Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments

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We report a technique to selectively and continuously label the proteomes of individual cell types in coculture, named cell type–specific labeling using amino acid precursors (CTAP). Through transgenic expression of exogenous amino acid biosynthesis enzymes, vertebrate cells overcome their dependence on supplemented essential amino acids and can be selectively labeled through metabolic incorporation of amino acids produced from heavy isotope–labeled precursors. When testing CTAP in several human and mouse cell lines, we could differentially label the proteomes of distinct cell populations in coculture and determine the relative expression of proteins by quantitative mass spectrometry. In addition, using CTAP we identified the cell of origin of extracellular proteins secreted from cells in coculture. We believe that this method, which allows linking of proteins to their cell source, will be useful in studies of cell-cell communication and potentially for discovery of biomarkers.

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We acknowledge E. Larsson, Y. Gruber, D.S. Marks, A. Arvey, J. Joyce and A. Koff for helpful discussions, H. Erdjument-Bromage for pilot MS/MS investigation, A.N. Miller, J. Cross and X. Jing for technical help, and E. Larsson, J. Gauthier, J. Joyce and A.M. Miller for helpful comments on the manuscript. This work was funded in part by US National Cancer Institute grant U54 CA148967.

Author information

Author notes

    • David Y Gin



  1. Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Nicholas P Gauthier
    • , Chris Sander
    •  & Martin L Miller
  2. Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Nicholas P Gauthier
    •  & William E Walkowicz
  3. Proteome Center Tübingen, University of T¨bingen, Tübingen, Germany.

    • Boumediene Soufi
    •  & Boris Macek
  4. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • William E Walkowicz
    •  & David Y Gin
  5. Department of Immunology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Virginia A Pedicord
  6. Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Konstantinos J Mavrakis


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N.P.G. and M.L.M. designed and performed experiments and analyzed data. W.E.W. generated reagents. B.S., K.J.M. and V.A.P. contributed to experiments. N.P.G. and M.L.M. wrote the manuscript. B.S., W.E.W., B.M., K.J.M., V.A.P., D.Y.G. and C.S. contributed to discussions and editing of the manuscript. N.P.G. conceived the hypothesis. N.P.G., C.S. and M.L.M. developed the concept and managed the project.

Competing interests

A provisional patent application (US 61/697,584) relating to the use of exogenous enzymes for proteomic labeling in multicellular culture has been filed by Memorial Sloan-Kettering Cancer Center with N.P.G., C.S. and M.L.M. listed as inventors.

Corresponding authors

Correspondence to Nicholas P Gauthier or Chris Sander or Martin L Miller.

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