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Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome.

ABSTRACT: Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Described here are easily synthesized isotopically labeled desthiobiotin azide (isoDTB) tags that shortened the chemoproteomic workflow and allowed an increased coverage of cysteines in bacterial systems. They were used to quantify 59?% of all cysteines in essential proteins in Staphylococcus aureus and enabled the discovery of 88 cysteines that showed high reactivity, which correlates with functional importance. Furthermore, 268 cysteines that are engaged by covalent ligands were identified. Inhibition of HMG-CoA synthase was verified and will allow addressing the bacterial mevalonate pathway through a new target. Overall, a broad map of the bacterial cysteinome was obtained, which will facilitate the development of antibiotics with novel modes-of-action.

SUBMITTER: Zanon PRA

PROVIDER: S-EPMC7027453 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Project description:Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-?-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard.

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Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome.

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