Comparative glycoproteomics of N-linked complex-type glycoforms containing sialic acid in human serum.
This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein.
Qiu
Ruiqing
R
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Regnier
Fred E
FE
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000097:blood / Q000737:chemistry; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000097:blood* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
D006020:Glycopeptides; D006023:Glycoproteins; D019158:N-Acetylneuraminic Acid