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

ABSTRACT: Udeshi ND, Mani DC, Satpathy S, Fereshetian S, Gasser JA, Svinkina T, Olive ME, Ebert BL, Mertins P, Carr SA. 2019. Abstract Protein ubiquitylation is involved in a plethora of cellular processes. Defects in the ubiquitin system are at the root of many acquired and hereditary diseases. While antibodies directed at ubiquitin remnants (K-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 ug peptide per sample from cells or tissue in a TMT10 plex in ca. 5 hr. 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.

INSTRUMENT(S): QExactive Plus, Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000084650 | MassIVE | Tue Dec 03 08:00:00 GMT 2019

REPOSITORIES: MassIVE

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