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Palmitoylated proteins: purification and identification

Nature Protocols volume 2, pages 1573–1584 (2007)Cite this article

Abstract

This proteomic protocol purifies and identifies palmitoylated proteins (i.e., S-acylated proteins) from complex protein extracts. The method relies on an acyl-biotinyl exchange chemistry in which biotin moieties are substituted for the thioester-linked protein acyl-modifications through a sequence of three in vitro chemical steps: (i) blockade of free thiols with N-ethylmaleimide; (ii) cleavage of the Cys-palmitoyl thioester linkages with hydroxylamine; and (iii) labeling of thiols, newly exposed by the hydroxylamine, with biotin–HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide. The biotinylated proteins are then affinity-purified using streptavidin–agarose and identified by multi-dimensional protein identification technology (MuDPIT), a high-throughput, tandem mass spectrometry (MS/MS)–based proteomic technology. MuDPIT also affords a semi-quantitative analysis that may be used to assess the gross changes induced to the global palmitoylation profile by mutation or drugs. Typically, 2–3 weeks are required for this analysis.

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Figure 1: Schematic of the proteomic acyl-biotinyl exchange methodology.
Figure 2: SDS–polyacrylamide gel electrophoresis (SDS-PAGE) of purified parallel plus- and minus-hydroxylamine (HA) yeast samples just before proteomic multi-dimensional protein identification technology (MuDPIT) analysis.
Figure 3: An x, y-scatter plot depiction of yeast proteins identified by multi-dimensional protein identification technology (MuDPIT) of plus- and minus-hydroxylamine (HA) samples.
Figure 4: Colorimetric depiction of palmitoylation changes seen in strains deficient for the different yeast DHHC protein acyl transferases (PATs).
Figure 5: Example spectral count analysis.

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Acknowledgements

We thank John R. Yates 3rd (The Scripps Research Institute, La Jolla, CA) for his help in the original development of this protocol. The MuDPIT proteomic technology developed by his laboratory proved indispensable to this analysis in terms of its high capacity and the facile quantitative analysis that it affords. The development of this technology was supported by NIH GM065525 (N.G.D.) and NIH RR11823 (John R. Yates 3rd).

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  1. Aaron O Bailey

    Present address: Present address: Program in Cell and Developmental Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.,

Authors and Affiliations

  1. Department of Pharmacology, Wayne State University School of Medicine, Detroit, 48201, Michigan, USA

    Junmei Wan, Amy F Roth & Nicholas G Davis

  2. Department of Cell Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Aaron O Bailey

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Correspondence to Nicholas G Davis.

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Wan, J., Roth, A., Bailey, A. et al. Palmitoylated proteins: purification and identification. Nat Protoc 2, 1573–1584 (2007). https://doi.org/10.1038/nprot.2007.225

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