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The incongruity of validating quantitative proteomics using western blots

Nature Plants volume 8, pages 1320–1321 (2022)Cite this article

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Similar to the age-old reviewer request for quantitative PCR validation of RNA-sequencing data, nearly every researcher who uses proteomics technologies has at one time or another been asked by a reviewer to provide ‘western blot validation’ of their mass-spectrometry-based protein abundance data. We believe that this request demonstrates a lack of awareness amongst the plant biology community about the extraordinary improvements in cost, sensitivity and reliability that the field of mass-spectrometry-based proteomics has made in recent years. Here, as a group of experts in different domains of quantitative plant proteomics, we explain why western blot validations of quantitative proteomics data are both unnecessary and invalid. Furthermore, we invite our colleagues in the plant science community to update their perception of quantitative mass spectrometry as a sensitive and reliable method of protein identification and quantification.

Quantitative proteomics, in its many flavours, offers plant science researchers the ability to quantify thousands of proteins in a single experiment with much greater precision and reliability than western blotting. Both intra- and inter-laboratory studies have established the reproducibility of protein quantification by both targeted1,2 and untargeted (particularly data-independent acquisition) proteomics3,4. By contrast, western blotting has a high rate of failure owing to the inherent stochasticity of in-gel separation, membrane transfer and unspecific antibody binding. Furthermore, these processes — in conjunction with variability in blot development using luminol-based chemiluminescent reagents — render western blot results semiquantitative at best. Mass-spectrometry-based protein quantification is highly reproducible1,2,3,4 and reliable because quantification is typically based on the measurement of multiple spectra and peptides per protein and is accompanied by statistical confidence measures for each peptide and protein.

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Acknowledgements

We thank Z.-Y. Wang for editorial input and helpful comments.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

    Devang Mehta & R. Glen Uhrig

  2. Department of Biosystems, KU Leuven, Leuven, Belgium

    Devang Mehta

  3. Plant Cell Atlas Proteomics Committee, Stanford, CA, USA

    Devang Mehta, Amir H. Ahkami, Justin Walley, Shou-Ling Xu & R. Glen Uhrig

  4. Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratories, Richland, WA, USA

    Amir H. Ahkami

  5. School of Biological Science (SBS), Washington State University (WSU), Pullman, WA, USA

    Amir H. Ahkami

  6. Iowa State University, Ames, IA, USA

    Justin Walley

  7. Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA

    Shou-Ling Xu

  8. Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

    R. Glen Uhrig

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  1. Devang Mehta
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  2. Amir H. Ahkami
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  4. Shou-Ling Xu
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Correspondence to Devang Mehta or R. Glen Uhrig.

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Mehta, D., Ahkami, A.H., Walley, J. et al. The incongruity of validating quantitative proteomics using western blots. Nat. Plants 8, 1320–1321 (2022). https://doi.org/10.1038/s41477-022-01314-8

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