GLYCOPEPTIDE ANALYSIS OF HIV-1 ENVELOPE PROTEIN USING HPLC/ESI-FTICR MS AND MALDI-TOF MS WITH ASIALOFETUIN ENRICHMENT AND SEPARATION TECHNIQUES

Abstract

Glycosylation on proteins serve many biological roles and their heterogeneity can affect various biological functions. The site occupancy of the glycans as well as their various glycoforms makes glycans unique to analyze due to the fact that the variation in structure is not based on a genomic code. Glycosylation is a post-translational modification that provides diverse glycoforms. Mass spectrometry has become a standard technique for characterizing the site occupancy and glycoforms that a protein exhibits. Methodologies in characterization of glycopeptides often include a proteolytic digest followed by purification of the digested protein with liquid chromatography. Mass spectrometers are sensitive to salts and buffer contents, thus a purification of the sample before analysis is vital for the detection of the sample. The research presented herein studies the glycosylation coverage of two HIV-1 envelope proteins. The intention of this study is to determine the specific sites on the HIV-1 protein that are heavily masked/protected by glycans or are free of glycans, in order for future studies to correlate if exposed regions on the protein are favorable regions for vaccine design. Another study was performed to aid in the glycosylation analysis of the HIV-1 proteins. Asialofetuin was used as a model protein to scrutinize the separation efficiencies of three various capillary columns. Specifically the separation of glycopeptides from a peptide mixture was conducted on the three columns in order to determine which platform would optimize the separation of N-linked HIV glycopeptides from a peptide mixture.