Analysis of human haptoglobin , digest with trypsin and Glu -C – six putative N-motif peptides.

1. Automated N-Glycopeptide Lookup from Glycan Databases using Tandem Mass Spectra MS/MS Spectra 3288 Kevin B. Chandler1, PetrPompach1, 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 GlycomeDB1 534 Results Results References Methods Background Distinct Glycopetpides 11 x LC-MS/MS • 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). 116 • Automated lookup of N-glycopeptidesmatched most of those identified by expert manual curation. • Identified haptoglobinglycopeptides are consistent with published reports2. • 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 Cartoonist3. • 116 distinct glycan-peptide pairsmatched to 534 spectrain less than 5 minutes. • Open format (XML) spectra inputand Excel output. • 56% (299) of filtered spectra matched a single glycan-peptide pair (< 0.2 Da). • Ninespectra matched glycan-peptide pairs representing more than one peptide. • 60 distinct non-isobaric glycans are represented. • 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. • 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. • Detection: UV 214 nm. HILIC fractions 16 to 26. 800.78, 5+ VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK (2+) VVLHPNYSQVDIGLIK Match: [GlcNAc]+ [GlcNAc+ Man ]+ - Sample Glycopeptide Matches [NeuAc - H2O ]+ [GlcNAc – 2H2O ]+ Haptoglobin [NeuAc]+ [GlcNAc – H2O ]+ [GlcNAc+Gal+NeuAc]+ Sample GlycomeDB1 Link 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. NLFLNHSENATAK KVVLHPNYSQVDIGLIK MVSHHNLTTGATLINE