Visualization and Identification of Fatty Acylated Proteins Using Chemical Reporters.
Protein fatty-acylation is the covalent addition of a lipid chain at specific amino acids. This modification changes the inherent hydrophobicity of a protein, often targeting it to cellular membrane compartments. Acylation may also regulate protein activity, stability, and protein-protein interactions. Its study is therefore critical to understanding the biology of the hundreds of proteins described to be lipid-modified, as well as those that are continually being discovered. Fatty-acylation can be analyzed using chemical reporters that mimic natural lipids and contain bioorthogonal chemical handles allowing them to be reacted with detection tags such as fluorophores or affinity tags. Our laboratory has successfully utilized alkynyl-chemical reporters of protein myristoylation, S-palmitoylation, prenylation and acetylation. Protocol 1 describes metabolic incorporation of these chemical reporters onto proteins in living cells. Protocol 2 describes the global visualization of reporter-labeled proteins by selectively reacting alkyne-containing chemical reporter-labeled proteins in cell lysates with azido-rhodamine via the click chemistry and fluorescence gel scanning. Protocol 3 describes analysis of protein acylation on individual candidate proteins using immunoprecipitation, click chemistry and fluorescence gel scanning. Finally, Protocol 4 allows identification of novel fatty acylated proteins by reacting chemical reporter-labeled proteins with azido-biotin via click chemistry and selective retrieval using streptavidin beads. This may be particularly valuable for the examination of S-palmitoylomes in different cell types or activation states, as these modifications do not occur on readily predicted consensus amino acid motifs. Overall, these techniques provide robust, non-radioactive methods for examining the acylation states of full cellular proteomes and individual proteins of interest.
Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, 1230 York Avenue, New York, NY 10065.
Jacob S
JS
Yount
Mingzi M
MM
Zhang
Howard C
HC
Hang