title: Robust fluorescent detection of protein fatty-acylation with chemical reporters.
abstract: Fatty-acylation of proteins in eukaryotes is associated with many fundamental cellular processes but has been challenging to study due to limited tools for rapid and robust detection of protein fatty-acylation in cells. The development of azido-fatty acids enabled the nonradioactive detection of fatty-acylated proteins in mammalian cells using the Staudinger ligation and biotinylated phosphine reagents. However, the visualization of protein fatty-acylation with streptavidin blotting is highly variable and not ideal for robust detection of fatty-acylated proteins. Here we report the development of alkynyl-fatty acid chemical reporters and improved bioorthogonal labeling conditions using the Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition that enables specific and sensitive fluorescence detection of fatty-acylated proteins in mammalian cells. These improvements allow the rapid and robust biochemical analysis of fatty-acylated proteins expressed at endogenous levels in mammalian cells by in-gel fluorescence scanning. In addition, alkynyl-fatty acid chemical reporters enable the visualization of fatty-acylated proteins in cells by fluorescence microscopy and flow cytometry. The ability to rapidly visualize protein fatty-acylation in cells using fluorescence detection methods therefore provides new opportunities to interrogate the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.
authors.affiliation: The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Charron
authors.forename: Mingzi M
authors.initials: MM
authors.lastname: Zhang
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Yount
authors.forename: John
authors.initials: J
authors.lastname: Wilson
authors.forename: Anuradha S
authors.initials: AS
authors.lastname: Raghavan
authors.forename: Eliah
authors.initials: E
authors.lastname: Shamir
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
mesh_terms: D000215:Acylation; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D003584:Cytological Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005227:Fatty Acids / Q000032:analysis*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence / Q000379:methods; D011506:Proteins / Q000737:chemistry*
chemical_list: D005227:Fatty Acids; D005456:Fluorescent Dyes; D011506:Proteins; D003300:Copper