| Quantitative proteome and phosphoproteome analyses of cultured cells based on SILAC labeling without requirement of serum dialysis. | |
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MedLine Citation:
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PMID: 20174688 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Koshi Imami; Naoyuki Sugiyama; Masaru Tomita; Yasushi Ishihama |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-01-12 |
Journal Detail:
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Title: Molecular bioSystems Volume: 6 ISSN: 1742-2051 ISO Abbreviation: Mol Biosyst Publication Date: 2010 Mar |
Date Detail:
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Created Date: 2010-02-22 Completed Date: 2010-05-24 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101251620 Medline TA: Mol Biosyst Country: England |
Other Details:
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Languages: eng Pagination: 594-602 Citation Subset: IM |
Affiliation:
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Institute for Advanced Biosciences, Keio University, Daihoji, Tsuruoka, Yamagata 997-0017, Japan. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Breast Neoplasms
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metabolism Carbon Isotopes / metabolism Cell Line, Tumor Culture Media Dialysis / methods Female Gene Expression Regulation, Neoplastic Hela Cells Humans Isotope Labeling / methods* Nitrogen Isotopes / metabolism Phosphoproteins / analysis*, metabolism Phosphorylation Proteome / analysis*, metabolism Serum / chemistry* Signal Transduction |
| Chemical | |
Reg. No./Substance:
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0/Carbon Isotopes; 0/Culture Media; 0/Nitrogen Isotopes; 0/Phosphoproteins; 0/Proteome |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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