The mass spectrometric detection was carried out using a SCIEX 4500 or a SCIEX 5500 triple quadrupole mass spectrometer operating in positive electrospray ionization utilizing multiple reaction monitoring (MRM) mode. The LC and the mass spectrometer were controlled by Analyst software (version 1.6.2 and greater). The settings for the SCIEX 4500 or the SCIEX 5500 mass spectrometer are in Table 1 .
Sciex 5500
Manufactured by AB Sciex
The SCIEX 5500 is a mass spectrometer that provides high-resolution, accurate mass detection for various analytical applications. It is designed to deliver reliable and sensitive performance for a wide range of analytes.
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6 protocols using sciex 5500
1
Mass Spectrometric Detection Protocol
2
Targeted Metabolomic Analysis of Macrophages
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Targeted metabolomics analysis of the metabolites was performed with assistance from the Wekemo Tech Group Co., Ltd. (Shenzhen, China). Briefly, the metabolites were extracted from RAW 264.7 cell macrophages transducted with adv-NC or adv-PRDX3. Analyses were performed using an Ultra High Performance Liquid Chromatography (UHPLC) (Agilent 1290 Infinity LC) coupled to a QTRAP (SCIEX 5500).
3
Quantitation of PPCS Biomarker in DBS
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The quantitation of the biomarker PPCS (C24H50O7N2P—legacy name lyso SM-509) [14 (link)] in DBS was performed by multiple reaction monitoring mass spectrometry (MRM-MS) in positive ion mode on a triple quadrupole mass spectrometer (Sciex 5500) with an ultra-performance liquid chromatography unit (Waters Acquity). The diagnostic cut-off was calculated to be 655 ng/ml, which corresponds to a PPCS/internal standard peak area ratio of 0.9. An extended methods description can be found in the Supplementary information.
4
Lipidomic Analysis of HAEC Lysates and Conditioned Medium
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Lipidomic analysis was performed at the University of California Los Angeles (UCLA) Lipidomic Core. A modified Bligh and Dyer extraction (93 (link)) was carried out on HAEC lysates or conditioned medium aliquots. Before biphasic extraction, a standard mixture of 75 lipid standards (Avanti, 330820, 861809, 330729, 330727, and 791642) was added to each sample. Following two successive extractions, pooled organic layers were dried down in a Thermo SpeedVac SPD300DDA using ramp setting 4 at 35°C for 45 min with a total run time of 90 min. Lipid samples were resuspended in 1:1 methanol:dichloromethane with 10 mM ammonium acetate and transferred to robovials (Thermo Fisher Scientific, 10800107) for analysis.
Samples were analyzed by direct infusion on a Sciex 5500 with Differential Mobility Device (DMS) (comparable to Sciex Lipidyzer platform) with a targeted acquisition list consisting of 1450 lipid species across 17 subclasses. The DMS was tuned with EquiSPLASH LIPIDOMIX (Avanti, 330731). Data analysis was performed with in-house data analysis workflow. Instrument settings, multiple reaction monitoring (MRM) lists, and analysis method are previously described (94 (link)). Quantitative values were normalized to cell counts or medium volume.
Samples were analyzed by direct infusion on a Sciex 5500 with Differential Mobility Device (DMS) (comparable to Sciex Lipidyzer platform) with a targeted acquisition list consisting of 1450 lipid species across 17 subclasses. The DMS was tuned with EquiSPLASH LIPIDOMIX (Avanti, 330731). Data analysis was performed with in-house data analysis workflow. Instrument settings, multiple reaction monitoring (MRM) lists, and analysis method are previously described (94 (link)). Quantitative values were normalized to cell counts or medium volume.
5
Lipidomics Extraction and Profiling
6
Targeted Metabolite Extraction and LC-MS/MS Analysis
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Take the sample stored at −80°C for 30 min at −20°C, and then place it in the refrigerator at 4°C to melt it; weigh 25 mg of each sample and put it into EP tube; add two small steel balls and 800 μl precipitant (methanol: acetonitrile: pure water = 2:2:1) into each EP tube; grind in a grinder (50 Hz, 4 min); crush the cells and then ultrasonic the sample in ice bath. After centrifugation for 15 min (25,000g, 4°C), 500 μl of supernatant was taken; 500 μl supernatant was added into freeze-drying machine; 500 μl 10% methanol solution was added into ice bath ultrasound for 10 min (power 80 Hz), and centrifuged for 15 min (25,000g, 4°C); the supernatant was detected on the LC-MS/MS thermo license II liquid phase SCIEX 5500, which is detected by MRM mode. The data is processed by multiquant software (SCIEX, Framingham, MA, United States), and the absolute content of the target compound in the sample is calculated.
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