Palmitoylated proteins: purification and identification

Nat Protoc. 2007;2(7):1573-84. doi: 10.1038/nprot.2007.225.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acylation
  • Animals
  • Biotin / chemistry
  • Biotinylation
  • Brain Chemistry
  • Mice
  • Palmitic Acid / analysis*
  • Palmitic Acid / chemistry
  • Protein Conformation
  • Proteins / chemistry*
  • Proteins / isolation & purification*
  • Proteomics / methods*
  • Saccharomyces cerevisiae / chemistry
  • Tandem Mass Spectrometry


  • Proteins
  • Palmitic Acid
  • Biotin