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  • Primer
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Top-down proteomics

Abstract

Proteoforms, which arise from post-translational modifications, genetic polymorphisms and RNA splice variants, play a pivotal role as drivers in biology. Understanding proteoforms is essential to unravel the intricacies of biological systems and bridge the gap between genotypes and phenotypes. By analysing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and can decipher protein function, uncover disease mechanisms and advance precision medicine. This Primer explores TDP, including the underlying principles, recent advances and an outlook on the future. The experimental section discusses instrumentation, sample preparation, intact protein separation, tandem mass spectrometry techniques and data collection. The results section looks at how to decipher raw data, visualize intact protein spectra and unravel data analysis. Additionally, proteoform identification, characterization and quantification are summarized, alongside approaches for statistical analysis. Various applications are described, including the human proteoform project and biomedical, biopharmaceutical and clinical sciences. These are complemented by discussions on measurement reproducibility, limitations and a forward-looking perspective that outlines areas where the field can advance, including potential future applications.

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Fig. 1: Proteoforms and the top-down approach.
Fig. 2: The pillars of top-down proteomics.
Fig. 3: Top-down proteomics sample preparation.
Fig. 4: Tandem mass spectrometry techniques for top-down proteomics.
Fig. 5: Fundamental concepts in protein analysis by top-down proteomics.
Fig. 6: Overview of top-down proteomics quantification methods.
Fig. 7: Biological applications for top-down proteomics.

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Acknowledgements

Y.G. acknowledges support from the NIH R01 HL096971, HL109810, GM117058 and GM125085. J.A.L. was supported by the NIH under award R35GM145286 and the Department of Energy under award DE-FC02-02ER63421. L.M.S. was supported by the NIGMS under the award R35GM126914. J.N.A. was supported by ALSA 508452. J.C.-R. and Y.O.T. were supported by the European Horizon 2020 programme under award 829157, and J.C.-R. was also supported by the Institut Pasteur, the CNRS and EPIC-XS under award 823839. S.W. was supported by OCAST HR23-169, NIH NIAID R01AI141625 and NIH/NIAID2U19AI062629. S.W. was also supported by the University of Alabama startup grant. X.L. was supported by the NIH under awards R01GM118470, R01CA247863 and R01AI141625 and the NSF under award 2307573. L.P.-T. was supported by the NIH under the award UH3CA256959. The authors acknowledge K. Brown for helpful discussions on surfactant-aided proteomics and R. Luo for the assistance and helpful discussion on clinical top-down proteomics.

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Authors and Affiliations

Authors

Contributions

Introduction (D.S.R., J.A.L., Y.O.T., J.N.A., L.P.-T. and Y.G.); Experimentation (D.S.R., J.A.L., Y.O.T., S.W., J.N.A. and Y.G.); Results (D.S.R., J.A.L., Y.O.T., X.L., S.W., J.C.-R., J.N.A., L.P.-T., L.M.S. and Y.G.); Applications (D.S.R., Y.O.T., S.W., J.C.-R., L.P.-T. and Y.G.); Reproducibility and data deposition (D.S.R., Y.O.T., X.L., S.W. and Y.G.); Limitations and optimizations (D.S.R., J.A.L., Y.O.T., X.L. and Y.G.); Outlook (D.S.R., Y.O.T., L.P.-T., L.M.S. and Y.G.); overview of the Primer (all authors).

Corresponding authors

Correspondence to David S. Roberts or Ying Ge.

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Competing interests

J.A.L., J.C.-R., J.N.A., L.P.-T., L.M.S. and Y.G. are currently board members of Consortium for Top-down Proteomics. Y.O.T. is an employee of Spectroswiss, a company that develops data acquisition systems and data processing software for mass spectrometry. X.L. has a project contract with Bioinformatics Solutions Inc., a company that develops data processing software for mass spectrometry. D.S.R. and Y.G. are named as inventors for the patent application US Patent App. 17/786,482. L.P.-T. is named as an inventor for the US Patent App. 17/954,834. Y.G. is named as an inventor for the US Patent App. 18/069,005; US Patent App. 17/978,793; US Patent App. 18/451,614; and US Patent 11,567,085. S.W. declares no competing interests.

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Nature Reviews Methods Primers thanks Federica Iavarone, Liangliang Sun, Jennifer Brodbelt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

National Resource for Translational and Developmental Proteomics: http://nrtdp.northwestern.edu/protocols/

Proteoform repository: http://repository.topdownproteomics.org/

Glossary

Bottom-up proteomics

A technique used to analyse peptide fragments from the proteolytic digestion of intact proteins by mass spectrometry, enabling sensitive and high-throughput identification of proteins.

Convoluted mass spectra

Refers to the potential overlap of two or more peaks with similar mass-to-charge (m/z) ratios. This can lead to incomplete separation of two or more mass spectral peaks owing to resolution limits and complicated mass spectral identification.

Data-dependent acquisition

Refers to the tandem mass spectrometry technique that involves specific selection of precursor ions before MS2 fragmentation. This technique commonly selects several of the most intense peaks observed in a single MS1 survey scan for fragmentation and only fragmenting a small subset of the total ions present.

Data-independent acquisition

Refers to the tandem mass spectrometry technique that forgoes specific selection of precursor ions and instead fragments all ions present in an MS1 survey scan.

Monoisotopic peak

The exact mass of a molecule, represented by the sum of the masses of the atoms in the molecule using the principal (most abundant) isotope for each element.

Post-translational modifications

All covalent processing events and modifications to the amino acid sequence of a given protein occurring after protein biosynthesis.

Proteoforms

A term used to describe all the different molecular forms of a protein product from a single gene. This includes changes from genetic variations, alternatively spliced RNA transcripts and post-translational modifications such as protein phosphorylation, glycosylation and protein truncations.

Tandem mass spectrometry

A technique performed using one or more mass analysers, involving multiple consecutive stages of mass spectrometry analysis — typically two, MS/MS, also known as MS2 — to fragment selected precursor ions in the MS1 spectrum and generate product ions that can elucidate the structure and chemical composition of a molecule.

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Roberts, D.S., Loo, J.A., Tsybin, Y.O. et al. Top-down proteomics. Nat Rev Methods Primers 4, 38 (2024). https://doi.org/10.1038/s43586-024-00318-2

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