ArticleMethods in molecular biology (Clifton, N.J.)2025
Activity-Based Profiling of Reactive Nucleophilic Cysteine Sites from Tissue Proteomes for Drug Discovery Applications.
Article in Methods in molecular biology (Clifton, N.J.), 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Identifying engaged nucleophilic sites in a proteome-wide manner has become a powerful means to leverage activity-based protein profiling toward assessing the target landscape of covalent compounds ranging from fragments to approved drugs. In this chapter, we provide a detailed protocol for reactive cysteine profiling from tissue lysates using a low-input high throughput-compatible workflow based on multiplexing via TMT-16pro. We apply this method to a representative biological context, human heart tissue lysates treated with highly reactive and promiscuous electrophilic scout fragments. We detect over 11,000 cysteine sites and multiple engaged ligandable sites specific to each scout fragment, revealing the quantitative accuracy of this method. Due to the low input material requirements, this protocol can be applied to many different contexts relevant to drug discovery, including the profiling of tissues from animal models after in vivo treatment with covalent drugs.
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