PXD057394

PXD057394 is an original dataset announced via ProteomeXchange.

Title	A mass spectrometry-based proteomics strategy to detect long-chain S-acylated peptides
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.
HostingRepository	PRIDE
AnnounceDate	2025-09-22
AnnouncementXML	Submission_2025-09-22_09:14:33.417.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Samiksha Sardana
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	farnesylated residue; palmitoylated residue; geranylgeranylated residue
Instrument	Orbitrap Fusion Lumos; Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2024-10-31 06:52:22	ID requested
⏵ 1	2025-09-22 09:14:33	announced

Dataset with its publication pending

submitter keyword: liquid chromatography, hydrophobicity, thioester stability, tandem mass spectrometry,Long-chain S-acylation

Marc Pieter
contact affiliation	Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, Utrecht 3584 CH The Netherlands Netherlands Proteomics Center, Padualaan 8, Utrecht 3584 CH, The Netherlands
contact email	m.p.baggelaar@uu.nl
lab head
Samiksha Sardana
contact affiliation	Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, Utrecht 3584 CH, The Netherlands Netherlands Proteomics Center, Padualaan 8, Utrecht 3584 CH, The Netherlands
contact email	s.sardana@uu.nl
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/09/PXD057394

PRIDE project URI

• PRIDE
  1. PXD057394
     1. Label: PRIDE project
     2. Name: A mass spectrometry-based proteomics strategy to detect long-chain S-acylated peptides