Robust fluorescent detection of protein fatty-acylation with chemical reporters.
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.
Charron
Guillaume
G
The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, USA.
Zhang
Mingzi M
MM
Yount
Jacob S
JS
Wilson
John
J
Raghavan
Anuradha S
AS
Shamir
Eliah
E
Hang
Howard C
HC
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*
D005227:Fatty Acids; D005456:Fluorescent Dyes; D011506:Proteins; D003300:Copper