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.
Proteomics
; 1(6): 778-86, 2001 Jun.
Article
en En
| MEDLINE
| ID: mdl-11677785
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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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Electroforesis en Gel Bidimensional
/
Alfa 1-Antitripsina
/
Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
/
Isoformas de Proteínas
Idioma:
En
Revista:
Proteomics
Asunto de la revista:
BIOQUIMICA
Año:
2001
Tipo del documento:
Article
Pais de publicación:
Alemania