Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins

A robust method has been developed that allows analysis of both N- and O-linked oligosaccharicles released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharicle profiling applicable to glycoproteornic analysis. The method involves the enzymatic release of N-linked oligosaccharicles using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-IVIS. Oligosaccharides from the major plasma glycoproteins with a p/ between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha(2)-HS-glycoprotein, serotransferrin, alpha(1)-antitrypsin, and alpha(1)-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccha rides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with 0-linked oligosaccha rides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccha rides.