Methods

1. Automated N-Glycopeptide Lookup using Tandem Mass Spectra and Glycan Databases MS/MS Spectra 3288 Kevin B. Chandler1, Petr Pompach1, Marshall Bern2, Radoslav Goldman1, Nathan J. Edwards1 w/ glycan oxonium ion (204, 366) peaks 2303 1 Georgetown University Medical Center, Washington, DC; 2 Palo Alto Research Center, Palo Alto, CA w/ “peptide” peaks Glycopeptide Discovery Workflow 885 (600/285)1 GlycomeDB1 534 (299/193/33/9)2 Results Conclusions References Background Methods 11 x LC-MS/MS Distinct Glycopeptides 116 • 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 GlycomeDB1 (1,289 of 34,457 glycans at glycome-db.org) • 116 distinct glycan-peptide pairsmatched to 534 spectrain 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. • Automated lookup of N-glycopeptides matched most of the glycopeptides identified by expert manual curation. • Identified haptoglobin glycopeptides are consistent with published reports2. • 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 Cartoonist3. • 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. • Ranzinger, Herget, von der Lieth, Frank. Nucleic Acids Res. 39(Database issue):D373-376 (2011). • Fujimura, Shinohara, Tossot, Pang, Kurogochi, Saito, Arai, Sadilek, Murayama, Dell, Nishimura, Hakomori. Int. J. Cancer 122:39–49 (2008). • Goldberg, Sutton-Smith, Paulson, Dell. Proteomics 5:865-875 (2005). • Detection: UV 214 nm. HILIC fractions 16 to 26 analyzed. 1Total spectra (1 peptide/2+ peptides) 2Total spectra (1 pep, 1 gly(topo)/1 pep, 1 gly(mass)/1 pep, 2+ glys/2+ peps) 800.78, 5+ VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK Match: [GlcNAc]+ [GlcNAc+ Man ]+ - [NeuAc - H2O ]+ [GlcNAc – 2H2O ]+ Haptoglobin [NeuAc]+ [GlcNAc – H2O ]+ [GlcNAc+Gal+NeuAc]+ Acknowledgements Kevin B. Chandler is supported by a Graduate Research Fellowship from the National Science Foundation. NLFLNHSE NATAK K VVLHPNYSQVD IGLIK MVSHHNLTTGATLINE