A Chemical Proteomics Method to Quantify Cysteine <i>S</i> -Acylation

<i>S</i>-acylation, often referred to as <i>S</i>-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 <i>S</i>-acylation proteome, notably by identifying hundreds of <i>S</i>-acylated proteins, sometimes with the modified Cys. However, the precise quantification of <i>S</i>-acylation levels for each Cys within a single sample remains challenging at the proteome level. Quantification of <i>S</i>-acylation levels is critical to further our understanding of protein <i>S</i>-acylation in cellular function and its role in health and diseases. We report here the development of an <i>S</i>-acylation quantification workflow based on the sequential labeling of free Cys and <i>S</i>-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 <i>S</i>-acylation quantification workflow to cellular proteomes allowed for the quantification of <i>S</i>-acylation levels in a HeLa proteome. We also identified dynamic <i>S</i>-acylation changes in response to autophagy induction.