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Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS

Nature Protocols volume 6, pages 859–869 (2011)Cite this article

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Abstract

Targeted quantification of proteins is a daily task in biological research but often relies on techniques such as western blotting that are only barely quantitative. Here we present a broadly applicable workflow for protein quantification from unpurified whole-cell extracts that can be completed in less than 3 d. Without prefractionation or affinity enrichment, a whole-cell extract is trypsin-digested in an acetonitrile-containing ammonium carbonate buffer and high-molecular-weight compounds are removed by filtration. A normalization strategy, which involves endogenous reference proteins, facilitates the determination of relative changes in protein expression without requiring isotope labeling or standard addition. On a triple-quadrupole mass spectrometer, we demonstrate standard-free quantification of yeast proteins present over five orders of magnitude and present at ≥500 copies per cell. Liquid chromatography/multiple reaction monitoring (LC-MRM)–based proteomics is therefore a next-generation alternative to western blotting, as it allows simultaneous and reliable quantification of multiple endogenous proteins without the need for enrichment, isotope labeling or use of antibodies.

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Figure 1: Workflow.
Figure 2: MRM-based detection of tryptic peptides for proteins present at different cellular concentrations in whole-proteome digests.
Figure 3
Figure 4: Linearity of quantification over several orders of magnitude.
Figure 5: Induction of galactose structural proteins studied in whole-cell extracts by standard-free and label-free MRM.

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Acknowledgements

We thank B. Lukaszewska-McGreal for help with sample preparation, our lab members for critical discussions and the Max Planck Institute for Molecular Genetics for funding. M.R. is a Wellcome Trust Research Career Development and Wellcome-Beit prize Fellow.

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Authors and Affiliations

  1. Mass Spectrometry Unit, Max Planck Institute for Molecular Genetics, Berlin, Germany

    Katharina Bluemlein & Markus Ralser

  2. Cambridge Systems Biology Centre & Department of Biochemistry, University of Cambridge, Cambridge, UK

    Markus Ralser

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  1. Katharina Bluemlein
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Contributions

K.B. designed and conducted experiments and contributed to the writing of the paper. M.R. designed and conducted experiments and contributed to the writing of the paper.

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Correspondence to Markus Ralser.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures 1 and 2

Supplementary Figure 1: Relative protein abundance in incomplete tryptic digests (PDF 644 kb)

Supplementary Figure 2: Detection of Cps1p in high concentrated protein extract.

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Bluemlein, K., Ralser, M. Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS. Nat Protoc 6, 859–869 (2011). https://doi.org/10.1038/nprot.2011.333

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