Simplification of mass spectral analysis of acidic glycopeptides using GlycoPep ID

Mass spectral analysis is an increasingly common method used to characterize glycoproteins. When more than one glycosylation site is present on a protein, obtaining MS data of glycopeptides is a highly effective way of obtaining glycosylation information because this approach can be used to identify not only what the carbohydrates are but also at which glycosylation site they are attached. Unfortunately, this is not yet a routine analytical approach, in part because data analysis can be quite challenging. We are developing strategies to simplify this analysis. Presented herein is a novel mass spectrometry technique that identifies the peptide moiety of either sulfated, sialylated, or both sialylated and sulfated glycopeptides. This technique correlates product ions in collision-induced dissociation (CID) experiments of suspected glycopeptides to a peptide composition using a newly developed web-based tool, GlycoPep ID. After identifying the peptide portion of glycopeptides with GlycoPep ID, the process of assigning the rest of the glycopeptide composition to the MS data is greatly facilitated because the "unknown" portion of the mass assignment that remains can be directly attributed to the carbohydrate component. Several examples of the utility and reliability of this method are presented herein.