mapping Complex O-Glycosylation

ECD Fragmentation Site-Mapping on an LTQ-FT

N-linked glycosylation on Secreted Adipocyte Proteome Site mapping using PNGaseF + 18O Water 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 Cyclophilin C-associated protein MAMA/CyCAP gi|6755144 (K)GLN#LTEDTYKPR (R)ALGYEN#ATQALGR RIKEN cDNA 9330129D05 gene gi|30520293 (K)MELKN#QSRLQEPAAR Cathepsin L gi|929719 (R)AEFAVAN#DTGFVDIPQQEK PPBG/ Cathepsin A gi|12860234 (R)LDPPCTN#TTAPSNYLNNPYVR Relative Quantification Possible by 16O and 18O Water

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

Glycan Release/Permethylation of Glycans Trypsin digestion of a-DG b-elimination- 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.

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

Permethylated Glycans from wildtype Drosophila embryos

Site Mapping and Characterization -How unsatisfying for complex glycoproteins! 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

Alpha-Dystroglycan a test case for mapping sites to structures

Fragmentation of O-glycans -SA -Hex -Hex -Hex-HexNAc + Na

Glycan Structures Observed

MS3 – Pseudo Neutral Loss Overall survey scan 8 most intense peaks were selected Fragmentation was induced upon these peaks If result from fragmentation produced neutral loss, additional fragmentation was induced when the loss of a neutral from table was observed Fragmentation was repeated resulting in MS3 Taking a different approach, our laboratory recently described a new strategy for ion dissociation of phosphopeptides in an ion trap mass spectrometer.25 The method, termed Pseudo MSn, induces constitutive collisional-activation of common neutral loss fragments following activation of the precursor ion, converting the dominant neutral loss product ions into a variety of structurally informative fragments. The Pseudo MSn approach is distinguished from the MS3 method, since product ions produced during the initial activation of isolated precursor ion and all subsequent neutral loss activations are simultaneously stored. Thus, the resulting Pseudo MSn mass spectrum comprises fragments derived from multiple precursor activations (a “composite” spectrum). In this report, we further characterize the method and its utility as applied to phosphopeptide ion dissociation.

Pseudo Neutral Loss Activated Data Dependant MS3 MS Survey Scan MS survey scan MS/MS scan Neutral loss? No Yes Top N peaks? MS/MS/MS scan SEQUEST ID http://www.thermo.com

Use of Pseudo Neutral Loss Method Full Scan at 5.81 minutes

MS/MS of 740.25 Neutral loss of SA

Identification of O-GalNAc Glycosylated Peptides - SA - Hex - HexNAc (R)TPRPVPR(V) SA Hex HexNAC [M+2H]2+ [M+H]+

Assignment of Isobaric Glycans - SA - Gal - GalNAc (R)TPRPVPR(V) SA Gal GalNAC [M+2H]2+ [M+H]+

Full Scan at 52.38 Full Scan at 52.38 minutes

MS/MS of 895.6 -SA

Identification of O-Man Glycosylated Peptides -Hex -HexNAc [M+2H]2+ [M+H]+ (R)LETASPPTR(I) SA Hex HexNAc

Assignment of O-Man Isobaric Glycans -Gal -Man -GalNAc [M+2H]2+ [M+H]+ (R)LETASPPTR(I) SA Gal GalNAc Man

How can we isolate the exact site of glycosylation? For peptide with only one Ser or Thr residue within sequence site of glycosylation is obvious For peptides containing more that one Ser or Thr residue assignment is difficult… Identify glycosylated peptide based upon addition of parent mass and combined neutral losses Observe glycosylation of b & y ions BEMAD-b-elimination and Michael addition of DTT

Identifying the Site O-GalNAc Glycosylation by b & y ions b2+HexNAc b3+HexNAc [M+H]+ [M+2H]2+ SA Hex HexNAC b5 b6 b2 b3 b4 (R)T P R P V P R (V) y4 y6 y5 y3 y2 y1

Identifying the Site O-Man Glycosylation by b & y ions SA Hex HexNAc -Hex (R)LETASPPTR(I) [M+H]+ b5+Hex [M+2H]2+ b6+Hex -HexNAc -Hex

Assignment of Sites of Glycosylation a-DG contains 114 Ser/Thr residues which are potential sites of O-linked glycosylation Observed peptides that contained 72 possible acceptor sites of glycosylation Confidently assigned glycan structures to 22 of the observed glycan receptor sites Out of the sites identified as being glycosylated 10 O-Man & 15 O-GalNAc sites