Abstract
The organization of eukaryotic cells into distinct subcompartments is vital for all functional processes, and aberrant protein localization is a hallmark of many diseases. Microscopy methods, although powerful, are usually low-throughput and dependent on the availability of fluorescent fusion proteins or highly specific and sensitive antibodies. One method that provides a global picture of the cell is localization of organelle proteins by isotope tagging (LOPIT), which combines biochemical cell fractionation using density gradient ultracentrifugation with multiplexed quantitative proteomics mass spectrometry, allowing simultaneous determination of the steady-state distribution of hundreds of proteins within organelles. Proteins are assigned to organelles based on the similarity of their gradient distribution to those of well-annotated organelle marker proteins. We have substantially re-developed our original LOPIT protocol (published by Nature Protocols in 2006) to enable the subcellular localization of thousands of proteins per experiment (hyperLOPIT), including spatial resolution at the suborganelle and large protein complex level. This Protocol Extension article integrates all elements of the hyperLOPIT pipeline, including an additional enrichment strategy for chromatin, extended multiplexing capacity of isobaric mass tags, state-of-the-art mass spectrometry methods and multivariate machine-learning approaches for analysis of spatial proteomics data. We have also created an open-source infrastructure to support analysis of quantitative mass-spectrometry-based spatial proteomics data (http://bioconductor.org/packages/pRoloc) and an accompanying interactive visualization framework (http://www.bioconductor.org/packages/pRolocGUI). The procedure we outline here is applicable to any cell culture system and requires ∼1 week to complete sample preparation steps, ∼2 d for mass spectrometry data acquisition and 1–2 d for data analysis and downstream informatics.
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Acknowledgements
C.M.M. and L.M.B. were supported by a Wellcome Trust Technology Development Grant (grant no. 108467/Z/15/Z). L.G. was supported by the BBSRC Strategic Longer and Larger grant (grant no. BB/L002817/1). A.G. was funded through the Alexander S. Onassis Public Benefit Foundation, the Foundation for Education and European Culture (IPEP) and the Embiricos Trust Scholarship of Jesus College Cambridge. D.J.H.N. was supported by a BBSRC grant (grant no. BB/LOO2817). K.S.L. is a Wellcome Trust Joint Senior Investigator (grant no. 110170/Z/15/Z).
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C.M.M., L.M.B. and K.S.L. wrote the manuscript with contributions from M.J.D. and A.G. Figures were prepared by L.M.B., A.G. and C.M.M. L.G., D.J.H.N., N.K.B., M.E. and A.C. advised on the content and layout of the protocol.
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Mulvey, C., Breckels, L., Geladaki, A. et al. Using hyperLOPIT to perform high-resolution mapping of the spatial proteome. Nat Protoc 12, 1110–1135 (2017). https://doi.org/10.1038/nprot.2017.026
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DOI: https://doi.org/10.1038/nprot.2017.026
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