Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation.
Proteomics
; 16(13): 1858-62, 2016 07.
Article
em En
| MEDLINE
| ID: mdl-27130503
ABSTRACT
Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large-scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large-scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two-step IMAC enrichment protocol under these conditions led to the identification of â¼100 phosphorylation sites from the tryptic digest of as little as 40 µL human serum.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfopeptídeos
/
Proteínas Sanguíneas
/
Compostos Férricos
/
Cromatografia de Afinidade
Limite:
Humans
Idioma:
En
Revista:
Proteomics
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Hungria