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

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Item Type:

Article

Title:

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

Creators Name:

Udeshi, N.D. and Mani, D.C. and Satpathy, S. and Fereshetian, S. and Gasser, J.A. and Svinkina, T. and Olive, M.E. and Ebert, B.L. and Mertins, P. and Carr, S.A.

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.

Keywords:

Breast Neoplasms, HeLa Cells, Ikaros Transcription Factor, Mass Spectrometry, Multiple Myeloma, Post-Translational Protein Processing, Proteins, Proteome, Proteomics, Sensitivity and Specificity, Staining and Labeling, Translational Medical Research, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, Animals, Mice

Source:

Nature Communications

ISSN:

2041-1723

Publisher:

Nature Publishing Group

Volume:

Number:

Page Range:

359

Date:

17 January 2020