A Practical Approach for Isolation and Characterization of Glycosylation Sites of Glycoproteins Bearing N- and/or O-Linked Carbohydrate Chains

Abstract

This chapter presents a practical approach for isolation and characterization of glycosylation sites of glycoproteins bearing N- and/or O-linked carbohydrate chains. Two glycoproteins were chosen as model glycoproteins for this study, namely, tissue plasminogen activator (t-PA) and human chorionic gonadotropin (HCG). t-PA bears three different N-linked carbohydrate chains, and hCG has several N-linked and O-linked carbohydrate chains attached. Chemicals were either reagent or HPLC grade, as appropriate. Sequence analysis was performed either on the ABI Model 477A or 473A Sequencer using standard cycles. Mass analysis was carried out on a prototype matrix-assisted laser desorption time-of-flight mass spectrometer. Peptides were separated on an ABI Model 172 HPLC System that consisted of a 140B Solvent Delivery System, a 785A Programmable Absorbance Detector, and a 112A Injector, using a solid-particle, nonporous column.