Quantitative analysis of a proteome by N-terminal stable-isotope labelling of tryptic peptides.
Rapid Commun Mass Spectrom
; 21(16): 2671-9, 2007.
Article
em En
| MEDLINE
| ID: mdl-17659651
ABSTRACT
Covalent modification of peptides and proteins with compounds containing stable isotopes (isotope tagging) has become an essential tool to detect dynamic changes in the proteome following external or internal influence; however, using terminal amino groups for global isotope labelling of tryptic peptides is challenged by the similar reactivity of the amino groups of lysine residues. We describe a new quantitative method based on selective tagging of the terminal amino groups of tryptic peptides with pentafluorophenyl esters containing stable isotopes. The labelled peptides were resolved by two-dimensional nanoflow liquid chromatography on weak anion-exchange and reversed-phase columns and then identified and quantified by tandem mass spectrometry. The method was applied to compare the proteomes of plasma membranes from proliferating and differentiated human colorectal adenocarcinoma (Caco-2) cells and endosomes purified from the livers of rats stimulated with insulin and epidermal growth factor. The comparison of the results obtained by isotope tagging and biochemical assays demonstrate that global isotope tagging with pentafluorophenyl esters allows accurate quantification of complex protein samples.
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Base de dados:
MEDLINE
Assunto principal:
Tripsina
/
Biomarcadores Tumorais
/
Cromatografia Líquida de Alta Pressão
/
Proteoma
/
Espectrometria de Massas por Ionização por Electrospray
/
Fígado
/
Proteínas de Neoplasias
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article