Targeted glycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome.
Although lectin selection is gaining increasing acceptance as a tool for targeting glycosylation in glycoproteomics, 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 glycoproteomics.
Durham
Malaika
M
Department of Chemistry and Cancer Center, Purdue University, West Lafayette, IN 47907, United States. modurham@msn.com <modurham@msn.com>
Regnier
Fred E
FE
D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D006023:Glycoproteins / Q000097:blood*; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D006023:Glycoproteins; D037102:Lectins; D020543:Proteome