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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.

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Figure 1: Workflow of the proposed approach.
Figure 2
Figure 3: Comparison of Coomassie blue staining and Nile red staining.
Figure 4: In-gel fluorescent imaging of zebrafish liver proteome.
Figure 5: Stable iFAT and quantitative analysis of C16:0 methyl esters.
Figure 6: In-gel iFAT and quantitative analysis of C16:0 moieties of the zebrafish liver proteome in response to DDT exposure.
Figure 7: Confirmation of S-fatty acylation by hydroxylamine treatment.

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Acknowledgements

We greatly appreciate the support from Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, no. IRT0953), Hubei Natural Science Foundation Council (HBNSFC, 2009CDA001), Research Funds of Central China Normal University from the Ministry of Education (120002040270) and the Research Platform of Hubei Province for Monitoring of Pesticide Residues and Agricultural Products Safety.

Author information

Authors and Affiliations

Authors

Contributions

L.D. conducted the experiments of sample preparation, gel electrophoresis, fluorescent and Coomassie blue staining, in-gel stable isotope–coded transmethylation and other related laboratory work. J.L. conducted the experiments for the development and optimization of the iFAT strategy. L.L. repeated part of this work and performed the quantification of changes in protein concentration. T.L. was involved in the development and optimization of the iFAT strategy. H.Z. developed the original concept, designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to Hongying Zhong.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Coomassie blue staining of the liver proteome of female Danio rerio upon 60 days exposure of DDT (PDF 144 kb)

Supplementary Figure 2

Mass spectra of d0- and d3- methanol derived fatty acids (PDF 118 kb)

Supplementary Figure 3

Mass spectra of d0- and d3-derived C16:0 methyl esters obtained from Danio rerio that have been raised in water containing DDT or normal water (PDF 176 kb)

Supplementary Figure 4

TIC (Total Ion Chromatograms) of gel bands (PDF 424 kb)

Supplementary Figure 5

Related total ion chromatograms (PDF 128 kb)

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Dong, L., Li, J., Li, L. et al. Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope–coded fatty acid transmethylation and mass spectrometry. Nat Protoc 6, 1377–1390 (2011). https://doi.org/10.1038/nprot.2011.358

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