title: Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope-coded fatty acid transmethylation and mass spectrometry.
abstract: Covalent attachment of palmitic acid or other fatty acids to the thiol groups of cysteine residues of proteins through reversible thioester bonds has an important role in the regulation of diverse biological processes. We describe here the development of a mass spectrometry protocol based on stable isotope-coded fatty acid transmethylation (iFAT) for qualitative and comparative analysis of protein S-fatty acylation under different experimental conditions. In this approach, cellular proteins extracted from different cell states are separated by SDS-PAGE and then the gel is stained with either Coomassie blue or Nile red for improved sensitivity. Protein bands are excised and then an in-gel stable iFAT procedure is performed. The fatty acid methyl esters resulting from derivatization with d0- and d3-methanol are identified by mass spectrometry. By measuring the intensities of labeled and unlabeled fragment ion pairs of fatty acid methyl esters, the levels of S-fatty acylation in different cells or tissues can be compared. This approach has been applied to monitor the changes of S-fatty acylation of zebrafish liver proteome in response to environmental dichlorodiphenyltrichloroethane exposure. Compared with the approach using metabolic incorporation of radioactive fatty acid analogs, it is not only simple and effective but also eliminates the hazards of handling radioactive isotopes.
authors: Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei, China.
authors: Linjie
authors: L
authors: Dong
authors: Jianjian
authors: J
authors: Li
authors: Lun
authors: L
authors: Li
authors: Tingting
authors: T
authors: Li
authors: Hongying
authors: H
authors: Zhong
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004952:Esters / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D020543:Proteome; D014240:Trichloroethanes / Q000633:toxicity; D029961:Zebrafish Proteins / Q000737:chemistry*
chemical_list: D004952:Esters; D005227:Fatty Acids; D020543:Proteome; D014240:Trichloroethanes; D029961:Zebrafish Proteins