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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Smotrys, J.E. & Linder, M.E. Palmitoylation of intracellular signaling proteins: regulation and function. Annu. Rev. Biochem. 73, 559–587 (2004).
Resh, M.D. Trafficking and signaling by fatty-acylated and prenylated proteins. Nat. Chem. Biol. 2, 584–590 (2006).
Resh, M.D. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1415, 1–16 (1999).
Degtyarev, M.Y., Spiegel, A.M. & Jones, T.L.Z. Increased plamitoylation of the GS protein α subunit after activation by the β adrenergic receptor or cholera toxin. J. Biol. Chem. 268, 23769–23772 (1994).
Papac, D.I., Thornburg, K.R., Bullesbach, E.E., Crouch, R.K. & Knapp, D.R. Palmitoylation of a G protein coupled receptor. Direct analysis by tandem mass spectrometry. J. Biol. Chem. 267, 16889–16894 (1992).
Misaki, R. et al. Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis. J. Cell Biol. 191, 23–29 (2010).
Hackett, M., Guo, L., Shabanowitz, J., Hunt, D.F. & Hewlett, E.L. Internal lysine palmitoylation in adenylyl cyclase toxin from Bordetella pertussis. Science 266, 433–435 (1994).
Wei, X. et al. De Novo lipogenesis maintains vascular homeostasis through endothelial nitric-oxide synthase(eNOS) palmitoylation. J. Biol. Chem. 286, 2933–2945 (2011).
Robbins, S.M., Quintrell, N.A. & Bishop, M. Yristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae. Mol. Cell. Biol. 15, 3507–3515 (1995).
Mumby, S.M. Reversible palmitoylation of signaling proteins. Curr. Opion. Cell Biol. 9, 148–154 (1997).
Baekkeskov, S. & Kanaani, J. Palmitoylation cycles and regulation of protein function. Mol. Membr. Biol. 26, 42–54 (2009).
Hallak, H. et al. Covalent binding of arachidonate to G protein α subunits of human platelets. J. Biol. Chem. 269, 4713–4716 (1994).
Kordyukova, L.V., Serebryakova, M.V., Baratova, L.A. & Veit, M. S acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine. J. Virol. 82, 9288–9292 (2008).
Shene, J.H. & Virag, I. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein GAP-43. J. Cell Biol. 108, 613–624 (1989).
Brown, D.A. & London, E. Functions of lipid rafts in biological membranes. Annu. Rev. Cell Dev. Biol. 14, 111–136 (1998).
Rocks, O. et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752 (2005).
Resh, M.D. Membrane targeting of lipid modified signal transduction proteins. Subcell. Biochem. 37, 217–232 (2004).
Dunphy, J.T. & Linder, M.E. Signaling functions of protein palmitoylation. Biochem. Biophy. Acta 1436, 245–261 (1998).
Lobo, S., Greentree, W.K., Linder, M.E. & Deschenes, R.J. Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J. Biol. Chem. 277, 41268–41273 (2002).
Zhou, F. et al. CSS-Palm: palmitoylation site prediction with a clustering and scoring strategy. Bioinformatics 22, 894–896 (2006).
Hemsley, P.A. et al. The tip growth defective1 S-acyl transferase regulates plant cell growth in Arabidopsis. Plant Cell 17, 2554–2563 (2005).
Berthiaume, L. et al. Synthesis and use of iodo-fatty acid analogs. Methods Enzymol. 250, 454–466 (1995).
Drisdel, R.C. & Green, W.N. Labeling and quantifying sites of protein palmitoylation. Biotechniques 36, 276–285 (2004).
Roth, A.F. et al. Global analysis of protein palmitoylation in yeast. Cell 125, 1003–1013 (2006).
Martin, B.R. & Cravatt, B.F. Large-scale profiling of protein palmitoylation in mammalian cells. Nat. Methods 6, 135–138 (2009).
Wan, J., Roth, A.F., Bailey, A.O. & Davis, N.G. Palmitoylated proteins: purification and identification. Nat. Protoc. 2, 1573–1584 (2007).
Zhang, J. et al. Identification of CKAP4/p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics method. Mol. Cell. Proteomics 7, 1378–1388 (2008).
Hang, H.C. et al. Chemical probes for the rapid detection of fatty-acylated proteins in mammalian cells. J. Am. Chem. Soc. 129, 2744–2745 (2007).
Hannoush, R.N. & Arenas-Ramirez, N. Imaging the lipidome: ω-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins. Chem. Biol. 4, 581–587 (2009).
Charron, G. et al. Robust fluorescent detection of protein fatty-acylation with chemical reporters. J. Am. Chem. Soc. 131, 4967–4975 (2009).
Yount, J.S. et al. Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3. Nat. Chem. Biol. 6, 610–614 (2010).
Zhong, H., Marcus, S. & Li, L. Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification. J. Am. Soc. Mass Spectrom. 16, 471–481 (2005).
Coutre, J.L. et al. Proteomics on full-length membrane proteins using mass spectrometry. Biochem. 39, 4237–4242 (2000).
Wu, C.C., MacCoss, M.J., Howell, K.E. & Yates III, J.R. A method for the comprehensive proteomics analysis of membrane proteins. Nat. Biotechnol. 21, 532–538 (2003).
Reid, G.E. & McLuckey, S.A. 'Top-down' protein characterization via mass spectrometry. J. Mass Spectrom. 37, 663–675 (2002).
Bogdanov, B. & Smith, R.D. Proteomics by FTICR mass spectrometry: top down and bottom up. Mass Spectrom. Rev. 24, 168–200 (2004).
Zhong, H., Zhang, Y., Wen, Z. & Li, L. Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis. Nat. Biotechnol. 22, 1291–1296 (2004).
Lu, B., McClatchy, D.B., Kim, J.Y. & Yates III, J.R. Strategies for shotgun identification of integral membrane proteins by tandem mass spectrometry. Proteomics 8, 3947–3955 (2008).
Issaq, H.J. The role of separation in proteomics research. Electrophoresis 22, 3629–3636 (2001).
Zhong, H., Marcus, S. & Li, L. Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography by MALDI MS/MS for protein identification. J. Am. Soc. Mass Spectrom. 16, 471–481 (2004).
Roth, A.F., Wan, J., Green, W.N., Yates III, J.R. & Davis, N.G. Proteomic identification of palmitoylated proteins. Methods 40, 135–142 (2006).
Daban, J.R., Samso, M. & Bartolome, S. Use of Nile red as a fluorescent probe for the study of the hydrophobic properties of protein-sodium dodecyl sulfate complexes in solution. Anal. Bioanal. 199, 162–168 (1991).
Daban, J.R., Bartolome, S. & Samso, M. Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels. Anal. Bioanal. 199, 169–174 (1991).
Bermudez, A., Daban, J.R., Garcia, J.R. & Mendez, E. Direct blotting, sequencing and immonodetection of proteins after five-minute staining of SDS and SDS-treated IEF gels with Nile red. Biotechniques 16, 621–624 (1994).
Samso, M., Daban, J.R., Hansen, S. & Jones, G.R. Evidence for sodium dodecyl sulfate/protein complexes adopting a necklace structure. Eur. J. Biochem. 232, 818–824 (1995).
Li, J., Yue, Y., Hu, X. & Zhong, H. Rapid transmethylation and stable isotope labeling for comparative analysis of fatty acids by mass spectrometry. Anal. Chem. 81, 5080–5087 (2009).
Li, J., Yue, Y., Li, T. & Zhong, H. Gas chromatography-mass spectrometric analysis of bonded long chain fatty acids in a single zebrafish egg by ultrasound-assisted one-step transmethylation and extraction. Anal. Chim. Acta. 650, 221–226 (2009).
Reinders, J. & Sickmann, A. Modificomics: Posttranslational modifications beyond protein phosphorylation and glycosylation. Biomol. Eng. 24, 169–177 (2007).
Hill, A.J. & Teraoka, H. Zebrafish as a model vertebrate for investigate chemical toxicity. Toxicol. Sci. 86, 6–19 (2005).
Weis, P. Ultrastructural changes induced by low concentrations of DDT in the livers of zebrafish and the guppy. Chemico-Biol. Inter. 8, 25–30 (1974).
Tinsley, I.J. & Lowry, R.R. An interaction of DDT in the metabolism of essential fatty acids. Lipids 7, 182–185 (1972).
Li, T. et al. Typing of unknown microorganisms based on quantitative analysis of fatty acids by mass spectrometry and hierarchical clustering. Anal. Chim. Acta 684, 8–16 (2011).
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
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
Ethics declarations
Competing interests
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)
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2011.358
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
