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Sampling the proteome by emerging single-molecule and mass spectrometry methods

Nature Methods volume 20, pages 339–346 (2023)Cite this article

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Mammalian cells have about 30,000 times as many protein molecules as mRNA molecules, which has major implications in the development of proteomics technologies. We discuss strategies that have been helpful for counting billions of protein molecules by liquid chromatography–tandem mass spectrometry and suggest that these strategies can benefit single-molecule methods, especially in mitigating the challenges posed by the wide dynamic range of the proteome.

The ubiquitous roles of proteins in biomedicine are well appreciated and have motivated technologies seeking to advance two key parameters: the sensitivity and throughput of quantitative protein analysis. While proteomic technologies may use different approaches, they face similar challenges, such as quantifying proteins of vastly different abundances, some present in only a few copies and some present in tens of millions of copies per typical mammalian cell. This wide dynamic range poses a substantial challenge for investigating proteome biology.

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Fig. 1: Overview of RNA and protein statistics.
Fig. 2: A liquid chromatography-mass spectrometry experiment can count billions of peptide ions within 90 min.
Fig. 3: Fractionation before counting molecules improves the dynamic range in proteomics.

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Acknowledgements

The authors acknowledge discussions with Edward Marcotte and members of the Alfaro, MacCoss and Slavov labs. M.J.M. appreciates the constructive feedback provided by UW Genome Sciences faculty. This work was supported in part by US National Institutes of Health grants U19 AG065156, R24 GM141156 and F31 AG066318; an Allen Distinguished Investigator award through The Paul G. Allen Frontiers Group to N.S.; a Seed Networks Award from CZI CZF2019-002424 to N.S.; award R01GM144967 from the US National Institute of General Medical Sciences; award R01HG10087 from the US National Human Genome Research Institute to M.W.; and the project ‘International Centre for Cancer Vaccine Science’ carried out within the International Agendas Programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. We thank the PL-Grid and CI-TASK Infrastructure, Poland, for providing their hardware and software resources. This work is supported by the Knowledge At the Tip of Your Fingers: Clinical Knowledge for Humanity (KATY) project funded from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101017453.

Author information

Author notes

  1. These authors contributed equally: Michael J. MacCoss, Javier Antonio Alfaro, Nikolai Slavov.

Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Michael J. MacCoss, Danielle A. Faivre & Christine C. Wu

  2. International Centre for Cancer Vaccine Science, University of Gdańsk, Gdańsk, Poland

    Javier Antonio Alfaro

  3. Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada

    Javier Antonio Alfaro

  4. School of Informatics, University of Edinburgh, Edinburgh, UK

    Javier Antonio Alfaro

  5. Department of Physics, Northeastern University, Boston, MA, USA

    Meni Wanunu

  6. Departments of Bioengineering, Biology, Chemistry and Chemical Biology, Single Cell Proteomics Center and Barnett Institute, Northeastern University, Boston, MA, USA

    Nikolai Slavov

  7. Parallel Squared Technology Institute, Watertown, MA, USA

    Nikolai Slavov

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Contributions

M.J.M., J.A. and N.S. conceived the project and wrote an initial draft. D.A.F., M.J.M. and N.S. made figures. C.C.W. collected the plasma extracellular vesicle data. All authors contributed significantly to the content, edited, and approved the final manuscript.

Corresponding authors

Correspondence to Michael J. MacCoss, Javier Antonio Alfaro or Nikolai Slavov.

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Competing interests

The MacCoss laboratory at the University of Washington has a sponsored research agreement with Thermo Fisher Scientific, a manufacturer of mass spectrometry instrumentation. M.J.M. is a paid consultant for Thermo Fisher Scientific. The Slavov laboratory at Northeastern University has a research agreement with Bruker, a manufacturer of mass spectrometry instrumentation.

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Nature Methods thanks Tae-Young Yoon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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MacCoss, M.J., Alfaro, J.A., Faivre, D.A. et al. Sampling the proteome by emerging single-molecule and mass spectrometry methods. Nat Methods 20, 339–346 (2023). https://doi.org/10.1038/s41592-023-01802-5

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