Comparative RP-HPLC for rapid identification of glycopeptides and application in off-line LC-MALDI-MS analysis

Abstract

Despite the increasing attention being paid to the functions of glycoproteins, their structural analysis is still difficult and hinders functional investigations. Structural analysis of post-translationally modified proteins is thought to be achieved using methods frequently utilized in proteomics research; however, the same methods cannot be used for glycosylated proteins. One of the difficulties associated with the physiochemical properties of glycopeptides and peptides is that the detection of the former is considerably more difficult, because of the existence of glycoforms that increase molecular weight and reduces quantities of individual species. Thus, difficulties are often faced in finding glycopeptide(s) by using MS when analyzing peaks (or fractions) obtained after proteolytic digestion and HPLC. One simple yet difficult solution to this problem would be to develop a purification method that provides better resolution. Our intention has been to address this issue by using a combination of conventional methods. We found that a method consisting of a combination of rough fractionation using a reverse-phase cartridge column under acidic conditions and comparative RP-HPLC, where the two chromatograms obtained using phosphate and borate buffers under basic conditions were compared, is effective for MS-based structural analysis. The applicability of the method in glycoprotein analysis was examined using various samples including ribonuclease B (RNase B), IgG1, ovalbumin (OVA), and asialo fetuin (ASF). The results suggest that the method is useful in the analysis of glycoproteins.