Target and identify: triazene linker helps identify azidation sites of labelled proteins via click and cleave strategy

Abstract

A method for identifying probe modification of proteins via tandem mass spectrometry was developed. Azide bearing molecules are immobilized on functionalised sepharose beads via copper catalysed Huisgen-type click chemistry and selectively released under acidic conditions by chemical cleavage of the triazene linkage. We applied this method to identify the modification site of targeted-diazotransfer on BirA.

Highly Stable Copper(I)-Based Metal–Organic Framework Assembled with Resorcin[4]arene and Polyoxometalate for Efficient Heterogeneous Catalysis of Azide–Alkyne “Click” Reaction

“…Copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC), as a powerful one-pot “click” reaction, has attracted considerable attention and found potential applications in various research fields such as materials science, organic synthesis, medicinal chemistry, and chemical biology. − Thus, far, a large number of copper­(I) catalysts have been explored for the AAC reaction, including simple Cu­(I) salts, organometallic Cu­(I) precatalysts, and nanostructured copper­(I) catalysts et al − Among them, homogeneous copper­(I) catalysts have been widely developed to catalyze AAC reactions. , Nevertheless, in view of environment and economics, homogeneous catalysts may easily be destroyed during the catalytic reaction and not be easily recovered after the catalytic reaction for recycling use . In contrast, the utilization of heterogeneous copper­(I) catalysts can offer several advantages, for example, high reusability, easy catalyst/product separation, high catalytic performance, and improved stability. − In this regard, Cu­(I) species coating onto various supports have been well explored as heterogeneous catalysts for the CuAAC reactions. , However, metal–organic frameworks (MOFs), especially polyoxometalates (POMs)-based Cu­(I)-MOFs, were rarely employed as promising heterogeneous catalysts for the copper­(I)-catalyzed “‘click’” reactions …”

Unbiased Mass Spectrometry Elucidation of the Targets and Mechanisms of Activity-Based Probes: A Case Study Involving Sulfonyl Fluorides

“…9 The primary endpoint assay coupled to ABPP workflows is liquid chromatography tandem mass spectrometry (LC-MS/MS) since it provides unbiased identification of the protein targets and can, at least in principle, identify the specific residue associated with the ABP interaction based upon the mass shift of the probe. Despite recent advancements in the development of cleavable linkers for selective enrichment, 10 the relative number of unambiguous MS/MS identifications of ABP/peptide adducts reported in this field is disproportionately low. [11][12][13][14][15] A possible explanation for the general lack of ABP/peptide MS/MS annotations is that the adduct may undergo further chemical modification in the gas phase and/or during sample processing, resulting in an unanticipated change in its total mass.…”

“…The primary end point assay coupled to ABPP workflows is liquid chromatography–tandem mass spectrometry (LC-MS/MS) since it provides unbiased identification of the protein targets and can, at least in principle, identify the specific residue associated with the ABP interaction based upon the mass shift of the probe. Despite recent advancements in the development of cleavable linkers for selective enrichment, the relative number of unambiguous MS/MS identifications of ABP/peptide adducts reported in this field is disproportionately low. − A possible explanation for the general lack of ABP/peptide MS/MS annotations is that the adduct may undergo further chemical modification in the gas phase and/or during sample processing, resulting in an unanticipated change in its total mass. , This represents a major hurdle in the analysis of mass spectrometry data, as peptide identification algorithms are reliant upon some form of a priori knowledge regarding what protein modifications are expected to be present, the amino acids they target, and their exact elemental composition. Recent studies utilizing mass-tolerant search strategies have highlighted that more than half of the MS/MS spectra collected in a standard shotgun proteomics experiment remain unannotated, partly owing to these search constraints.…”

An in situ combinatorial methodology to synthesize and screen chemical probes

“…This essential biotin ligase has emerged as a target for the development of antibacterial agents 10 and we recently showed that a biotin-diazotransfer reagent modifies purified BirA. 11 Unfortunately, this reagent did not label BirA efficiently in more complex samples. To identify new probe leads, we derivatized biotin ligands L1, L4 and L5 with reactive groups R1-R3 and R5-R9 and incubated cell lysate of BirA-overexpressing E. coli with 22.2 mM of the resulting probes for 2 hours.…”