Lipid Mediator Metabololipidomics

Un-perfused lung tissue was snap-frozen in liquid nitrogen, prior to addition of ice-cold liquid chromatography/mass spectrometry-grade methanol (Thermo Fisher Scientific) containing 500 pg of each of the following deuterium-labeled internal standards: d₈-5S-HETE (Cayman Chemical), d₄-LTB₄ (Cayman Chemical), d₅-LTC₄ (Cayman Chemical), d₅-LTD₄ (Cayman Chemical), ¹³ C315N-MCTR3, and ¹³ C215N-PCTR1 for calculating extraction and recovery of endogenous material. Lungs were gently dispersed using a glass tissue grinder (Kimble Chase Life Science and Research Products, Vineland, NJ) and protein precipitation occurred at -20°C for 30 minutes. Lung suspensions were centrifuged at 1000 g for 10 minutes at 4°C, supernatants were collected, and products were solid phase extracted per optimized methods using an automated extractor (Extrahera, Biotage, Charlotte, NC) as in reference (8). Samples were brought to an apparent pH 3.5 with acidified water (9 mL), and rapidly loaded onto 3 mL-SPE Isolute C18 100 mg cartridges (Biotage) and neutralized with double-distilled water (4 mL). The columns were washed once with hexane (Supelco, Bellefonte, PA) (4 mL). Next, the methyl formate fraction (Sigma-Aldrich) (4 mL) eluted SPMs, prostaglandins, leukotrienes, and thromboxane. The methanol fraction (Thermo Fisher Scientific) (4 mL) eluted cys-SPMs and cys-LTs. Both the methyl formate and methanol fractions were separately brought to dryness with a gentle stream of nitrogen gas using an automated evaporation system (TurboVap LV, Biotage), and immediately suspended in a methanol-water mixture (50:50, v/v) for injection on liquid chromatography tandem mass spectrometer (LC-MS/MS). Samples were injected and data acquired using a LC-MS/MS 6500⁺ QTRAP in low mass mode (Sciex, Framingham, MA) equipped with an ExionLC (Shimadzu, Tokyo, Japan).

A Kinetex Polar C18 column (100 mm x 4.6 mm x 2.6 µm; Phenomenex, Torrance, CA, USA) was kept in a column heat jacket maintained at 50°C. (Table S2) specifies polarity, retention time (min), Q1 (m/z), Q3 (m/z), dwell time (msec), declustering potential (DP, V), entrance potential (EP, V), collision energy (CE, V), collision cell exit potential (CXP, V), calibration correlation coefficient (r²), and lower limit of detection (LLOD, pg) for each mediator. The mobile phase gradient, multiple reaction monitoring (MRM), and enhanced product ion (EPI) mode settings are described in (Table S3). For each mediator, linear calibration curves were obtained using synthetic material with r² values of ≥0.98. Identification of each mediator included unbiased MS/MS matching (>70% fit score) to authentic and synthetic material in a MS/MS library (library matching parameters: precursor mass tolerance ± 0.8 Da, fragment mass tolerance ± 0.4 Da, collision energy ± 5 eV, use polarity, intensity threshold = 0.05, minimal purity = 5.0%, and intensity factor = 100) and a matching retention time to those of the authentic and synthetic material. Data was acquired with Analyst 1.7.1 software (Sciex). The MS/MS spectral library was created in LibraryView version 1.4.0 (Sciex). LC-MS/MS MRM trace data and EPI spectral data are shown as screen captures from Sciex OS version 1.7.0.36606 (Sciex).