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.

Profiling the proteome-wide selectivity of diverse electrophiles

“…2,3 We have recently built on this platform by developing isotopically labelled desthiobiotin azide (isoDTB) tags to streamline the experimental procedure. 4 In the underlying isoDTB-ABPP workflow 4 (Fig. 1a), two samples of a proteome-of-interest are treated with a covalent ligand or the corresponding solvent as a control.…”

“…a, Workflow for competitive, residue-specific chemoproteomic experiments using the isoDTB-ABPP workflow. 4 RG = reactive group; D = desthiobiotin. b, Unbiased workflow to comprehensively investigate electrophile reactivity in the proteome using the MSFragger-based FragPipe computational platform.…”

Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries

“…Chemically cleavable biotin linkers have been utilized to isolate conjugated cysteine-containing peptides, bypassing on-bead digestion [23][24][25][26] . In addition, desthiobiotin-containing enrichment probes have recently been shown to be more efficient at recovering conjugated peptides from avidin, simplifying workflows [27][28][29] . To address throughput, tandem mass tags (TMT), which can quantify 11 samples simultaneously, have been reported 26,30-32 but are not yet fully optimized for profiling of reactive cysteine sites.…”

AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins

“…However, perhaps due to the low sequence coverage (53%) it did not lead to identification of the modification site. In order to specifically enrich only the modified peptides from lysates, we performed enrichment using the desthiobiotin linker, which allows one to selectively elute modified peptides after the enrichment and tryptic digestion ( Zanon et al., 2020 ). Unfortunately, spectra analysis did not lead to identification of either PLD3 or any other site including T518 on the HSPA5.…”