1 / 1

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

Automated N-Glycopeptide Lookup from Glycan Databases using Tandem Mass Spectra. MS/MS Spectra. 3288 . Kevin B. Chandler 1 , Petr Pompach 1 , Marshall Bern 2 , Radoslav Goldman 1 , Nathan J. Edwards 1. w/ glycan oxonium ion (204, 366) peaks. 2303.

tariq
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related