A Chemical Proteomics Method to Quantify Cysteine S-Acylation

Area of Science:

• Biochemistry
• Proteomics
• Cellular Biology

Background:

• S-acylation (S-palmitoylation) is a reversible posttranslational modification involving fatty acid addition to cysteine residues.
• While mass spectrometry has identified many S-acylated proteins, precise quantification of S-acylation levels at the proteome-wide scale remains challenging.
• Accurate quantification is vital for understanding S-acylation's role in cellular processes, health, and disease.

Purpose of the Study:

• To develop and optimize a robust workflow for the precise, quantitative analysis of S-acylation across the proteome.
• To enable the quantification of S-acylation levels for individual cysteine residues within biological samples.
• To apply the developed workflow to investigate dynamic S-acylation changes in response to cellular stimuli.

Main Methods:

• Developed a quantitative workflow utilizing sequential labeling of free and S-acylated cysteine residues with isotopic reagents.
• Optimized the workflow by comparing alkyne-tagged probes and azido-tagged capture reagents for site identification.
• Integrated the workflow with high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled with LC-MS/MS for peptide separation.

Main Results:

• Quantified over 17,000 unique cysteine residues in biological samples using the enhanced workflow.
• Successfully applied the workflow to quantify S-acylation levels in a HeLa cell proteome.
• Identified dynamic alterations in S-acylation in response to autophagy induction.

Conclusions:

• The developed S-acylation quantification workflow significantly advances the ability to measure S-acylation levels proteome-wide.
• This method provides a powerful tool for dissecting the functional roles of S-acylation in cellular signaling and disease pathogenesis.
• The workflow revealed dynamic S-acylation changes associated with autophagy, highlighting its utility in studying dynamic posttranslational modifications.