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Originally published In Press as doi:10.1074/mcp.M400021-MCP200 on April 21, 2004.
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Molecular & Cellular Proteomics 3:729-735, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Quantitative Cancer Proteomics: Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) as a Tool for Prostate Cancer Research*,S

Patrick A. Everley{ddagger},§, Jeroen Krijgsveld{ddagger},¶, Bruce R. Zetter{ddagger},§ and Steven P. Gygi{ddagger},||

From the {ddagger} Department of Cell Biology, Harvard Medical School, Boston, MA 02115; § Program in Vascular Biology, Department of Surgery, Children’s Hospital, Boston, MA 02115; and Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, Utrecht, The Netherlands

Microarrays have been the primary means for large-scale analyses of genes implicated in cancer progression. However, more recently a need has been recognized for investigating cancer development directly at the protein level. In this report, we have applied a comparative proteomic technique to the study of metastatic prostate cancer. This technology, termed stable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment. SILAC makes use of 12C- and 13C-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either "light" or "heavy" proteins, respectively. Upon mixing lysates collected from these cells, proteins can be identified by tandem mass spectrometry. The incorporation of stable isotopes also allows for a quantitative comparison between the two samples. Using this method, we compared the expression levels for more than 440 proteins in the microsomal fractions of prostate cancer cells with varying metastatic potential. Of these, 60 were found elevated greater than 3-fold in the highly metastatic cells, whereas 22 were reduced by equivalent amounts. Western blotting provided further confirmation of the mass spectrometry-based quantification. Our results demonstrate the applicability of this novel approach toward the study of cancer progression using defined cell lines.

|| To whom correspondence should be addressed: Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, MA 02115. Tel.: 617-432-3155; Fax: 617-432-1144; E-mail: steven_gygi{at}hms.harvard.edu


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