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

Namrata D. Udeshi (), Deepak C. Mani, Shankha Satpathy, Shaunt Fereshetian, Jessica A. Gasser, Tanya Svinkina, Meagan E. Olive, Benjamin L. Ebert, Philipp Mertins and Steven A. Carr ()
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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
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
Philipp Mertins: Broad Institute of MIT and Harvard
Steven A. Carr: Broad Institute of MIT and Harvard

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-019-14175-1

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