Document Detail

Quantitative proteome and phosphoproteome analyses of cultured cells based on SILAC labeling without requirement of serum dialysis.
MedLine Citation:
PMID:  20174688     Owner:  NLM     Status:  MEDLINE    
The use of dialyzed serum is essential in the application of the conventional stable isotope labeling by amino acids in cell culture (SILAC) approach to achieve complete labeling of proteins for quantitative proteomics. Here, we first evaluated the impact of dialyzed serum on the proteome and phosphoproteome of hormone-sensitive breast cancer MCF-7 cells and found that dialyzed serum influenced the expression of proteins related to signaling systems via hormone receptors, inducing a marked change of the phosphoproteome compared with the use of non-dialyzed serum. We also evaluated 9 other cell lines, including HeLa, HEK293 and Panc1, and found that the influence of serum dialysis on the expression profiles of the proteome and phosphoproteome varied, depending on the cell type. To avoid these problems, we established a SILAC-based quantification approach without the requirement of serum dialysis. Our simple approach is based on dual labeling of two populations of cells with two kinds of heavy amino acids of different mass, using non-dialyzed serum. Using our SILAC approach with non-dialyzed serum, we successfully quantified the phosphoproteome of MCF-7 cells induced by lapatinib, an EGFR1/Her2 dual kinase inhibitor. Because of the dual labeling approach, our method is widely applicable to cultured cells in which protein labeling is incomplete for any reason, e.g., owing to the use of non-dialyzed serum or a low growth rate.
Koshi Imami; Naoyuki Sugiyama; Masaru Tomita; Yasushi Ishihama
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-01-12
Journal Detail:
Title:  Molecular bioSystems     Volume:  6     ISSN:  1742-2051     ISO Abbreviation:  Mol Biosyst     Publication Date:  2010 Mar 
Date Detail:
Created Date:  2010-02-22     Completed Date:  2010-05-24     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101251620     Medline TA:  Mol Biosyst     Country:  England    
Other Details:
Languages:  eng     Pagination:  594-602     Citation Subset:  IM    
Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata 997-0017, Japan.
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MeSH Terms
Breast Neoplasms / metabolism
Carbon Isotopes / metabolism
Cell Line, Tumor
Culture Media
Dialysis / methods
Gene Expression Regulation, Neoplastic
Hela Cells
Isotope Labeling / methods*
Nitrogen Isotopes / metabolism
Phosphoproteins / analysis*,  metabolism
Proteome / analysis*,  metabolism
Serum / chemistry*
Signal Transduction
Reg. No./Substance:
0/Carbon Isotopes; 0/Culture Media; 0/Nitrogen Isotopes; 0/Phosphoproteins; 0/Proteome

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