Palmitoylated proteins: purification and identification.
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.
Wan
Junmei
J
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Roth
Amy F
AF
Bailey
Aaron O
AO
Davis
Nicholas G
NG
D000215:Acylation; D000818:Animals; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D001923:Brain Chemistry; D051379:Mice; D019308:Palmitic Acid / Q000032:analysis* / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D053719:Tandem Mass Spectrometry
D011506:Proteins; D019308:Palmitic Acid; D001710:Biotin