Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures.
The structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapie-associated prion protein, PrP(Sc). These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results.
D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000032:analysis* / Q000302:isolation & purification; D006868:Hydrolysis; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000032:analysis; D011328:Prions / Q000032:analysis; D011492:Protein Hydrolysates; D014357:Trypsin / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000032:analysis
D006020:Glycopeptides; D011134:Polysaccharides; D011328:Prions; D011492:Protein Hydrolysates; D000509:alpha-Fetoproteins; D014357:Trypsin