Isoform differentiation of intact AGP from human serum by capillary electrophoresis-mass spectrometry.
Human AGP is an acidic glycoprotein mainly produced by liver that presents a high degree of heterogeneity. It can present different amino acid sequences and has five N-glycosylation sites leading to a wide range of different protein isoforms. AGP structure and composition has been widely studied due to its drug-binding behavior and relation with disease. However, so far, the characterization has been performed only on protein fragments, i.e., the peptide or glycan level. Here, the analysis of intact human AGP purified from human serum is performed by capillary electrophoresis-time-of-flight mass spectrometry. In this way, it is possible to characterize more than 150 human AGP isoforms, differing both in the amino acid sequence and in the glycosylation. The detected masses could be attributed unequivocally to an overall composition based on the combination of the analysis of the released glycans and the characterization of the deglycosylated protein. Different AGP samples purified from human serum were characterized and compared. High inter-individual variability among AGP isoforms expression was observed. The presented method enables for the first time clinical studies based on detailed isoform distribution of intact glycoproteins.
Ongay
Sara
S
Institute of Organic Chemistry (C.S.I.C.), Juan de la Cierva 3, 28006, Madrid, Spain.
Neusüss
Christian
C
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D044967:Serum / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D009961:Orosomucoid; D011134:Polysaccharides; D020033:Protein Isoforms