Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1132, Issue 1-2, Pages 165-173Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2006.07.070
Keywords
proteomics; serial lectin affinity chromatography; O-glycosylation; mass spectrometry
Funding
- NIGMS NIH HHS [GM59996] Funding Source: Medline
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Although lectin selection is gaining increasing acceptance as a tool for targeting glycosylation in glycoproteornics, most of the work has been directed at N-glycosylation. The work reported here focuses on the use of lectins in the study of O-glycosylation. The problem with using lectins for studying O-glycosylation is that they are not sufficiently specific. This paper reports that through the use of serial lectin affinity chromatography (SLAC) it is possible to select predominantly O-glycosylated peptides from tryptic digests of human serum. Jacalin is relatively specific for O-glycosylation but has the problem that it also selects high mannose N-type glycans. This problem was addressed by using a concanavalin A affinity column to first remove high mannose, hybrid-type and biantennary complex-type N-type glycans before application of the Jacalin columns. When used in a serial format, concanavalin A and Jacalin together provide essentially O-glycosylated peptides. The glycoprotein parents of glycopeptides were identified by deglycosylating the selected O-glycopeptides by oxidative elimination. These peptides were then separated by RPC and further analyzed using ESI-MS/MS and MALDI-MS/MS. Using this approach all the O-glycosylated sites in a model protein (fetuin) and over thirty glycoprotein parents from human serum were identified. It is concluded that a serial combination of Con A and Jacalin can be of utility in the study of O-glycosylation in glycoproteornics. (c) 2006 Published by Elsevier B.V.
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