Mass spectrometry grade acetonitrile

VICs were isolated from AVs removed for severe stenosis using tissue lysis with 2% collagenase (Sigma-Aldrich, St Louis, MO, USA). Protein extraction and proteolysis were performed with the methanol-chloroform method and trypsin [(Gold Grade; Promega, Wisconsin)/RapiGest procedure (Waters, Milford, MA, USA)], respectively, as previously published (18 (link)). Fifteen micrograms of protein was used per sample. The tryptic peptides were desalted using Oasis Hlb 1cc (10 mg) columns (Waters, Milford, MA, USA), and dried with a tabletop speed vacuum (SPD1010, Thermo Fisher Scientific, USA). After resuspension in 40 μl of 5% mass spectrometry grade acetonitrile (Thermo Fisher Scientific, USA) and 5% formic acid (Sigma-Aldrich, St Louis, MO, USA), the tryptic peptide samples were analyzed by liquid chromatography–mass spectrometry.

MgT was purchased from Shanghai Yuanye Biotechnology Co., CAS number is 778571–57-6, molecular formula is C8H14MgO10, purity is more than 95%. Trypsin Golden (Promega, United States), dithiothreitol (DTT, Sigma, Germany), iodoacetamide (IAA, Sigma, Germany), ammonium bicarbonate NH4HCO3 (Sigma, Germany), urea (Sigma, Germany), pure water (China Wahaha Company), mass spectrometry grade methanol (Thermo Fisher Scientific, United States), mass spectrometry grade acetonitrile (Thermo Fisher Scientific, United States), mass spectrometry grade purified water (Thermo Fisher Scientific, United States), Tris-base (Promega, United States), thiosulfate (Sigma, Germany), thiamine (Sigma, Germany), thiophene (Promega, United States), and thiourea (Sigma, Germany) were obtained.

Leucine enkephalin was purchased from Waters Corporation (Milford, MA, USA). Chromatography-grade methanol, mass-spectrometry-grade acetonitrile, and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Folin & Ciocalteu phenol reagent was bought from Sigma-Aldrich, Co., Ltd. (St. Louis, MO, USA). Ferrous sulfate, ferric chloride, and analytical-grade ethyl alcohol were procured from Tianjin Xinbo Chemical Co., Ltd. (Tianjin, China). Analytical-grade anhydrous sodium carbonate (Na₂CO₃), aluminum chloride (AlCl₃), potassium acetate, and gallic acid monohydrate (purity 99.0%) were obtained from Tianjin Yongsheng Fine Chemical Co., Ltd. (Tianjin, China). The phenolic standards such as rutin, quercetin, ferulic acid, caffeic acid, isorhamnetin, kaempferol 3-O-beta-rutinoside, p-coumaric acid, chlorogenic acid, and isoquercetin were purchased from Anpel Laboratory Technologies Inc. (Shanghai, China). Hydrochloric acid was purchased from Xi’an Chemical Reagent Factory (Xi’an, China). The 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), and sodium acetate were provided by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Ultrapure water was purchased from Watsons Group Ltd. (Hong Kong, China).