Tripletof 5600 quadrupole time of flight mass spectrometer

Chromatographic separation was performed as follows: 0.00–0.10 min 99% eluent B, 0.10–7.00 min 30% eluent B, 7.00–7.10 min 99% eluent B, and 7.10–10.00 min 99% eluent B, using a Waters Xbridge BEH amide column (2.1 × 150 mm, 2.5 µm). Column temperature was maintained at 45 ºC. The elution flow rate was 400 µL/min. Agilent Series 1290 was used as LC instrument.
Mass detection was carried out using an SCIEX Triple TOF 5600 quadrupole-time-of-flight mass spectrometer in ESI (-) mode (SCIEX, Concord, ON, Canada) for HILIC analysis. Fragmentation and mass spectra were obtained by operating the TripleTOF 5600 using a TOF method and an information-dependent acquisition (IDA) technique to simultaneously collect full-scan HRMS and MS/MS information.
The IDA technique was used to fragment the eight most intense ions. Exact mass calibration was automatically performed every eight injections. An MP sample was run every 50 samples to identify impurities from the solvents or extraction procedure and to test carryover contamination from intense peaks. A QC sample was injected every 10 chromatographic runs to check variability in the analysis.

The cells were submitted to metabonomic analysis 48 h after transfection. The ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry analysis was performed on Nexera X2 system (Shimadzu, Japan) coupled with a TripleTOF 5600 quadrupole-time-of-flight mass spectrometer (AB SCIEX, United States). Liquid chromatography separation was performed on a ZORBAX Eclipse Plus C18 column (2.1 × 100 mm, 3.5 μm, Agilent, United States) maintained at 45°C. Independent reference lock mass ions via Analyst TF 1.6 and MarkerView 1.2.1 were used to ensure mass accuracy during data acquisition. The assigned modified metabolite ions were identified by database searches in the HMDB (http://www.hmdb.ca/spectra/ms/search) databases. The mass tolerance for the HMDB database search was set at 0.05 Da.

One milliliter of MeOH:ACN:H₂O (2:2:1, v/v) solvent mixture was added into the cell samples followed by sonification. Cells were prepared as previously described.⁷ Liquid chromatographic separation for processed samples was achieved on a ZORBAX Eclipse Plus C18 column (2.1 × 100 mm, 3.5 μm, Agilent, USA) maintained at 45°C, whereas mass spectrometry was performed on a Nexera X2 system (Shimadzu, Japan) coupled with a Triple TOF 5600 quadrupole‐time‐of‐flight mass spectrometer (AB SCIEX, USA). All the steps for LC/MS analysis and data preprocessing were depicted in our previous study.⁷We used partial least squares discriminant analysis (PLS‐DA) to distinguish the overall difference in metabolic profile between rhPDGF‐BB treated‐ and control hPASMCs. Variables with a variable weight value (Variable Important in Projection, VIP) >1 were considered to be distinguishing among groups. The enriched metabolic pathways of metabolites with VIP >1 and fold change (FC) >2 or <0.5 and corresponding p < .05 between rhPDGF‐BB treated‐ versus vehicle treated hPASMCs were further analyzed by Metaboanalyst²² (v5.0, https://www.metaboanalyst.ca). The top 10 enriched metabolite pathways were selected as key word for analysis in Genecards²³ (https://www.genecards.org). Genes with relevance score >8 were defined as metabolism associated genes (MAGs).

Liquid chromatographic separation for processed cell samples was achieved on a ZORBAX Eclipse Plus C18 column (2.1 × 100 mm, 3.5 μm, Agilent, United States) maintained at 45°C, whereas mass spectrometry was performed on a Nexera X2 system (Shimadzu, Japan) coupled with a Triple TOF 5600 quadrupole-time-of-flight mass spectrometer (AB SCIEX, United States). The temperature of the sample chamber was maintained at 7°C. The gradient elution steps were shown in Supplementary Table S1. The injected sample volume was 10 μL for each run in the full loop injection mode, and the flow rate of the mobile phase was 0.5 ml/min. The mobile phase A was mainly composed of water and contains 0.1% formic acid. The mobile phase B was mainly composed of acetonitrile and contains 0.1% formic acid. Water (LC-MS grade) and acetonitrile (LC-MS grade) were purchased from Fisher Scientific. The purity of formic acid purchased from Acros was greater than 98%.