Acetonitrile

Metabolite extraction and ratiometric profiling utilized acetonitrile (ACN), isopropanol (IPA), and methanol (MeOH) that were purchased from Fisher Scientific. High-purity Millipore filtered and deionized water (ddH2O; >18 MOhm) was used in extraction media and chromatography mobile phases. OmniTrace glacial acetic acid and ammonium hydroxide were obtained from EMD Chemicals. Ammonium acetate, ammonium formate, and all other chemicals and standards were obtained from Sigma-Aldrich in the best available grade.
Left ventricle was isolated from mice post-sham, 1hr I/R, or 4hr I/R, N=5 each condition and snap frozen in liquid nitrogen. Once received, tissue samples were washed with ice-cold PBS, followed by metabolite extraction using −70°C 80:20 methanol:water (LC/MS grade methanol, Fisher Scientific). The tissue–methanol mixture was subjected to bead-beating for 45 s using a Tissuelyser cell disrupter (Qiagen). Extracts were centrifuged for 5 min at 18,500 × g to pellet insoluble protein and supernatants were transferred to clean tubes. The extraction procedure was repeated two additional times and all three supernatants were pooled, dried in a Vacufuge (Eppendorf), and stored at −80°C until analysis. The methanol-insoluble protein pellet was solubilized in 0.2 M NaOH at 95°C for 20 min and protein was quantified using a BioRad DC assay. On the day of metabolite analysis, dried cell extracts were reconstituted in 70% acetonitrile at a relative protein concentration of 4 μg/ml, and 4 μl of this reconstituted extract was injected for LC/MS-based targeted and untargeted metabolite profiling.
Tissue extracts were analyzed by LC/MS as described previously (117 (link)), using a platform comprised of an Agilent Model 1290 Infinity II liquid chromatography system coupled to an Agilent 6550 iFunnel time-of-flight MS analyzer. Chromatography of metabolites utilized aqueous normal phase (ANP) chromatography on a Diamond Hydride column (Microsolv, cat# 70000–15D-2, 4um, 2.1mm ID × 150mm Length, 100A). Mobile phases consisted of (A) 50% isopropanol, containing 0.025% acetic acid, and (B) 90% acetonitrile containing 5 mM Ammonium acetate. To eliminate the interference by metal ions on chromatographic peak integrity and electrospray ionization, EDTA was added to the mobile phase at a final concentration of 5 μM. The following gradient was applied: 0–1.0 min, 99% B; 1.0–15.0 min, to 20% B; 15.0 to 29.0, 0% B; 29.1 to 37 min, 99% B. Raw data were analyzed using MassHunter Profinder 8.0 and MassProfiler Professional (MPP) 15.1 software (Agilent). Student’s t-tests (P < 0.05) were performed to identify significant differences between groups.
Peripheral blood was isolated fresh from murine IV post-sham, 1hr I/R, or 4hr I/R, N=5 each condition, into BD microtainer (cat#365967; BD) tubes for blood separation after 30 min room-temperature incubation followed by centrifugation at 1500 g for 5 min. Plasma was then snap-frozen and sent for LC/MS analysis. Plasma metabolites were extracted by the addition of 1 part plasma to 20 parts 70% acetonitrile in ddH₂O (vol:vol). The mixture was briefly vortexed and then centrifuged for 5 min at 16,000 g to pellet precipitated proteins. An aliquot of the resulting extract (3 μl) was subjected to LC/MS-based untargeted metabolite profiling in both positive and negative ion modes.

Reagents: The total phenol detection kit and anthocyanin detection kit were purchased from Biosystems, Spain. Anhydrous ethanol (chromatographically pure) was purchased from Shanghai Aladdin Reagent Co., Ltd. Hydrochloric acid was purchased from Tianjin Miou Reagent Co., Ltd., Tianjin, China, and the monomeric phenolic substance standard (chromatographic purity) was obtained from the National Institutes of Food and Drug Control, Beijing, China. In addition, formic acid, methanol, acetonitrile (chromatographic purity), and ethanol (chromatographic grade) were obtained from Fisher Corporation, USA. NaCl was purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.
Standards: A total of 65 volatile compound standards (see Supplementary Table S3), including ethyl acetate, ethyl isobutyrate, and ethyl butyrate, were obtained from Sigma-Aldrich, St. Louis, MO, with purities of over 90 %, whereas monomeric phenol standards (Table S3) were obtained from the National Institutes for Food and Drug Control, Beijing, China, with a purity of over 98 % (see Supplementary Table S4).

SDS-PAGE (10% polyacrylamide) was run until the entire sample had migrated into the resolving gel, approximately 1 cm. The gel was then stained using the Colloidal Blue Staining Kit (Invitrogen). The entire proteome was excised, cut into small pieces, and subjected to in-gel digestion with trypsin (1). The gel was destained using 50 mM ammonium bicarbonate (ABC) (Sigma-Aldrich, Darmstadt, Germany) and 50% acetonitrile (ACN) (Fisher Chemical, Waltham, MA, USA). The sample was reduced with 10 mM dithiothreitol (Bio-Rad, Hercules, CA, USA) in 50 mM ABC and alkylated with 55 mM iodoacetamide (GE Healthcare Life Sciences, Pittsburgh, PA, USA) in 50 mM ABC. Gel pieces were then digested with porcine trypsin (Thermo-Fisher Scientific) at a final concentration of 12.5 ng/mL in 50 mM ABC overnight at 37 °C. Peptides were extracted using 100% ACN and 0.5% trifluoroacetic acid (Sigma-Aldrich), purified with a Zip Tip (Millipore, Sigma-Aldrich), and dried. The sample was reconstituted in 20 μL of 0.1% formic acid prior to analysis by nanoscale liquid chromatography–tandem mass spectrometry (nLC-MS/MS).

The quantification of PHB was determined by measuring the absorbance of crotonic acid at 215 nm using HPLC. The pellet was harvested from 20 mL of cell culture by centrifugation (15,317× g, 10 min, 4 °C) and washed twice with equal volumes of acetone and ethanol. The conversion of P(3HB) to crotonic acid was achieved by digesting the pellet in 1 mL of concentrated sulfuric acid (Merck, Darmstadt, Germany) for 30 min at 105 °C. Following digestion, the samples were diluted with nanopure H₂O in a 1:5 volume ratio and filtered using 0.22 μm filters. Subsequently, the Agilent 1260 Infinity II LC System (Agilent, Santa Clara, CA, USA) was used to analyze the filtered samples. The samples were loaded onto the reversed-phase column InfinityLab Poroshell 120 EC-C18 (4 μm pore size, 4.6 × 150 mm, Agilent, Santa Clara, CA, USA) and eluted with a solution of 0.1 M 85% phosphoric acid (Honeywell, NC, USA) and 15% (v/v) acetonitrile (Fisher Scientific, Portsmouth, NH, USA) at a flow rate of 0.5 mL/min and a temperature of 30 °C. Crotonic acid in the samples was detected by a diode array detector at 215 nm and quantified based on a standard curve.