Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment.

Autor:	Rivera KD; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Olive ME; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Bergstrom EJ; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Nelson AJ; Cell Signaling Technology, Danvers, Massachusetts, USA., Lee KA; Cell Signaling Technology, Danvers, Massachusetts, USA., Satpathy S; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Carr SA; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address: scarr@broad.mit.edu., Udeshi ND; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Electronic address: udeshi@broadinstitute.org.
Jazyk:	angličtina
Zdroj:	Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2021; Vol. 20, pp. 100154. Date of Electronic Publication: 2021 Sep 27.
DOI:	10.1016/j.mcpro.2021.100154
Abstrakt:	Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites are 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-ϵ-GG peptides are enriched with anti-K-ε-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-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 μg input per sample processed in ∼2 h. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility, and significantly reduced processing time. The automated method also significantly reduced variability across process replicates compared with the manual method. 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 the method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples. Competing Interests: Conflict of interest S. A. C. is a member of the scientific advisory boards of Kymera, PTM Biolabs, and Seer and an ad hoc scientific advisor to Pfizer and Biogen. All other authors declare no competing interests. (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu