Work

A Chemical Proteomics Method to Quantify Cysteine S -Acylation

Year: 2026

Type: article

Abstract: S -acylation, often referred to as S -palmitoylation, is a reversible and dynamic posttranslational modification that corresponds to the addition of a long-chain fatty acid to cysteine (Cys) residues. Established mass spectrometry-based chemoproteomics methods have improved our understanding of the S -acylation proteome, notably by identifying hundreds of S -acylated proteins, sometimes with the modified Cys. However, the precise quantification of S -acylation levels for each Cys within a single sample remains challenging at the proteome level. Quantification of S -acylation levels is critical to further our understanding of protein S -acylation in cellular function and its role in health and diseases. We report here the development of an S -acylation quantification workflow based on the sequential labeling of free Cys and S -acylated Cys with isotopic labeling reagents. The workflow was extensively optimized, notably by comparing the number of sites identified with two alkyne-tagged Cys-reactive isotopic probes and four azido-tagged biotin-based capture reagents. By integrating this enhanced workflow with high-field asymmetric waveform ion mobility spectrometry (FAIMS) on LC–MS/MS instruments for the separation of labeled peptides, over 17,000 unique Cys could be quantified in biological samples. Application of the S -acylation quantification workflow to cellular proteomes allowed for the quantification of S -acylation levels in a HeLa proteome. We also identified dynamic S -acylation changes in response to autophagy induction.

Source: ACS Chemical Biology

DOI: 10.1021/acschembio.5c00824

Authors: Chloé Freyermuth, Jean-William Dupuy, Thibaud T. Renault, Stéphane Claverol, Anne-Aurélie Raymond, Emmanuelle Thinon