Figure 1. Flow chart of the sequence analysis of a glycoprotein.

The purified glycoprotein is denatured and alkylated prior to tryptic digestion. The digests are subsequently subjected to guanidination in order to overcome the problem of ion suppression caused by peptides lacking arginines. Following the enrichment of glycopeptides using a cellulose cartridge column, the guanidinated tryptic digests were divided into 2 portions. One portion was subjected to PNGase F treatment followed by reverse-phase cartridge purification to determine the sites of N-glycosylation. The fraction containing multiple potential N-glycosylation sites was further treated with endoproteinase AspN. The other portion was fractionated by reverse-phase HPLC followed by MS and MS/MS analysis to obtain the glycan sequence.