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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.

Authors
Type
Published Article
Journal
Proteomics
Publication Date
Volume
1
Issue
6
Pages
778–786
Identifiers
PMID: 11677785
Source
Medline
License
Unknown

Abstract

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 alpha 1-antitrypsin was achieved by using narrow range (4.5-5.5) p/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 alpha 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 alpha 1-antitrypsin were obtained by MALDI-TOF.

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