Uhplc system

The DAN was characterized by the following methods chemically: 20 mg sample was weighted into an EP tube accurately, and 1 mL extract solution (water:methanol:acetonitrile = 1:2:2) containing 1 μg/mL internal standard was added. The samples were homogenized at 35 Hz for 4 min and sonicated for 5 min with a 3 times repetition in an ice-water bath. Then all samples were incubated for 1 h and centrifuged at 12,000 rpm for 15 min. The last supernatant fluid was transferred into a fresh glass vial for LC/MS analysis. The analysis was performed by a UHPLC system (Agilent Technologies, Santa Clara, CA, USA) with a UPLC HSS T3 column connected to a QE mass spectrometer (Santa Clara, CA, USA). The injection volume was 2 μL. The QE mass spectrometer was used because it can acquire MS/MS spectra in information-dependent acquisition (IDA) mode under the control of the acquisition software (Xcalibur 4.0.27, Thermo, Waltham, MA, USA). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: Aux gas flow rate was 15Arb, sheath gas flow rate was 45 Arb, capillary temperature was 400 °C, MS/MS resolution was 17,500, full MS resolution was 70,000, spray voltage was 4.0 kV (positive) or −3.6 kV (negative), and collision energy was 20/40/60 in NCE mode, respectively.

400 μL MeOH and 400 μL ACN were added to 100 μL serum. Incubated for 1 h at −20 °C, centrifuged for 15 min at 13,000 rpm and 4 °C. Took the supernatant and evaporated to dryness at 4 °C. Reconstituted with 100 μL ACN/H2O (volume/volume, 1:1), vortexed for 30 s, sonicated for 10 min (4 °C water bath). Centrifuged for 15 min at 13,000 rpm and 4 °C. Supernatants were analyzed by LC-MS. The LC–MS analysis was performed using an UHPLC system (1,290 series, Agilent Technologies) coupled to a triple quadrupole mass spectrometer (Agilent 6,495 QqQ, Agilent Technologies). Chromatographic separation was performed on the Merck ZIC-pHILIC column (100 × 2.1 mm, 5 μm). The column was maintained at room temperature, and the flow rate was 0.2 ml/min. Mobile phase A was 100% H2O, 25 mM CH3COONH4, and 25 mM NH4OH, and mobile phase B was 100% acetonitrile. The column was eluted with a linear gradient system (B %): 0 min, 80%; 1 min, 80%; 3 min, 65%; 7 min, 50%; 7.1, 20%; 9.5 min, 20%; 9.6 min, 80%; 13 min, 80%. Optimized MRM transition 123.1/53.1 in positive mode was utilized for quantifying NAM.

Cell growth was determined by measuring the OD₆₀₀ using a Beckman Coulter DU370 spectrophotometer. Methanol concentrations were measured using an Agilent 1290 Infinity System (Santa Clara, CA, USA) equipped with an Aminex HPX-87H column. l-Lysine was determined using SBA-40E immobilized enzyme biosensor (Shandong, China) [21 (link)]. NADH and NADPH were assayed by means of the NAD/NADH quantitation Kit and the NADP/NADPH quantitation Kit (Sigma-Aldrich).
LC–MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies) equipped with a UPLC BEH Amide column (1.7 μm, 2.1*100 mm, Waters) coupled to Triple TOF 6600 (Q-TOF, AB Sciex). The mobile phase consisted of 25 mM NH₄OAc and 25 mM NH₄OH in water (pH = 9.75) (A), and acetonitrile (B) was carried with elution gradient as follows: 0 min-95% B, 0.5 min-95% B, 7 min-65% B, 8 min-40% B, 9 min-40% B, 9.1 min-95%, and B, 12 min-95% B, which was delivered at 0.5 mL/min. The injection volume was 1 μL. The Triple TOF mass spectrometer was used to assess its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during a LC/MS experiment. ESI source conditions were set as follows: Ion source gas 1 as 60, Ion source gas 2 as 60, Curtain gas as 35, source temperature 550 °C, Ion Spray Voltage Floating (ISVF) 5500 V or − 4500 V in positive or negative modes, respectively.

LC-MS/MS measurements were performed on a QTrap 4000 (Sciex, Foster City, CA, USA) equipped with a TurboV electrospray ionization source and coupled to a 1290 ultra-high performance liquid chromatography (UHPLC) system (Agilent Technologies, Waldbronn, Germany). A generic water-methanol gradient with 5 mM ammonium acetate as modifier in both eluents was applied for chromatographic separation. Either a Gemini C-18 column (150 × 4.6 mm, 5 µm, Phenomenex, Aschaffenburg, Germany) or a Kinetex C-18 (150 × 2.1 mm, 2.6 µm, Phenomenex) column was used. The selected reaction monitoring transitions of the aglycons and the later optimized glucosides are provided in Table 6. Theoretical selected reaction monitoring transitions from the m/z-values of the trichothecene-glucosides to the aglycons were applied before standards became available. The instrument was operated and data were evaluated with Analyst 1.6.2 (Sciex, Foster City, CA, USA). Alternatively, in some cases the LC-HRMS method described below for the characterization of the glucosides was also used in the screening process.

The metabolites from T. mesophilum SP-7-OMVs were extracted, and six parallel samples (labeled EV-1-1, EV-1-2, EV-1-3, EV-2-1, EV-2-2, and EV-2-3) were analyzed by ultrahigh-performance LC (UHPLC) coupled with Q Exactive (QE) Orbitrap/MS utilizing a nontargeting approach (Guangzhou Genedenovo Biotechnology Co., Ltd.). LC-tandem MS (MS/MS) analyses were performed by using a UHPLC system (1290, Agilent Technologies) with an Acquity HSS T3 column (2.1 × 100 mm; 1.7 μm) coupled with QE (Orbitrap MS, Thermo Fisher Scientific). The acquisition software (Xcalibur 4.0.27, Thermo Fisher Scientific) continuously evaluated the full-scan survey MS data as it collected and triggered the acquisition of MS/MS spectra depending on the preselected criteria. MS raw data (.raw) files were converted into the mzML format using ProteoWizard and processed via R package XCMS (version 3.2). OSI-SMMS (version 1.0, Dalian Chem Data Solution Information Technology Co., Ltd) was used for peak annotation after data processing with the in-house MS/MS database. Metabolites were matched with MS2 (the second-order mass spectrum), in which these metabolites could be defined as a “superclass”. Metabolites were mapped to the KEGG metabolic pathways for further shared pathway analysis (Supplementary material 1.1).

Blood samples were taken from 4-month-old mice with 3 months of swim training and 15-month-old mice with 3 months of swim training and 11 months of detraining. Liver samples were collected from 15-month-old mice with 3 months of swim training and 11 months of detraining. Metabolomic profiling was conducted by liquid chromatography tandem mass spectrometry (LC-MS) on the UHPLC system (Agilent Technologies, Santa Clara, CA, USA, #1290) by Gene Denovo Biotechnology Co. (Guangzhou, China). After data pre-processing and annotation, multivariate statistical analysis was performed. Orthogonal projection to latent structures-discriminant analysis (OPLS-DA) was used in comparison groups with R package models. A variable importance in projection (VIP) score was used to rank the metabolites distinguished between the two groups. A t-test was employed to screen differential metabolites, with a p-value < 0.05 and VIP ≥ 1 considered as significant criteria. The VIP score of OPLS-DA was used to create a graph. And the top 20 metabolites, ranked in descending order, were depicted in the VIP score plot.

Metabolomics analysis was performed using an UHPLC system (1,290, Agilent Technologies, USA) connected to a UPLC BEH Amide column (1.7 μm 2.1 × 100 mm, Waters, USA), and coupled to both TripleTOF 6600 (Q-TOF, AB Sciex, USA) and QTOF 6550 (Agilent, USA). The R package XCMS (Version 3.2) was used to process the MS raw data files. The preprocessing results generated a data matrix containing retention time (RT), mass-to-charge ratio (m/z) values, and peak intensity. The R package CAMERA was used for peak annotation. Bioinformatics analysis was performed using the MetaboAnalyst platform (https://www.metaboanalyst.ca/) [23 (link)]. Metabolite functional prediction was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (https://www.kegg.jp/).