Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.
Proteomics
; 9(2): 299-309, 2009 Jan.
Article
em En
| MEDLINE
| ID: mdl-19142957
ABSTRACT
The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Receptores de Antígenos de Linfócitos T
/
Linfócitos T CD4-Positivos
/
Análise Serial de Proteínas
Limite:
Humans
Idioma:
En
Revista:
Proteomics
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
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