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A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry2. This ‘bottom-up’ process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous2 characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species3. ‘Top-down’ interrogation of whole proteins can overcome these problems for individual proteins4,5, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database6, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research7.

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  • 07 December 2011

    Acknowledgements were corrected.


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We thank all members of the group who contributed to development of top-down mass spectrometry over the years along with several private foundations: The Searle Scholars Program, The Burroughs Wellcome Fund, The David and Lucile Packard Foundation, The Richard and Camille Dreyfus Foundation, and The Chicago Biomedical Consortium with support from The Searle Funds at The Chicago Community Trust. We further acknowledge the Department of Chemistry at the University of Illinois, the Neuroproteomics Center on Cell to Cell Signaling supported through the National Institute on Drug Abuse (P30DA 018310), the National Institute for General Medical Sciences (GM 067193-08) and the National Science Foundation (DMS 0800631), whose combined investment in basic research over the past decade made this work possible. We dedicate this work in memory of Jonathan Widom.

Author information

Author notes

    • Adaikkalam Vellaichamy
    •  & Nertila Siuti

    Present addresses: Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, Texas 77030, USA (A.V.) ; Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA (N.S.).


  1. Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • John C. Tran
    • , Leonid Zamdborg
    • , Dorothy R. Ahlf
    • , Ji Eun Lee
    • , Adam D. Catherman
    • , Kenneth R. Durbin
    • , Adaikkalam Vellaichamy
    • , John F. Kellie
    • , Mingxi Li
    • , Cong Wu
    • , Steve M. M. Sweet
    • , Bryan P. Early
    • , Nertila Siuti
    • , Paul M. Thomas
    •  & Neil L. Kelleher
  2. Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA

    • John C. Tran
    • , Dorothy R. Ahlf
    • , Adam D. Catherman
    • , Kenneth R. Durbin
    • , Jeremiah D. Tipton
    • , John F. Kellie
    • , Mingxi Li
    • , Steve M. M. Sweet
    • , Bryan P. Early
    • , Philip D. Compton
    • , Paul M. Thomas
    •  & Neil L. Kelleher
  3. Doping Control Center and Center for Theragnosis, Korea Institute of Science and Technology, Seoul, South Korea

    • Ji Eun Lee
  4. Department of Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA

    • Richard D. LeDuc


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Project design: J.C.T., L.Z., P.M.T., N.L.K. Cell culture and biology: J.C.T., J.E.L., A.D.C., D.R.A., M.L., C.W., S.M.M.S., N.S. Separations: J.C.T., J.E.L., A.D.C., D.R.A. Mass spectrometry: J.C.T., J.E.L., A.D.C., D.R.A., J.D.T., A.V., J.F.K., P.D.C. Data analysis and statistics: J.C.T., L.Z., K.R.D., B.P.E., R.D.L., P.M.T., N.L.K. Writing: J.C.T., N.L.K.

Competing interests

Some components of the separations and software (ProSight) are available commercially.

Corresponding author

Correspondence to Neil L. Kelleher.

Supplementary information

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    Supplementary Information

    The file contains Supplementary Text, Supplementary References and Supplementary Figures 1-14 with legends.

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    Supplementary Tables

    This file contains Supplementary Tables 1-8 with detailed reports on protein identifications.

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