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Analysis by matrix assisted laser desorption/ ionisation‐time of flight mass spectrometry of the post‐translational modifications of α 1 ‐antitrypsin isoforms separated by two‐ dimensional polyacrylamide gel electrophoresis
Author(s) -
Mills Philippa B.,
Mills Kevin,
Johnson Andrew W.,
Clayton Peter T.,
Winchester Bryan G.
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200106)1:6<778::aid-prot778>3.0.co;2-h
Subject(s) - glycosylation , chemistry , mass spectrometry , time of flight mass spectrometry , glycoprotein , n linked glycosylation , matrix assisted laser desorption/ionization , sialic acid , glycan , gel electrophoresis , polyacrylamide gel electrophoresis , peptide mass fingerprinting , gene isoform , biochemistry , peptide , chromatography , molecular mass , proteomics , enzyme , desorption , ionization , ion , organic chemistry , adsorption , gene
The state of protein glycosylation in terms of occupation of potential N ‐linked glycosylation sites (macroheterogeneity) and type of glycosylation at that site (microheterogeneity) is important when investigating the consequences of aberrant glycosylation in the pathophysiology of disease. Protocols have been developed to permit characterisation of the site‐specific glycosylation of individual isoforms of glycoproteins after separation by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and analysis of the peptide mixture by peptide mass fingerprinting using matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry (MALDI‐TOF). High resolution of the individual isoforms of α 1 ‐ antitrypsin was achieved by using narrow range (4.5–5.5) p I strips. The individual isoforms were then subjected to sequential digestion with a recombinant N ‐glycanase followed by a protease. Using this strategy it was possible not only to increase the coverage of the amino acid sequence but also to monitor the occupancy of all three putative N ‐linked glycosylation sites. Glycans were enzymatically released from α 1 ‐antitrypsin which had been separated in gels formed with a low percentage of bis ‐acrylamide cross‐linker and analysed. Profiles of the N ‐linked glycans of the individual isoforms of α 1 ‐antitrypsin were obtained by MALDI‐TOF.