Analysis by matrix assisted laser desorption/ionisation-time of flight mass spectrometry of the post-translational modifications of alpha(1)-antitrypsin isoforms separated by two-dimensional polyacrylamide gel electrophoresis

The state of protein glycosylation in terms of occupation of potential N-li nked glycosylation sites (macroheterogeneity) and type of glycosylation at that site (microheterogeneity) is important when investigating the conseque nces of aberrant glycosylation in the pathophysiology of disease. Protocols have been developed to permit characterisation of the site-specific glycos ylation of individual isoforms of glycoproteins after separation by two-dim ensional polyacrylamide gel electrophoresis (2D-PAGE) and analysis of the p eptide mixture by peptide mass fingerprinting using matrix-assisted laser d esorption/ionisation-time of flight mass spectrometry (MALDI-TOF). High res olution of the individual isoforms of alpha (1)-antitrypsin was achieved by using narrow range (4.5-5.5) pi strips. The individual isoforms were then subjected to sequential digestion with a recombinant N-glycanase followed b y 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 al l three putative N-linked glycosylation sites. Glycans were enzymatically r eleased from cll-antitrypsin which had been separated in gels formed with a low percentage of bis-acrylamide cross-linker and analysed. Profiles of th e N-linked glycans of the individual isoforms of alpha (1)-antitrypsin were obtained by MALDI-TOF.