1. Analysis of human haptoglobin, after digest with trypsin and Glu-C – six putative N-linked 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 (mzXML w/ msconvert) Spectra filtered for glycan oxonium ions and peptide + N- linked core fragments, then mass-lookup in N-linked human GlycomeDB 1 (1,289 of 34,457 glycans at glycome-db.org ) 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 using Tandem Mass Spectra and Glycan Databases Protein glycosylation is important! N-linked glycans are heterogeneous – glycan synthesis is affected by spatial, temporal, physiological, enzymatic effects Mass spectrometry can be used to study N-glycopeptides. Tools are poorly designed for large MS/MS datasets Glycopeptide identification from CID MS/MS spectra is challenging due to low glycopeptide abundance and dominant oxonium ion fragmention. Background 116 distinct glycan-peptide pairs matched to 534 spectra in less than 5 minutes (< 0.2 Da). 92% of matched spectra matched a single glycan-composition & peptide; 56% a single glycopeptide (up to glycan topology). Nine spectra matched glycan-peptide pairs representing more than one peptide; 42 spectra matched non-isobaric glycans. 60 distinct non-isobaric glycans are represented. Results Detection: UV 214 nm. HILIC fractions 16 to 26 analyzed. MS/MS Spectra w/ glycan oxonium ion (204, 366) peaks w/ “peptide” peaks GlycomeDB 1 Distinct Glycopeptides 3288 2303 885 (600/285) 1 534 (299/193/33/9) 2 116 Glycopeptide Discovery Workflow 11 x LC-MS/MS Haptoglobin MVSHHNLTTGATLINE K VVLHPNYSQVD IGLIK NLFLNHSE NATAK Automated lookup of N-glycopeptides matched most of the glycopeptides 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 for Fucose), match glycopeptide ions’ isotope clusters with MS spectra, and integrate with Cartoonist 3. Conclusions 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. 1 Total spectra (1 peptide/2+ peptides) 2 Total spectra (1 pep, 1 gly(topo)/1 pep, 1 gly(mass)/1 pep, 2+ glys/2+ peps) VVLHPNYSQVDIGLIK (2+) [GlcNAc – 2H 2 O ] + [GlcNAc – H 2 O ] + [NeuAc - H 2 O ] + [NeuAc] + [GlcNAc+ Man ] + [GlcNAc] + [GlcNAc+Gal+NeuAc] + Match: - 800.78, 5+