Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g. cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate, for the first time, more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction, and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Tuning
reactivity of sulfur electrophiles is key for advancing
click chemistry and chemical probe discovery. To date, activation
of the sulfur electrophile for protein modification has been ascribed
principally to stabilization of a fluoride leaving group (LG) in covalent
reactions of sulfonyl fluorides and arylfluorosulfates. We recently
introduced sulfur–triazole exchange (SuTEx) chemistry to demonstrate
the triazole as an effective LG for activating nucleophilic substitution
reactions on tyrosine sites of proteins. Here, we probed tunability
of SuTEx for fragment-based ligand discovery by modifying the adduct
group (AG) and LG with functional groups of differing electron-donating
and -withdrawing properties. We discovered the sulfur electrophile
is highly sensitive to the position of modification (AG versus LG),
which enabled both coarse and fine adjustments in solution and proteome
activity. We applied these reactivity principles to identify a large
fraction of tyrosine sites (∼30%) on proteins (∼44%)
that can be liganded across >1500 probe-modified sites quantified
by chemical proteomics. Our proteomic studies identified noncatalytic
tyrosine and phosphotyrosine sites that can be liganded by SuTEx fragments
with site specificity in lysates and live cells to disrupt protein
function. Collectively, we describe SuTEx as a versatile covalent
chemistry with broad applications for chemical proteomics and protein
ligand discovery.
Sulfonyl-triazole probes modified with a kinase recognition element are developed for live cell activity-based profiling to identify tyrosine sites located in catalytic and regulatory domains that are important for kinase function.
This review discusses the development of sulfonyl–triazoles and highlights the merits and opportunities for deploying this sulfur electrophile for biological discovery.
With continuously increasing scale and depth of coverage in affinity proteomics (AP-MS) data, the analysis and visualization is becoming more challenging. A number of tools have been developed to identify high confidence interactions; however, a cohesive and intuitive pipeline for analysis and visualization is still needed. Here, we present Automated Processing of SAINT Templated Layouts (APOSTL), a freely available, Galaxy-integrated software suite and analysis pipeline for reproducible, interactive analysis of AP-MS data. APOSTL contains a number of tools woven together using Galaxy workflows, which are intuitive for the user to move from raw data to publication-quality figures within a single interface. APOSTL is an evolving software project with the potential to customize individual analyses with additional Galaxy tools and widgets using the R web application framework, Shiny. The source code, data and documentation are freely available from GitHub (https://github.com/bornea/APOSTL) and other sources.
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