LC/MS analysis of complex multiglycosylated human alpha(1)-acid glycoprotein as a model for developing identification...

The site-specific characterization of the complex glycans in multiglycosylated proteins requires developing methods where the carbohydrates remain covalently bound to the protein. The complexity in the carbohydrate composition of alpha(1)-acid glycoprotein (AAG) makes it an ideal model protein for such development. AAG has five N-asparaginyl-linked glycosylation sites, each varying in its bi-, tri-, and tetraantennary glycan content. We present an on-line liquid chromatography/mass spectrometry (LC/MS) method that uses high-low cone voltage switching for in-source fragmentation to determine the structures of the complex glycans present on each site for the two gene products of AAG. High cone voltage caused carbohydrate fragmentation, leading to the generation of signature carbohydrate ions that we used as markers to identify the glycopeptides. Low cone voltage produced minimal carbohydrate fragmentation and enabled the identification and quantification of the intact oligosaccharide structures on each glycopeptide based on its monoisotopic mass and intensity. Quantitation was accomplished by using the intensities of peaks from deconvoluted and deisotoped mass spectra or from the areas of the extracted ion chromatograms from the tryptic peptide maps. The combined results from the two methods can be used to better characterize and quantitate site heterogeneity in multiglycosylated proteins.