C₆₀-SIMS studies of glycerophospholipid in a LIPID MAPS model system: KDO₂-Lipid A stimulated RAW 264.7 cells

Although secondary ion mass spectrometry (SIMS) has been successfully employed for mapping lipid distributions at the cellular level, the identification of intact lipid species in situ is often complicated by isobaric interference. The high mass resolution and tandem MS capabilities of a C ₆₀-QSTAR hybrid instrument has been utilized to identify over 50 lipid species from mouse macrophages (RAW 264.7). In this investigation, lipid assignments made based on mass accuracy were confirmed with tandem MS analyses. Data obtained from C₆₀-SIMS was compared to liquid chromatography (LC)-MS data obtained by the LIPID MAPS consortium. A majority of the lipids detected with LC-MS, but not detected with C₆₀-SIMS, were present at concentrations below 2.0 pmol/μg of DNA. Matrix-related effects prevented the detection of lipids with the glycerophosphoethanolamine (PE) headgroup, glycerophosphoserine (PS) headgroup and lipids with polyunsaturated fatty acyl chains in the C₆₀-SIMS analyses. Lipid distributions obtained from a lawn of RAW 264.7 cells stimulated with the endotoxin KDO₂-Lipid A were also studied. The results obtained with C₆₀-SIMS agreed with the established LC-MS data for the glycerophosphoinositol lipid class (PI) with adequate molecular sensitivity achieved with as few as 500 cells.