Identification of CL and MLCL in RAW264.7 cells

The CL and MLCL species in RAW264.7 cells were identified by LC-MS/MS. Based on the structure of CL and its charge, the extracted total lipid was analyzed by reverse-phase chromatography and mass spectrometry in negative mode. The methodology for CL analysis and identification has been described previously [6, 24]. In short, the fragmentation of CL by tandem mass spectrometry determined the fatty acyl compositions of each CL species. Internal CL standard CL(14:0)₄ was added before lipid extraction. The CL quantity was semi-quantified by the relative extract ion current (XIC) of the target CL to the XIC of the internal standard. In our measurement, RAW264.7 cells contain 55 CL species with 42 types of molecular weights, and the acyl chains of each species were identified. For MLCL analysis, the signal of extracted ion chromatogram of MLCL standard (C14:0)₃ appeared by around 24 min in our liquid chromatography gradient program. The retention time of MLCL in samples was around 26–30 min. MLCL species showed in five clusters in m/z range of 1100–1300. There are 33 MLCL species with 16 molecular weights. All of the identification of CL and MLCL was carried out by secondary fragmentation.