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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kislinger, T. et al. PRISM, a Generic Large Scale Proteomic Investigation Strategy for Mammals. Mol. Cell. Proteomics 2, 96–106 (2003).
Mootha, V.K. et al. Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 115, 629–640 (2003).
Krapfenbauer, K., Fountoulakis, M. & Lubec, G. A rat brain protein expression map including cytosolic and enriched mitochondrial and microsomal fractions. Electrophoresis 24, 1847–1870 (2003).
Dreger, M., Bengtsson, L., Schoneberg, T., Otto, H. & Hucho, F. Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane. Proc. Natl. Acad. Sci. USA 98, 11943–11948 (2001).
Kislinger, T. et al. Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling. Cell 125, 173–186 (2006).
Hoffmann, K. et al. New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues. Anal. Bioanal. Chem. 381, 1138–1144 (2005).
Foster, L.J. et al. A mammalian organelle map by protein correlation profiling. Cell 125, 187–199 (2006).
Reifschneider, N.H. et al. Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE. J. Proteome Res. 5, 1117–1132 (2006).
Holden, M.J. & Colombini, M. The outer mitochondrial membrane channel, VDAC, is modulated by a protein localized in the intermembrane space. Biochim. Biophys. Acta 1144, 396–402 (1993).
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.273
This article is cited by
-
High-throughput and proteome-wide discovery of endogenous biomolecular condensates
Nature Chemistry (2024)
-
In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility
Cell and Tissue Research (2023)
-
Characterisation of a novel OPA1 splice variant resulting in cryptic splice site activation and mitochondrial dysfunction
European Journal of Human Genetics (2022)
-
A new strategy to uncover fragile X proteomic biomarkers using the nascent proteome of peripheral blood mononuclear cells (PBMCs)
Scientific Reports (2021)
-
Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity
Nature Immunology (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
