Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media.
J Proteome Res
; 8(10): 4779-88, 2009 Oct.
Article
en En
| MEDLINE
| ID: mdl-19711906
ABSTRACT
Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Sanguíneas
/
Proteínas
/
Medios de Cultivo Condicionados
/
Proteómica
/
Suero
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Proteome Res
Asunto de la revista:
BIOQUIMICA
Año:
2009
Tipo del documento:
Article
País de afiliación:
Suiza