Abstract

We are continuing to develop procedures for unambiguously identifying those serine and threonine residues of glycopeptides that have glycosyl chains attached. In this approach, the glycopeptide is digested with pronase, which cleaves the peptide backbone in a random fashion. The digestion mixtures are analyzed by MALDI-MS after digestion for 5, 10, and 15 min. These spectra contain a series of peptide and glycopeptide peaks that identify the glycosylation site. The primary advantages of our pronase digestion/MALDI-TOF procedure for identifying glycosylation sites is its speed (less than 30 min) and sensitivity (requiring only 1-10 picomoles of glycopeptide). Furthermore, this approach is suitable for determining the sites of other post-translational modifications, including phosphorylation, sulfation, disulfide bonds, and acetylation. This project is aimed at filling the current void of analytical procedures capable of determining O-linked glycosylation sites from sub-nanomole quantities of a glycopeptide.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR005351-08
Application #
5225019
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
1996
Total Cost
Indirect Cost

  Publications

Hannides, Angelos K; Aller, Robert C (2016) Priming effect of benthic gastropod mucus on sedimentary organic matter remineralization. Limnol Oceanogr 61:1640-1650
Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul et al. (2016) Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family. Glycobiology 26:360-76
Zhao, Wujun; Zhu, Taotao; Cheng, Rui et al. (2016) Label-Free and Continuous-Flow Ferrohydrodynamic Separation of HeLa Cells and Blood Cells in Biocompatible Ferrofluids. Adv Funct Mater 26:3990-3998
Wu, Liang; Viola, Cristina M; Brzozowski, Andrzej M et al. (2015) Structural characterization of human heparanase reveals insights into substrate recognition. Nat Struct Mol Biol 22:1016-22
Qiu, Hong; Xiao, Wenyuan; Yue, Jingwen et al. (2015) Heparan sulfate modulates Slit3-induced endothelial cell migration. Methods Mol Biol 1229:549-55
Li, Zixuan; Moniz, Heather; Wang, Shuo et al. (2015) High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface. J Biol Chem 290:10729-40
Czuchry, Diana; Desormeaux, Paul; Stuart, Melissa et al. (2015) Identification and Biochemical Characterization of the Novel ?2,3-Sialyltransferase WbwA from Pathogenic Escherichia coli Serotype O104. J Bacteriol 197:3760-8
Liu, Lin; Zha, Jingying; DiGiandomenico, Antonio et al. (2015) Synthetic Enterobacterial Common Antigen (ECA) for the Development of a Universal Immunotherapy for Drug-Resistant Enterobacteriaceae. Angew Chem Int Ed Engl 54:10953-7
Zhang, Fuming; Moniz, Heather A; Walcott, Benjamin et al. (2014) Probing the impact of GFP tagging on Robo1-heparin interaction. Glycoconj J 31:299-307
Zarnowski, Robert; Westler, William M; Lacmbouh, Ghislain Ade et al. (2014) Novel entries in a fungal biofilm matrix encyclopedia. MBio 5:e01333-14

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