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


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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Figure 1: Overview of CTAP.
Figure 2: Vertebrate cell lines expressing L-lysine biosynthesis enzymes grow on and incorporate L-lysine produced from their precursors.
Figure 3: Limited gene expression changes observed when growing cells in precursor versus L-lysine.
Figure 4: Using two distinct enzyme-precursor pairs, cocultured cells exhibit precursor-based differential proteome labeling.
Figure 5: Application of CTAP for determining cell of origin for secreted factors.

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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.

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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.

Corresponding authors

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

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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.

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Gauthier, N., Soufi, B., Walkowicz, W. et al. Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments. Nat Methods 10, 768–773 (2013). https://doi.org/10.1038/nmeth.2529

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