A Fully Automated Workflow for Glycopeptide Analysis Julian Saba1, Rosa Viner1, Paul Shan2, Lei Xin2, Sergei Snovida3, Edward Bodnar4, Kay-Hooi Khoo3 and Hélène Perreault4 1Thermo Fisher Scientific, San Jose, CA, USA; 2Bioinformatics Solutions Inc., Waterloo, ON, Canada; 3Institute of Biological Chemistry, Academia Sinica; Taiwan; 4University of Manitoba, Winnipeg, MB, Canada

Introduction: Why Are We Interested? 50% of all mammalian proteins are glycosylated Glycopeptides play important roles in cell development, differentiation and tumorigenesis Despite its ubiquity and biomedical importance, only a small fraction of O- and N-linked glycosylation sites have been mapped and characterized to date

Why Are Glycopeptides So Difficult To Analyze By Mass Spectrometry? Oligosaccharides can be much larger than the peptide backbone Glycosylation influences chromatograpihc elution properties compared to nonglycosylated peptides Using standard HPLC solid phases may not result in chromatographic resolution (i.e. co-eluters) Conventional CID preferentially cleaves glycosidic bonds or peptide-glycan linkages CAD

Why Are Glycopeptides So Difficult To Analyze By Mass Spectrometry? Modifications are often highly heterogeneous May dilute the peptide signal compared to nonglycosylated peptides There are no clearly defined mass shifts as those for acetylation, oxidation, and phosphorylation Hard to search for peptides in protein databases using MS/MS spectra

Glycopeptide Identification Requires CAD and ETD Serotransferrin T421-433 CAD: glycan composition identification ETD: peptide/glycosylation site identification

Localization of the Glycosylation Site by ETD Challenges: ETD performance decreases for ions with m/z >1000 N-linked glycopeptides are often beyond the range unless the peptide backbone is short asialylated glycan ETD fragmentation efficiency is low It’s sequence and charge dependent

Our Strategy: How to Improve ETD Performance for Gycopeptides Dynamic range Sample enrichment Multiple strategies Optimize HPLC conditions Sample specific LC column Increase precursor charge state- Introduce additional charge groups TMT labeling Metal adducts 7

Glycoproteomic Strategies Taking from what is already out there and combining Pan, S. et al., MCP

Our Glycoproteomics Workflow

Glycopeptide Enrichment Strategies

Glycopeptide Enrichment Strategies Graphite SAX TiO2 ZIC-HILIC Cellulose Amide 80 Poly- Hydroxyethyl A TiO2-Graphite

Comparison of ZIC-HILIC vs TiO2 for Enrichment of Human Serum Sialylated Glycopeptides *PNGaseF/H2O18 Treated

Enrichment Efficiency of TiO2 vs ZIC-HILIC for Sialylated N-Linked Glycopeptides T340-353 from Histidine-Rich Glycoprotein in Human Serum NL: 2.53E4 1. Hex5HexNAc4Neu5Ac2 2. dHexHex5HexNAc4Neu5Ac2 3. dHex2Hex6HexNAc5Neu5Ac2 4. dHexHex6HexNAc5Neu5Ac3 5. Hex5HexNAc4Neu5Ac1 6. dHex2Hex5HexNAc4Neu5Ac1 7. dHex3Hex5HexNAc4Neu5Ac1 100 958.1336 90 R=43614 z=4 1407.5337 80 R=35905 B=47.95 z=2 70 7 B=107.24 60 1244.1884 R=34109 1328.5154 50 z=? R=36682 40 B=86.22 z=2 B=97.07 30 1 1174.4867 2 1039.5395 R=38281 R=42792 z=1 6 20 1496.1092 1607.8599 z=? B=76.67 R=31405 R=30808 10 B=58.13 z=? z=? Relative Abundance B=118.64 B=130.74 957.884 1 R=41325 NL: 3.60E4 100 z=4 B=17.93 90 80 4 70 60 3 7 2 1158.455 50 R=38541 40 z=3 1122.191 1427.8766 994.397 B=20.50 1326.1959 1496.2544 R=38520 R=35548 R=34985 30 R=40047 R=33713 z=4 5 z=3 z=3 20 z=4 B=20.08 6 z=3 B=16.48 B=10.47 1681.6612 B=18.59 B=6.43 R=32244 10 z=2 B=0.89 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z

Identification of TiO2 Enriched N-Linked Glycopeptides T340-353 from Histidine-Rich Glycoprotein in Human Serum HCD ETD

T274 is a novel glycosylation site Identification of TiO2 Enriched O-Linked Glycopeptides T271-284 from Histidine-Rich Glycoprotein in Human Serum ETD ETD T274 is a novel glycosylation site

Enrichment of N- and O-Linked Sialylated Glycopeptides from Histidine-Rich Glycoprotein (P04196) in Human Serum Peptide ZIC-HILIC TiO2 SAX HSHNNNSDLHPHK 4 10 SSTTKPPFKPHGSR 3 VENTTVYYLVLDVQESDCSVLSRK 2 VIDFNCTTSSVSSALANTK # Glycopeptides

Summary of All Enrichment Strategies Sixteen different enrichment strategies on protein/peptide level were evaluated: Lectins: WGA, ConA ( at the protein level) HILIC: ZIC, Cellulose, Amide80, PolyHydroxyethyl A Miscellaneous: TiO2, Graphite, TiO2-Graphite, SAX Total number of glycoproteins identified 101 Total number of glycopeptides identified 706 Total number of sites 307 All strategies provide some complementarities, most orthogonal are SAX and TiO2

Improving ETD Fragmentation with Isobaric Labeling Approach via Tandem Mass Tags (TMT)

Tandem Mass Tags - TMT A family of amine reactive isobaric MS/MS tags based on identical chemical structure * 126 Da * 127 Da` The addition of the basic TMT groups increases the average charge state of the precursor ion improves ionization chromatographic separation improves ETD fragmentation of acidic glycopeptides. type of isobaric mass tag. Several different forms of a molecule have the same mass and can be independently attached to a peptide or protein via a reactive group. Mass Reporter (Tag) and the Mass Normaliser are connected via a Cleavable Linker, which has the tendency to break preferentially during MS/MS fragmentation conditions. By selective placement of isotopes such as 13C, 15N or 18O at certain positions of the Tag and the Mass Normaliser, the mass of the Tag can be varied to produce series of Mass Reporters, typically in 1 Dalton increments, while keeping the mass of the entire label constant. Mass Reporters: m/z 126, 127, 128, 129, 130, 131

Increases the Average Charge State of the Precursor Ions +5 +4 Fetuin T54-85 -TMT +6 +7 +TMT

- TMT( m/z 920.89, 4+) Serotransferrin T421-433 CID ETD

+ TMT( m/z 1036.41, 4+)- Improves ETD Fragmentation Even for the Same Charge State Precursor Serotransferrin T421-433 CID ETD Same peptide but with TMT label, cysteine was alkylated with iodoacetic acid.

Identified TiO2 Enriched Human Serum Glycosites With and Without TMT-Labeling 151 71

Mass Spectrometry Acquisition Strategy Thermo Scientific LTQ Orbitrap Velos with ETD

Countless Possibilities for PTM Analysis +HCD/ETD, NL(product) ETD ‘Pick and Mix’ method setup Instrument Fragmentation Method Acquisition Method LTQ Family CID/PQD MSn NL MS3, MSA LTQ ETD Family + ETD +NL ETD, CID/ETD, DDDT Orbitrap ETD Family + HCD +HCD/ETD, NL(product) ETD

Discovery of Glycopeptides Traditional approaches: In source decay- Pseudo-MS3 of Oxonium ions Peterman, S. et al., JASMS (2006) 17, 168-179 Low mass oxonium ions from HCD(PQD) MS/MS Spectra Snovida, S. et al., Carbohydr Res. (2010) 345, 792-801 Kuster, B. et al., JASMS (2011) 22, 931-942 Glycopeptide’s precursors identified in a post-acquisition fashion !

The Utility of Oxonium Ions – Importance of Mass Accuracy Huddleston, M.J. et al., Analytical Chem. (1993) 65, 877-874. Jebanathirajah, J. et al., JASMS (2003) 14, 777–784

Our Approach - On the Fly Identification of Glycopeptides HCD Accurate Mass Product Dependent ETD (HCD-PD-ETD)

Our Approach- On the Fly Identification of Glycopeptides Streamlines data analysis Improves dynamic range and duty cycle

Hex4HexNAc4Neu5AcNeu5Gc dHexHex4HexNAc4Neu5Gc2 Looking for Needle in a Haystack – Targeting Low-Abundance Glycopeptides 36.71 481.799 Unenriched 12 protein mixture digest 100 Base Peak Chromatogram 47.22 90 425.287 80 55.74 34.05 498.311 317.277 70 47.85 672.879 60 32.03 57.23 369.214 578.836 75.13 736.982 Relative Abundance 50 62.83 26.75 40 761.094 303.261 78.58 811.488 85.47 105.65 30 613.885 306.292 20 1.94 25.27 88.68 111.47 10 19.96 105.06 525.379 444.246 599.978 338.344 124.15 301.687 732.104 408.791 Hex4HexNAc4Neu5AcNeu5Gc dHexHex4HexNAc4Neu5Gc2 bAGP QNGTLSK 40.11 XIC for m/z 204.087 from HCD spectra 204.084 100 Ovomucoid 90 80 dHex2Hex7HexNAc6Neu5Ac2 70 60 CNFCNAVVESNGTLTLSHFGK 34.69 50 204.083 40 30 40.67 204.088 20 48.51 10 204.085 89.36 204.088 10 20 30 40 50 60 70 80 90 100 110 120 Time (min)

Looking for Needle in a Haystack – Targeting Low-Abundance Glycopeptides Number of glycopeptides identified Total number of ETD spectra acquired Percentage of ETD spectra identified 224 160 Higher-Energy Collisional Dissociation Accurate Mass Product Dependent Electron Transfer Dissociation – HCD-PD-ETD

Data analysis

Current Solution

Proteome Discoverer Software + GlycoMaster = Solution for Glycopeptide Identification

=V*VLHPN(2204.57)YSQVDIGLIK*(P00738:HPT_HUMAN) Sequence tag: SQVDIGLIK Charge 4 precursor 1112.7736

Software Evaluation: Training Set Results Sample 10 standard protein digest mixture containing following glycoproteins human serotransferrin, chicken ovalbumin; and chicken ovomucoid as contaminant protein 25 6

Summary A complementary of ZIC-HILIC and TiO2enrichment strategies for glycopeptide analysis was demonstrated Introduction of a novel instrumental control within the LTQ Velos Orbitrap software called HCD-PD-ETD analysis. This approach increases the overall productivity for MS analysis of glycopeptides Streamlines data analysis Improves dynamic range and duty cycle GlycoMaster automates the analysis of the data acquired from the combined fragmentation techniques A fully automated workflow has been presented that minimizes some of the pain points associated with enrichment, MS acquisition and data interpretation

Acknowledgments Thermo Fisher Scientific, San Jose, CA Terry Zhang Scott Peterman Eric Hemenway David Horn Bernard Delange John Syka Jae Schwartz Vlad Zabrouskov Amy Zumwalt Sucharita Dutta Thermo Fisher Scientific, Rockford, IL John Rogers