Proteoform-specific insights into cellular proteome regulation

Emma L. Norris, Madeleine J. Headlam, Keyur A. Dave, David D. Smith, Alexander Bukreyev, Toshna Singh, Buddhika A. Jayakody, Keith J. Chappell, Peter L. Collins, Jeffrey J. Gorman

    Research output: Contribution to journalArticle

    1 Scopus citations

    Abstract

    Knowledge regarding compositions of proteomes at the proteoform level enhances insights into cellular phenotypes. A strategy is described herein for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. The strategy was exemplified using five matched sets of lysates of uninfected and human respiratory syncytial virus-infected A549 cells. Template matching demonstrated that 67.3% of 10475 protein profiles identified focused to narrow pI windows indicative of efficacious focusing. Furthermore, correlation between experimental and theoretical pI gradients indicated reproducible focusing. Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. One component of this strategy involved examination of the focusing profiles for protein groups. A novel concordance analysis facilitated differentiation between proteoforms, including proteoforms generated by alternate splicing and proteolysis. Evaluation of focusing profiles and concordance analysis were applicable to cells from a single and/or multiple biological states. Statistical analyses identified proteoform variation between biological states. Regulation relevant to cellular responses to human respiratory syncytial virus was revealed. Western blotting and Protomap analyses validated the proteoform regulation. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. Novel truncated proteoforms of MX1 were identified in infected cells and phosphorylation driven regulation of HSPB1 proteoforms was correlated with infection. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysis of other biological systems.

    Original languageEnglish (US)
    Pages (from-to)3297-3320
    Number of pages24
    JournalMolecular and Cellular Proteomics
    Volume15
    Issue number10
    DOIs
    StatePublished - Oct 2016

    ASJC Scopus subject areas

    • Analytical Chemistry
    • Biochemistry
    • Molecular Biology

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  • Cite this

    Norris, E. L., Headlam, M. J., Dave, K. A., Smith, D. D., Bukreyev, A., Singh, T., Jayakody, B. A., Chappell, K. J., Collins, P. L., & Gorman, J. J. (2016). Proteoform-specific insights into cellular proteome regulation. Molecular and Cellular Proteomics, 15(10), 3297-3320. https://doi.org/10.1074/mcp.O116.058438