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. 2023 Sep 6;8(10):1263–1282. doi: 10.1016/j.jacbts.2023.06.006

Figure 3.

Figure 3

Global Profiling of Substrate Proteins of N-Myristoylation in Cardiac Myocytes by Click Chemistry–Based Quantitative Proteomics

(A) A schematic diagram for the workflow of proteomic strategy using click chemistry. Neonatal rat cardiomyocytes (NRCMs) and H9c2 myocytes were incorporated with myristic acid azide for the labeling. N-myristoylated proteins were selectively captured by Click-iT protein enrichment technology using a click reaction with alkyne-agarose resin and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). According to quantitative proteomics, 103 and 195 N-myristoylated–enriched proteins are identified in (B) NRCM and (C) H9c2 myocytes, respectively. The log quantitative value is plotted for each N-myristoylated substrate. The light green bars show proteins with the MG motif at the N-terminal, and the black bars show those with no MG motif. Data are presented as mean values from 5 and 3 independent experiments performed in triplicate, respectively. (D and E) Scatterplots for log quantitative value and log percentage of total spectra for N-myristoylated proteins according to the containing motif in (D) NRCM and (E) H9c2 myocytes. (F) Biological functions of 40 N-myristoylated proteins commonly detected in NRCM and H9c2 myocytes according to gene ontology annotation. (G) Chemical proteomic workflow with genetic inhibition of NMT2. NRCM were infected with Ad5-shNMT2 or LacZ (Ad5-shCTRL) at a multiplicity of infection of 100. H9c2 myocytes were transfected with small interfering RNA specific to NMT2 (siNMT2) or nontargeting control small interfering RNA (siCTRL). (H) Western blot analysis for NMT2 in NRCM infected with Ad5-shNMT2 for 48 hours (n = 3 in each). GAPDH was used as a loading control. The densitometric analysis is shown in the graph. (I) Volcano plots for N-myristoylated proteins in NRCM infected with Ad5-shNMT2 compared with those with Ad5-shCTRL from independent 3 experiments performed in triplicate. (J) Western blot analysis of NMT2 in H9c2 myocytes (n = 3 in each). (K) Volcano plots showing quantitative value and log P value for N-myristoylated proteins in H9c2 myocytes transfected with siNMT2 in comparison to siCTRL from independent 3 experiments performed in triplicate. ∗∗∗P < 0.001 by the unpaired Student’s t-test (2-sided). Abbreviations as in Figures 1 and 2.