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Lipid-specific metabolic labelling reveals site-specific heterogeneity in long-chain S-acylation

ABSTRACT: Long-chain S-acylation is a post-translational modification that regulates key cellular processes, including signal transduction and metabolic regulation. However, the dynamic nature, and site- and lipid-specific patterns of long-chain protein acylation remain poorly understood. Site- and lipid-specific metabolic labelling with various ω-alkynyl fatty acids uncovered the site-specific heterogeneity of long-chain protein S-acylation. Cells use various fatty acids for long-chain S-acylation, including C16:0, C18:0, and C18:1 on cysteines, while N-myristoylation preferentially incorporates C14:0 on N-terminal glycine residues. Our results demonstrate that long-chain S-acylation sites can exhibit both lipid heterogeneity and specificity and reveal that both enzymatic specificity and metabolic context can influence fatty acid incorporation. Exploration of the dy-namic protein long-chain S-acylation uncovered that acyl-protein thioesterases targeted by Pal-mostatin B regulate long-chain S-acylation involving various lipids, including C16:0, C18:0, and C18:1. Our approach represents a significant advancement in lipid metabolic labelling methodol-ogies, offering enhanced efficiency and sensitivity for studying S-acylation dynamics with lipid- and site-specificity.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Anneroos Nederstigt

LAB HEAD: Marc Pieter

PROVIDER: PXD066568 | Pride | 2026-06-30

REPOSITORIES: Pride

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Project description:Long-chain S-acylation is the addition of long-chain fatty acids to cysteine residues on proteins. This lipid modification is essential for protein membrane association and signalling but presents analytical challenges due to both its hydrophobicity and the labile nature of thioester bonds. We developed and optimised a bottom-up mass spectrometry workflow tailored for the detection of long-chain S-acylated peptides. Following liquid chromatography optimisation for improved separation and elution of long-chain S-acylated peptides from a C18 stationary phase, we investigated thioester stability under typical proteomics sample preparation conditions, including variations in pH, reducing agents, and trypsin digestion. Stability analyses revealed that long-chain S-acylated peptides were generally resistant to pH variations and reducing agents, while extended digestion times resulted in a loss of signal from some peptides. For MS/MS analysis, CID, HCD and ETD were applied to analyse long-chain S-acylated peptides. Neutral losses of the modification were observed with all these fragmentation methods. However, HCD proved to be the most effective, as the fragment ions resulting from the neutral losses provided sequence information, unlike those from CID and ETD. Applying this workflow to HEK293T cells overexpressing the long-chain S-acylated proteins GNA13 and RhoB, we detected dual acylation states of GNA13 and observed both long-chain S-acylation and prenylation on RhoB. Our optimised analytical strategy facilitates the identification and analysis of long-chain S-acylation on proteins without the need for chemical derivatization by alkyne-tagged probes or acyl-biotin exchange. Although recombinant overexpression of the long-chain S-acylated proteins was still required for long-chain S-acylation detection, this direct analysis strategy for protein long-chain S-acylation enables the study of lipid modifications with lipid-specific resolution, laying a foundation for deeper insights into the regulatory roles of these hydrophobic modifications in protein function and cellular signalling.

			Action	DRS
	1A_2h-alk16-R1.raw	Raw
	1A_Alk14_R1.raw	Raw
	1B_2h-alk16-R2.raw	Raw
	1B_Alk14_R2.raw	Raw
	1C_2h-alk16-R3.raw	Raw

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Lipid-specific metabolic labelling reveals site-specific heterogeneity in long-chain S-acylation

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