GLYCOPEPTIDE ISOLATION OF BOVINE FETUIN BY ELECTROSTATIC REPULSION-HYDROPHILIC INTERACTION CHROMATOGRAPHY

The pivotal role that glycosylation plays in cell signaling to disease processes in particular, has led to increasing interest in glycation site identification. Current "bottom-up" proteomic methods are complex and time-consuming, requiring a combination of affinity and reverse-phase chromatography prior to mass spectrometry. Moreover, it is difficult to optimize or compare these methods because a set of appropriate glycopeptide standards is not commercially available, nor is it practical to synthesize a set. Here we utilize electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to isolate glycopeptides selectively from a source in vivo: A tryptic digest of bovine fetuin. We have identified eight of the eleven published peptide sequence/glycan compositions, with the addition of five novel combinations . From these, a set of glycopeptide standards has been selected for use in the development of chromatographic methods that will reliably retain and deliver all glycopeptides in a digest.