Multidimensional mass spectrometry-based shotgun lipidomics analysis of lipids for macrophages samples was performed as described (33 , 34 (link)). In brief, premixed internal standard was added to 106 isolated macrophages for quantitation of lipid species and then normalized with the protein content (per mg protein), which was performed following the instruction of Pierce™ BCA protein assay kit (Cat #23225, Thermo Scientific). The lipids were extracted using a modified Bligh and Dyer procedure (35 ), and each lipid extract was reconstituted in chloroform/methanol (1:1, v/v) at a volume of 50 µL.
For shotgun lipidomics, lipid extracts were diluted to a final concentration of ~500 fmol total lipids µL-1. Mass spectrometric analysis was performed on a triple quadrupole mass spectrometer (TSQ Altis, Thermo Fisher Scientific, San Jose, CA) and a Q Exactive mass spectrometer (Thermo Scientific, San Jose, CA), both of which were equipped with an automated nanospray device (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY) as described (36 (link)). Identification and quantification of lipid species were performed using an automated software program (37 (link), 38 (link)). Data processing (ion peak selection, baseline correction, data transfer, peak intensity comparison and quantitation) was performed as described (38 (link)).
For shotgun lipidomics, lipid extracts were diluted to a final concentration of ~500 fmol total lipids µL-1. Mass spectrometric analysis was performed on a triple quadrupole mass spectrometer (TSQ Altis, Thermo Fisher Scientific, San Jose, CA) and a Q Exactive mass spectrometer (Thermo Scientific, San Jose, CA), both of which were equipped with an automated nanospray device (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY) as described (36 (link)). Identification and quantification of lipid species were performed using an automated software program (37 (link), 38 (link)). Data processing (ion peak selection, baseline correction, data transfer, peak intensity comparison and quantitation) was performed as described (38 (link)).
