Exploring long-chain S-acylation with mass spectrometry strategies

Protein long-chain S-acylation is a reversible lipid modification involving the covalent attachment of fatty acids to cysteine residues, enhancing protein hydrophobicity. This modification influences protein localisation, stability, interactions, and activity. Unlike other lipid modifications, S-acylation is uniquely dynamic, regulated by the interplay between protein acyltransferases and acyl protein thioesterases. It plays essential roles in cardiac function, immune regulation, and synaptic plasticity. Dysregulation of this modification has been linked to various diseases, including autoimmune disorders, cancer, cardiovascular dysfunction, and neurodegeneration. Consequently, elucidating the mechanisms and dynamics of long-chain S-acylation provides crucial opportunities for identifying novel therapeutic targets and disease biomarkers. Despite its importance, studying S-acylation remains analytically challenging. The lack of a conserved sequence motif complicates prediction, while the hydrophobicity of modified peptides poses difficulties for standard analytical workflows. Nevertheless, LC-MS-based proteomics combined with chemical biology strategies such as acyl-biotin exchange (ABE) and lipid metabolic labelling (LML) has emerged as a powerful platform for studying S-acylation. These indirect enrichment approaches enable large-scale mapping of S-acylated proteins, yet they present limitations in resolving modification sites and determining the specific lipid species involved. This thesis aims to overcome these limitations by: (1) enhancing the assignment of long-chain S-acylated proteins using the complementary chemical biology tools, and (2) refining detection strategies to achieve site- and lipid-specific characterisation, thereby enabling a dynamic and comprehensive understanding of long-chain S-acylation in both physiological and pathological contexts. Chapter 1 introduces the biological and regulatory principles of long-chain S-acylation and outlines current MS-based strategies for its analysis, including indirect enrichment and direct detection methods. Chapter 2 investigates S-acylation during retinoic acid-induced differentiation of SH-SY5Y cells. By optimising ABE with an enhanced double-blocking protocol and refining LML through selective hydroxylamine treatment, a robust and reproducible workflow was established. Integration of both methods identified 2,002 S-acylated proteins, with 650 confirmed by both techniques. Many were associated with neuronal differentiation, highlighting a potential regulatory role of S-acylation in neurodevelopment. Chapter 3 applies S-acylproteomics to post-mortem brain tissue from multiple sclerosis (MS) donors. While conventional proteomics revealed broad disease signatures, ABE-based S-acylproteomics uncovered an enrichment of immune-regulatory proteins, particularly those involved in T cell activation. These findings reveal an underappreciated role of long-chain S-acylation in neuroinflammatory mechanisms. Chapter 4 presents a lipid- and site-resolved metabolic labelling strategy using ω-alkynyl fatty acids (Alk-14, Alk-16, Alk-18, and Alk-18:1) to trace site-specific fatty acid incorporation and turnover. This approach revealed heterogeneity in chain length specificity and thioesterase-mediated turnover, providing time-resolved insights into the dynamics of S-acylation. Chapter 5 focuses on direct detection of endogenous S-acylated peptides. Synthetic S-palmitoylated peptides were used to optimise chromatographic behaviour, fragmentation efficiency, and stability under proteomics conditions. The improved workflow enabled direct detection of lipidation states in the proteins GNA13 and RhoB, uncovering unexpected lipid heterogeneity. Collectively, this work advances both the methodological and biological understanding of long-chain S-acylation. By improving detection sensitivity, lipid specificity, and site resolution, it establishes a foundation for future investigations into the regulation, dynamics, and dysfunction of this essential lipid modification in health and disease.