Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media.

Details

Serval ID
serval:BIB_7DDA894596D3
Type
Article: article from journal or magazin.
Collection
Publications
Title
Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media.
Journal
Journal of Proteome research
Author(s)
Colzani M., Waridel P., Laurent J., Faes E., Rüegg C., Quadroni M.
ISSN
1535-3907[electronic]
Publication state
Published
Issued date
2009
Volume
8
Number
10
Pages
4779-4788
Language
english
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.
Pubmed
Web of science
Create date
08/01/2010 15:58
Last modification date
03/03/2018 18:40
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