1. A Decade of Click Chemistry in Protein Palmitoylation: Impact on Discovery and New Biology 
Xinxin Gao, Rami N. Hannoush 
Cell Chemical Biology 
Volume 25, Issue 3, Pages (March 2018)
DOI: /j.chembiol
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2. Figure 1 Types of Cellular Protein Myristoylation and Palmitoylation
N-Myristoylation occurs at the N-terminal glycine, and lysine can be modified by side-chain myristoylation (on its N-ɛ-NH2 group). S-Palmitoylation occurs at cysteine and N-palmitoylation occurs at amino-terminal cysteine (which undergoes S-N acyl migration). O-Palmitoylation and O-palmitoleoylation occur at serine/threonine. The color on the chemical structures indicates the key residues for palmitoylation and the alkene bond in O-palmitoleoylation.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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3. Figure 2
Click-Chemistry-Based Probes for Detecting Protein Myristoylation and Palmitoylation
The azide (left) or alkyne (right) fatty acid probes label protein myristoylation (green) and palmitoylation (orange) and can be metabolically incorporated onto cellular proteins.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 3
Quantitative Analysis of Protein Palmitoylation Using Alkyne Palmitate Probes and SILAC
Cells are treated with isotopically labeled heavy and light amino acids followed by metabolic labeling using an alkyne palmitate probe and click chemistry. Cell lysates are mixed at a 1:1 ratio, and palmitoylated proteins are ligated to biotin-azide through their alkyne tags. After avidin enrichment and trypsin digestion, palmitoylated proteins are detected and further analyzed by tandem mass spectrometry (MS/MS)-based quantitative proteomics methods.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 4 Biochemical and Imaging Detection of Palmitoylated Proteins
Cells are metabolically labeled with Alk-C16. For biochemical detections (left), the cells are lysed, and the alkyne-labeled palmitoylated proteins are ligated to azide-tagged biotin by click chemistry. The target protein is immunoprecipitated from cell lysates using a primary antibody. Upon PAGE separation, the target protein is recognized by its primary antibody, and the palmitoylated forms of the target protein (with biotin-tagged palmitate groups) are detected using streptavidin-conjugated dye. For fluorescence imaging (right), cells are fixed, permeabilized, and processed for click chemistry with azide-tagged biotin and then visualized by PLA. Cells are incubated with two primary antibodies that recognize biotin and the target protein. The two primary antibodies are then recognized by two PLA secondary antibodies conjugated to oligonucleotides, which interact upon addition of two circle-forming oligonucleotides only when the two antibodies are in close proximity (<40 nm). After ligation of the two added oligonucleotides, the newly formed DNA circle can be amplified by a rolling circle amplification reaction, and the signal is developed using fluorescently labeled oligonucleotides complementary to the DNA circle. The readout, which is palmitoylation of the target protein, can be visualized using a fluorescence microscope.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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