Dataset Information

Direct identification of protein S-palmitoylation sites

ABSTRACT: S-fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human disease. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterization of endogenously attached fatty acids is still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Emmanuelle Thinon

LAB HEAD: Howard C Hang

PROVIDER: PXD008399 | Pride | 2018-03-28

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	MS173350QEP_Emmanuelle_Hang_T.raw	Raw
	MS173350QEP_Emmanuelle_Hang_T_IOAC.raw	Raw
	MS173539QEp_Emmanuelle_Hang_C.raw	Raw
	MS173539QEp_Emmanuelle_Hang_C_repeat.raw	Raw
	MS173539QEp_Emmanuelle_Hang_DC.raw	Raw

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Publications

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

Thinon Emmanuelle E Fernandez Joseph P JP Molina Henrik H Hang Howard C HC

Journal of proteome research 20180406 5

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification a ...[more]

PMID: 29575903

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Project description:Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate most physio-pathological states. Here, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by enrichment with titanium dioxide (TiO2) and mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to Hela cells lysate, achieving high specificity and enrichment efficiency. In particular,From 500µg of Hela cell lysate, the method led to the identification of 9229 unique Cys peptides starting from 500µg of Hela cell lysate . the CysPAT labelled Cys peptides showed increased hydrophobicity. Finally, the method was further developed to simultaneously enrich Cys peptides and phosphorylated peptidesThe method was further developed to simultaneously enrich Cys peptides and phosphorylated peptides from SILAC labelled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, of 22972 unique reversibly modified Cys peptides (3886 proteins) and 18549 unique phosphopeptides (2257 proteins) were simultaneously identified by Orbitrap Fusion. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins interacted with EGF receptor (EGFR)-binding proteins and proteins involved in EGFR signaling pathway. EGF stimulation can activate the phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), meanwhile, it also leads to the activation of multiple protein tyrosine phosphatases (PTPs) by reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif, Llater the activated PTPs would dephosphorylate and inactivate the molecules activated by EGF stimulation. Overall, the CysPAT strategy is a promising tool for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced cysteine modifications.