Document Detail


Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope-coded fatty acid transmethylation and mass spectrometry.
MedLine Citation:
PMID:  21886103     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
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:
Linjie Dong; Jianjian Li; Lun Li; Tingting Li; Hongying Zhong
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Publication Detail:
Type:  Journal Article     Date:  2011-08-18
Journal Detail:
Title:  Nature protocols     Volume:  6     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2011  
Date Detail:
Created Date:  2011-09-02     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  1377-90     Citation Subset:  IM    
Affiliation:
1] Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei, China. [2].
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