1. Analysis of human haptoglobin, digest with trypsin and Glu-C – six putative N-motif peptides. Glycopeptide separation by hydrophilic interaction liquid chromatography (HILIC) Eleven glycopeptide fractions analyzed by nanoC18 RP LC- MS/MS using a Q-STAR Elite mass-spectrometer. IDA: Four most abundant ions with 20 sec exclusion. 15,780 MS and 3,468 MS/MS spectra (msconvert) Spectra filtered for glycan oxinium ions and peptide + N- linked core fragments, then mass-lookup in GlycomeDB. Methods Kevin B. Chandler 1, Petr Pompach 1, Marshall Bern 2, Radoslav Goldman 1, Nathan J. Edwards 1 1 Georgetown University Medical Center, Washington, DC; 2 Palo Alto Research Center, Palo Alto, CA Automated N-Glycopeptide Lookup from Glycan Databases using Tandem Mass Spectra Detection: UV 214 nm. HILIC fractions 16 to 26. MS/MS Spectra w/ glycan oxonium ion (204, 366) peaks w/ “peptide” peaks GlycomeDB 1 Distinct Glycopetpides 3288 2303 885 534 116 Protein glycosylation is important! N-linked glycans are heterogeneous – glycan synthesis affected by spatial, temporal, physiological, enzymatic context Mass spectrometry is used to study N-glycosylation Tools are poorly designed for large MS/MS datasets Glycopeptide identification from CID MS/MS spectra is challenging due to low glycopeptide abundance and oxonium ion fragments. Background 116 distinct glycan-peptide pairs matched to 534 spectra in less than 5 minutes. Open format (XML) spectra input and Excel output. 56% (299) of filtered spectra matched a single glycan-peptide pair (< 0.2 Da). Nine spectra matched glycan-peptide pairs representing more than one peptide. 60 distinct non-isobaric glycans are represented. Results Glycopeptide Discovery Workflow 11 x LC-MS/MS VVLHPNYSQVDIGLIK (2+) [GlcNAc – 2H 2 O ] + [GlcNAc – H 2 O ] + [NeuAc - H 2 O ] + [NeuAc] + [GlcNAc+ Man ] + [GlcNAc] + [GlcNAc+Gal+NeuAc] + Match: - Haptoglobin MVSHHNLTTGATLINE KVVLHPNYSQVDIGLIK NLFLNHSENATAK Automated lookup of N-glycopeptides matched most of those identified by expert manual curation. Identified haptoglobin glycopeptides are consistent with published reports 2. In future work, we plan to identify more oxonium/characteristic ions (such as Fucose), match glycopeptide ions’ isotope clusters in MS spectra, and integrate with Cartoonist 3. Results 1. Ranzinger, Herget, von der Lieth, Frank. Nucleic Acids Res. 39(Database issue):D373-376 (2011). 2.Fujimura, Shinohara, Tossot, Pang, Kurogochi, Saito, Arai, Sadilek, Murayama, Dell, Nishimura, Hakomori. Int. J. Cancer 122:39–49 (2008). 3.Goldberg, Sutton-Smith, Paulson, Dell. Proteomics 5:865-875 (2005). References Acknowledgements Kevin B Chandler is supported by a Graduate Research Fellowship from the National Science Foundation. Nathan J Edwards is supported, in part, by NIH/NCI/CPTI grant CA126189. Sample GlycomeDB 1 Link 800.78, 5+ Sample Glycopeptide Matches Scanm/zΔmassPeptideGlycanID 13671062.11-0.003 VVLHPNYSQVD17745 15481004.920.007 MVSHHNLTTGATLINE18352 16691261.51-0.008 NLFLNHSE2113 13881060.090.014 NLFLNHSE4706 1407800.7810.089 VVLHPNYSQVDIGLIK4706