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Trans-SILAC: sorting out the non-cell-autonomous proteome

Abstract

Non-cell-autonomous proteins are incorporated into cells that form tight contacts or are invaded by bacteria, but identifying the full repertoire of transferred proteins has been a challenge. Here we introduce a quantitative proteomics approach to sort out non-cell-autonomous proteins synthesized by other cells or intracellular pathogens. Our approach combines stable-isotope labeling of amino acids in cell culture (SILAC), high-purity cell sorting and bioinformatics analysis to identify the repertoire of relevant non-cell-autonomous proteins. This 'trans-SILAC' method allowed us to discover many proteins transferred from human B to natural killer cells and to measure biosynthesis rates of Salmonella enterica proteins in infected human cells. Trans-SILAC should be a useful method to examine protein exchange between different cells of multicellular organisms or pathogen and host.

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Figure 1: Detecting non-cell-autonomous translated proteins by trans-SILAC.
Figure 2: Sorting of highly purified human CD56+ NK cells.
Figure 3: FACS-based validation of the trans-SILAC results.
Figure 4: Non-cell-autonomous proteins that form the 'cancer, immunological disease, hematological disease' network.

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Acknowledgements

O.R. was supported by a scholarship from the Clore Israel Foundation. M.K. was supported by the Edmond J. Safra Program in Bioinformatics at Tel Aviv University. Operating funds for this work came, in part, from the Prajs-Drimmer Institute for the Development of Anti-degenerative Disease Drugs to Y.K., from the Israel Cancer Association to I.G. and Y.K. and from a Canadian Institutes of Health Research Operating grant (MOP-77688) to L.J.F. Mass spectrometry infrastructure used in this project was supported by the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund and the British Columbia Proteomics Network. Y.F. is supported by a studentship from the Genome Sciences and Technologies graduate program. Expression vectors encoding for EGFP-tagged RALA and RALB proteins were a gift from A. Cox (The University of North Carolina at Chapel Hill) and vectors for Arf4, Rab10 and Rab11a were a gift from D. Cassel (Technion, Israel Institute of Technology).

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Authors and Affiliations

Authors

Contributions

O.R. jointly conceived the study with I.G., designed experiments, performed experiments, analyzed data and wrote the paper; M.K. developed analytical tools and analyzed data; Y.F. designed and performed experiments and analyzed data; H.V. performed experiments and analyzed data; J.J.-H. analyzed data; L.J.F. designed experiments, developed analytical tools, analyzed data and wrote the paper; Y.K. and I.G. jointly supervised the project, designed experiments, analyzed data and wrote the paper.

Corresponding authors

Correspondence to Yoel Kloog or Itamar Goldstein.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 3–5 (PDF 3283 kb)

Supplementary Table 1

Raw trans-SILAC data. (XLS 1145 kb)

Supplementary Table 2

All spectral counts. (XLS 566 kb)

Supplementary Table 6

Bacterial intracellular protein synthesis during early Salmonella invasion. (XLS 200 kb)

Supplementary Software

The in-house Perl and R scripts used to process the datasets and a readme file with detailed instructions for sorting out the positive hits. (ZIP 5846 kb)

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Rechavi, O., Kalman, M., Fang, Y. et al. Trans-SILAC: sorting out the non-cell-autonomous proteome. Nat Methods 7, 923–927 (2010). https://doi.org/10.1038/nmeth.1513

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