Abstract
We have shown that sample fractionation is an effective method for increasing the detection coverage of the proteome of complex samples, such as organs, by mass-spectrometric techniques. Further fractionating a sample based on subcellular compartments can generate molecular information on the state of a tissue and the distribution of its protein components. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs or tissues for subsequent analysis by mass spectrometry. It uses differential centrifugation in density gradients to isolate nuclear, cytosolic, mitochondrial and mixed microsomal (Golgi, endoplasmic reticulum, other vesicles and plasma membrane) fractions. Once the fractions are isolated, they are extracted for protein and the samples can then be frozen for processing and analysis at a later date. The procedure can typically be completed in 5 h.
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Acknowledgements
The authors would like to thank Dr. Thomas Kislinger for assistance in development of this protocol and the reviewers for their thoughtful comments on the protocol. Operating grants were awarded to A.E. from the McLaughlin Centre for Molecular Medicine, Genome Canada, the Ontario Genomics Institute (OGI) and the National Science and Engineering Council of Canada (NSERC). B.C. was supported by a Canadian Institutes of Health Research Doctoral fellowship.
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Cox, B., Emili, A. Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Nat Protoc 1, 1872–1878 (2006). https://doi.org/10.1038/nprot.2006.273
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DOI: https://doi.org/10.1038/nprot.2006.273
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