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High-throughput and site-specific N-glycosylation analysis of human alpha-1-acid glycoprotein

ntroduction Glycosylation is one of the most common yet diverse co- and post-translational modification essential for proper protein function and stability. Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, whose biological role is not completely understood. Carbohydrates represent about 45% of AGP’s molecular mass, attached in the form of five N- linked complex glycans. AGP exists in three main genetic variants and multiple genetic polymorphisms have been identified. Its glycosylation has been described before, but it was never analyzed on more than a few different samples or on the bigger cohort with specific experimental design addressing clinical issues. Development of a method for high-throughput glycosylation analysis of AGP would enable better understanding of its role and function in health and disease. Methods “Seromucoid” fraction was prepared from human plasma with perchlorate and phosphotungstic acid precipitation, containing 50-75% AGP. The fraction was reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin. Tryptic AGP glycopeptides were enriched with hydrophilic interaction chromatography solid phase extraction and analyzed with RP-LC-ESI-MS/MS. Various alterations of this method were tested. Preliminary data Acidic precipitation of “seromucoid” fraction proved to be an efficient approach for high- throughput AGP enrichment from human plasma. After tryptic digestion, AGP glycopeptides were successfully separated by RP-LC and detailed glycan information was obtained about each AGP glycosylation site by ESI-MS/MS. Glycopeptides were identified on the basis of their characteristic sugar oxonium ions and MS/MS peptide fragmentation patterns. Each AGP genetic variant and each glycosylation site were differently glycosylated. Multiple genetic polymorphisms of AGP were also observed, as it was previously reported. A Python script was used to automate relative quantification of the MS data. Our method allows detailed analysis of AGP glycosylation in hundreds of plasma samples in one week. It does not require isolation of AGP with antibodies and affinity chromatography, but AGP is analyzed directly from the “seromucoid” fraction of human plasma. Novel aspect To best of our knowledge, we describe for the first time a method for high-throughput detailed N- glycosylation analysis of AGP.

Alpha-1-acid glycoprotein ; AGP ; orosomucoid ; N-glycosylation ; high-throuhput

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