GSNOR drives age-related obesity by regulating the S-nitrosation of Beclin-1 to promote adipose tissue whitening
Description
To elucidate the potential mechanisms of GSNOR in age-related obesity, we performed protein quantitative proteomics and quantitative S-nitrosation proteomics to investigate the levels and sites of protein S-nitrosation in iWATs from control and KI mice. The abundance ratios of proteins and their S-nitrosation states (KI/WT) were assessed, with ratios greater than 1.2 and less than 0.8 considered statistically significant. The results indicated that the KI group exhibited a marked reduction in S-nitrosation modification targets, with approximately half of the identified targets showing significant downregulation. A clustering analysis of differentially expressed proteins between the two groups highlighted that molecular functions related to fatty acid metabolism and cellular components associated with mitochondria were enriched. We subsequently focused on the top-ranked downregulated (≤0.8) S-nitrosation proteins that were most closely associated with fatty acid metabolism and mitochondria. Beclin-1, a core member of autophagy, attracted our attention, and we determined that cysteine 351 of Beclin-1 was S-nitrosated.
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Mouse iWAT quantitative S-nitrosation proteomics and LC‒MS/MS analysis For quantitative S-nitrosation proteomic analysis, total SNO-modified proteins were prepared according to the above IBP procedures, and the biotin labeling reagent was replaced with iodoTMT reagent. The protein sample was subsequently alkylated by iodoacetamide incubation. The iWAT protein was digested with sequencing-grade modified trypsin (25 μg of trypsin to digest 1 mg of protein). After incubation at 37°C overnight, the protein digests were desalted using C18 SPE columns and dried in a centrifugal vacuum concentrator, and 200 μl of 1X TBS buffer was used to suspend the freeze-dried peptide. Two hundred microliters of settled resin was added to enrich the iodo-TMT-labeled proteins. The peptide was analyzed by mass spectrometry. All nano liquid chromatography tandem-mass spectrometry (LC‒MS/MS) experiments were performed on a Q Exactive instrument (Thermo Scientific) equipped with an Easy n-LC 1000 HPLC system (Thermo Scientific). The labeled peptides were loaded onto a 100 μm id × 2 cm fused silica trap column packed in-house with reversed-phase silica (Reprosil-Pur C18 AQ, 5 m, Dr. Maisch GmbH) and then separated on a 75 μm id × 20 cm C18 column packed with reversed-phase silica (Reprosil-Pur C18 AQ, 3 m, Dr. Maisch GmbH). Peptides bound to the column were eluted with a 78-min linear gradient. Solvent A consisted of 0.1% formic acid in water, and solvent B consisted of 0.1% formic acid in acetonitrile. The gradient used was as follows: 4–10% B, 5 min; 10–22% B, 53 min; 22–32% B, 12 min; 32–90% B, 1 min; and 90% B, 7 min at a flow rate of 310 nl/min. MS data were acquired in data-dependent acquisition mode at high resolution (70,000; m/z 200) across a mass range of 350–1600 m/z. The target value was 3.00E+06 with a maximum injection time of 60 ms. The top 20 precursor ions were selected from each MS full scan with an isolation width of 2 m/z for fragmentation in the HCD collision cell and a normalized collision energy of 32%. MS/MS spectra were subsequently acquired at a resolution of 17,500 (m/z 200). The target value was 5.00E+04 with a maximum injection time of 80 ms, and the dynamic exclusion time was 40 s. The spray voltage for the nanoelectrospray ion source setting was 2.0 kV, there was no sheath gas flow, and the heated capillary temperature was 320°C. For each analysis, 2 samples were injected, and each sample was measured in duplicate.