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Rapid and deep-scale ubiquitylation profiling for biology and translational research

Author

Listed:
  • Namrata D. Udeshi

    (Broad Institute of MIT and Harvard)

  • Deepak C. Mani

    (Broad Institute of MIT and Harvard)

  • Shankha Satpathy

    (Broad Institute of MIT and Harvard)

  • Shaunt Fereshetian

    (Broad Institute of MIT and Harvard)

  • Jessica A. Gasser

    (Broad Institute of MIT and Harvard
    Brigham and Women’s Hospital
    Dana-Farber Cancer Institute)

  • Tanya Svinkina

    (Broad Institute of MIT and Harvard)

  • Meagan E. Olive

    (Broad Institute of MIT and Harvard)

  • Benjamin L. Ebert

    (Broad Institute of MIT and Harvard
    Brigham and Women’s Hospital
    Dana-Farber Cancer Institute
    Dana-Farber Cancer Institute)

  • Philipp Mertins

    (Broad Institute of MIT and Harvard
    Max Delbrück Center for Molecular Medicine in the Helmholtz Society
    Berlin Institute of Health)

  • Steven A. Carr

    (Broad Institute of MIT and Harvard)

Abstract

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.

Suggested Citation

  • Namrata D. Udeshi & Deepak C. Mani & Shankha Satpathy & Shaunt Fereshetian & Jessica A. Gasser & Tanya Svinkina & Meagan E. Olive & Benjamin L. Ebert & Philipp Mertins & Steven A. Carr, 2020. "Rapid and deep-scale ubiquitylation profiling for biology and translational research," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-019-14175-1
    DOI: 10.1038/s41467-019-14175-1
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