Site-specific analysis of glycosylated proteins using mass spectrometry

Among the numerous post-translational modifications that a protein can undergo, glycosylation is by far the most common, having the most profound influence on the structural and functional properties of the protein. Therefore, profiling glycosylation patterns in glycosylated proteins and defining the structures and locations of these glycans is important in understanding the structure-function relationship of glycans in glycosylated proteins. The work presented herein focuses on applying different mass-spectrometric methods to profile glycosylation patterns in glycoprotein hormones and HIV envelope proteins. To determine the structures and locations of the glycans on these proteins, a glycopeptide-based mass mapping approach was employed. Glycoprotein hormones mainly contain acidic glycans that are highly sulfated and/or sialylated. These acidic functional groups affect the biological clearance of these proteins. To characterize the glycan structures on glycoprotein hormones, we used a non-specific enzyme to generate small glycopeptides that are easier to separate and analyze. However, analysis of these glycopeptides can be challenging since it involves simultaneous analysis of two unknowns; the peptide and the glycan portions. To facilitate identification of the peptide portion, we developed a web-based tool (GlycoPep ID) that utilizes a characteristic product ion observed in (-)MS/MS data of these glycopeptides. To identify the glycan portion, since (-) MS/MS analysis gives very minimal glycan structural information; we developed an ion-pairing approach, which provides a wealth of structural information on the glycan portion of these glycopeptides. Finally, an HIV envelope protein, CON-S gp140∆CFI, a potential vaccine candidate for HIV/AIDS, was characterized. This protein is extensively glycosylated with over 50% of its mass constituting of glycans. Although these glycans play a major role in viral defense mechanism against the host immune system, the structures and locations of these glycans are still not yet known. To develop an efficacious vaccine against this virus, a complete characterization of these glycans is required. A full glycosylation site-specific analysis of glycans in this protein was performed. This information provided biological insights into why CON-S gp140∆CFI is a good immunogen, thus a potential candidate for an HIV vaccine