Quantitative Lipidomics Analysis of Macrophage-Bacteria Interactions

Macrophages were routinely incubated at 5 × 10⁶ cells/ml of PBS with Ca²⁺ and Mg²⁺ (PBS + Ca/Mg). In experiments where macrophages were treated in the presence of 0.5 mM EDTA and 20 µM BAPTA/AM, cells were incubated in PBS. Freshly grown pathogenic E. coli (serotype O6:K2:H1, except stated otherwise) or non-pathogenic E. coli JM-109 or E. coli BL21 strains were added at a ratio of 1:50 (macrophages:E. coli), and incubated at 37 °C for the indicated times. The supernatants were transferred to 2 ml of ice-cold methanol containing the deuterium-labeled internal standards d₈-5S-HETE, d₄-LTB₄, d₅-LXA₄, d₅-RvD2, and d₄-PGE₂ (500 pg, each) to facilitate quantification and sample recovery. Samples were kept at −20 °C for 60 min to allow protein precipitation and then centrifuged (1200 × g, 4 °C, 10 min). Solid-phase C18 cartridges were equilibrated with 6 ml methanol before the addition of 6 ml H₂O. Next, 9 ml acidified H₂O (pH 3.5, HCl) was added to the samples, and loaded onto the conditioned C18 columns that were washed once with 6 ml H₂O, followed by 6 ml hexane. The products were eluted with 6 ml of methyl formate. Samples were brought to dryness using an evaporation system (TurboVap LV, Biotage) and immediately suspended in methanol–water (50/50 vol/vol) for LC–MS–MS automated injections.
The LC–MS–MS system employed was equipped with a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector (Shimadzu, Kyoto, Japan), coupled with a QTrap 5500 (ABSciex, Framingham, MA). An Eclipse Plus C18 column (100 × 4.6 mm × 1.8 μm; Agilent) was kept in a column oven maintained at 50 °C (ThermaSphere TS-130; Phenomenex, Torrance, CA), and LM were eluted with a mobile phase consisting of methanol–water–acetic acid of 55:45:0.01 (vol/vol/vol) that was ramped to 85:15:0.01 (vol/vol/vol) over 10 min and then to 98:2:0.01 (vol/vol/vol) for the next 8 min. This was subsequently maintained at 98:2:0.01 (vol/vol/vol) for 2 min, and the flow rate was maintained at 0.4 ml/min. The QTrap 5500 was operated in negative ionization mode using scheduled MRM coupled with information-dependent acquisition (IDA) and an enhanced product ion scan. The scheduled MRM window was 90 s, and each LM parameter was optimized individually.
To monitor each LM and their respective pathways, an MRM method was used with diagnostic ion fragments and identification using recently published criteria^{9 (link)}, including matching RT to synthetic and authentic materials and at least six diagnostic ions for each LM. Calibration curves were obtained for each using authentic compound mixtures and deuterium-labeled LM at 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg (e.g., d₈-5S-HETE, d₄-LTB₄, d₅-LXA₄, and d₅-RvD₂). Linear calibration curves were obtained for each LM, which gave r² values of 0.98–0.99. Internal standard recoveries, interference of the matrix, and limit of detection (range of 20–220 fg for the QTrap 5500 in tissue and in biological matrix) were determined.