MassIVE MSV000087363

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Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment

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Description

Rivera KD, Olive ME, Bergstrom EJ, Nelson A, Lee K, Satpathy S, Carr SA, Udeshi ND. 2021. Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites is necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-GG peptides are enriched with anti-K-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-GG antibody (mK-GG) and a magnetic particle processor. We report the identification of ~20,000 ubiquitylation sites from a TMT10-plex with 500 ug input per sample processed in ~2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of this method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples. [doi:10.25345/C56N7K] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: UbiFast ; Ubiquitin Enrichment ; TMT

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Principal Investigators:
(in alphabetical order) 		Steven A. Carr, Broad Institute of MIT and Harvard, United States
Submitting User: clauser
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