Comprehensive High Throughput One‐Pot Analysis of Glycoproteins through Tool for Rapid Analysis of Glycopeptide by Permethylation (TRAP) Method.

Journal Article · Sun Apr 01 00:00:00 EDT 2018 · FASEB Journal

DOI:https://doi.org/10.1096/fasebj.2018.32.1_supplement.673.25· OSTI ID:1797866

Shajahan, Asif ^[1]; Supekar, Nitin T. ^[1]; Heiss, Christian ^[1]; Azadi, Parastoo ^[1]

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1. Complex Carbohydrate Research Center The University of Georgia Athens GA

Glycoproteins plays key roles in various cellular functions such as protein folding, protein targeting, receptor function, cell–cell recognition, protein stability, signaling, and other cellular functions. Currently a comprehensive and universal tool for the automated analysis of glycopeptides through their tandem mass spectrometry and data processing is a tedious process. We recently developed a method, Tool for Rapid Analysis of glycopeptide by Permethylation (TRAP), that simplifies glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. We employed this approach for the detailed analysis of glycosidic linkages, composition, glycan sequencing, glycosylation site mapping and sequencing of peptide moiety of glycopeptides of glycoproteins from both prokaryotes and eukaryotes. Here we are presenting an easy to use, robust, suitable for large as well as small sample sets and also cost‐effective glycoprotein analysis tool through TRAP approach. A permethylation protocol which can be automated and suitable for reliable permethylation of glycopeptides from the protease digest of glycoproteins was developed. Initial studies on glycopeptides from a model glycoprotein bovine RNAse B indicates that improved sensitivity and throughput of permethylation reactions can be achieved in conducting the reaction in microscale. This approach would enable convenient, rapid, and reliable high‐throughput screening of protein glycosylation and can be employed as a cost‐effective kit for glycosylated biomarker discoveries. In order to improvise the comprehensive glycosylation analysis and throughput, we have also developed an LC‐MS/MS platform for the analysis of glycopeptides processed through TRAP. We have conducted the analysis of standard glycoproteins such as bovine RNase B, human transferrin, and bovine fetuin and our preliminary results indicate that TRAP hyphened with LC‐MS/MS enables characterization of isomers of each glycoform at each glycosylation sites of complex glycoprotein mixtures. Support or Funding Information This research was supported by the National Institutes of Health (NIH) funded research, Grant R21GM122633; resource for biomedical glycomics, Grant P41GM103490; instrument grant, Grant S10OD018530; and U.S. Department of Energy grant, Grant DE‐SC0015662, to the Complex Carbohydrate Research Center. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

• Sponsoring Organization: USDOE
• Grant/Contract Number: DE‐SC0015662
• OSTI ID: 1797866
• Journal Information: FASEB Journal, Journal Name: FASEB Journal Vol. 32 Journal Issue: S1; ISSN 0892-6638
• Publisher: FASEB
• Country of Publication: United States
• Language: English