Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by IEF and capillary HPLC-ESI-MS/MS

• Published in: Proteomics (Weinheim) 2005-10, Vol.5 (15), p.4025-4033
• Main Authors: Plematl, Alexander, Demelbauer, Uwe M., Josic, Djuro, Rizzi, Andreas
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Antithrombin; Antithrombin III - chemistry; Antithrombin III - isolation & purification; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Fundamental and applied biological sciences. Psychology; Glycopeptides - chemistry; Glycoprotein; Glycosylation; Humans; Isoelectric Focusing; Isoforms; Mass spectrometry; Miscellaneous; Monosaccharides - chemistry; Monosaccharides - isolation & purification; Peptide Mapping - methods; Polysaccharides - chemistry; Protein Isoforms - chemistry; Protein Isoforms - isolation & purification; Proteins; Site-specific glycosylation; Spectrometry, Mass, Electrospray Ionization
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200401238

The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP‐HPLC‐ESI‐MS equipped with a quadrupole ion‐trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of α‐AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF‐band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn135 and Asn155, alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main‐isoform of α‐AT but missing one terminal SA. There was no site specificity found for the mono‐sialo structure. The isoform at pI 5.31 is the major isoform of β‐AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than β‐AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance.