Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry

Megan C. Yap, Morris A. Kostiuk, Dale D O Martin, Maneka A. Perinpanayagam, Pieter G. Hak, Anjaiah Siddam, Janaki R. Majjigapu, Gurram Rajaiah, Bernd O. Keller, Jennifer A. Prescher, Peng Wu, Carolyn R. Bertozzi, J R Falck, Luc G. Berthiaume

Research output: Contribution to journalArticlepeer-review

88 Scopus citations


Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic ω-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3- methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fl uorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, ω-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with ω-alkynyl-myristate or ω-alkynyl-stearate, via an alkali sensitive thioester bond. Third, ω-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of ω-alkynylpalmitate and ω-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with ω-alkynyl- palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using ω-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The ω-alkynylmyristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.

Original languageEnglish (US)
Pages (from-to)1566-1580
Number of pages15
JournalJournal of lipid research
Issue number6
StatePublished - Jun 1 2010


  • Enzymes
  • Membranes
  • Mitochondria
  • Myristoylation
  • Palmitoylation

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology


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