Dataset Information

Traceless peptide and protein modification via rational tuning of halopyridiniums

ABSTRACT: Building on the insights gained from systematically investigating of aromatic nucleophilic substitution (SNAr) and thiol-exchange, we identified the highly electron-deficient halopyridiniums as a tunable and predictable reaction platform for adjusting electrophilicity and thiol-cleavability by following a positive Hammett correlation. This pyridinium-based platform was practical for both cleavable peptide stapling and protein functionalization. With this highly tunable and predictable toolkit, we successfully achieved GSH-response cleavable peptide stapling, antibody conjugation, enzyme masking/de-masking, and proteome labeling. Furthermore, the positively charged species exhibit improved cell-permeability and digestion-stability, making them attractive candidates for the manipulation of the functions and biophysical properties of biomolecules.

ORGANISM(S): Homo Sapiens

SUBMITTER: Rui Wang

PROVIDER: PXD045683 | iProX | Mon Sep 25 00:00:00 GMT+01:00 2023

REPOSITORIES: iProX

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Traceless Peptide and Protein Modification via Rational Tuning of Pyridiniums.

Wan Chuan C Zhang Yichi Y Wang Jinpeng J Xing Yun Y Yang Dongyan D Luo Qinhong Q Liu Jianbo J Ye Yuxin Y Liu Zhihong Z Yin Feng F Wang Rui R Li Zigang Z

Journal of the American Chemical Society 20240119 4

Herein, we report a versatile reaction platform for tracelessly cleavable cysteine-selective peptide/protein modification. This platform offers highly tunable and predictable conjugation and cleavage by rationally estimating the electron effect on the nucleophilic halopyridiniums. Cleavable peptide stapling, antibody conjugation, enzyme masking/de-masking, and proteome labeling were achieved based on this facile pyridinium-thiol-exchange protocol. ...[more]

PMID: 38239111

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Project description:Cysteine is unique among all protein-coding amino acids owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by both a broad range of redox mechanisms and various electrophiles derived from exogenous or endogenous sources. Measuring proteomic cysteines response to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling (ABPP) based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed as QTRP (Quantitative Thiol Reactivity Profiling), that relies on the ability of a commercially available thiol-reactive probe IPM to covalently label, enrich, and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g. control vs treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics, (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also outline here how to use QTRP for measuring the changes on proteomic cysteines upon redox perturbation and for identifying targeted cysteine sites of thiol-reactive electrophiles. We anticipate that this protocol should find broad applications in both redox/chemical biology and drug discovery. The protocol for sample preparation takes 3 days, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

Dataset Information

Traceless peptide and protein modification via rational tuning of halopyridiniums

Publications

Traceless Peptide and Protein Modification via Rational Tuning of Pyridiniums.

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