A TSQ Quantum Ultra Plus triple-quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an automated nanospray apparatus (i.e., Nanomate HD, Advion Bioscience Ltd., Ithaca, NY) and Xcalibur system software were utilized in the study13 (link). Ionization voltages of -1.1, -0.95, and +1.2 kV and gas pressures of 0.3, 0.15, and 0.3 psi were employed on the nanomate apparatus for the analyses of anionic lipids, PE, and PC, respectively. The nanomate was controlled by Chipsoft 7.2.0 software. Each lipid extract solution prepared above was diluted to less than 50 pmol of total lipids/μl with CHCl3/MeOH/isopropanol (1:2:4 by volume) prior to infusion to the mass spectrometer with the nanomate. This concentration was estimated based on the protein content and the total lipid content that is normalized to protein content from prior studies or previous experience. This procedure uses low concentrations of lipid to prevent lipid aggregation during analysis and to minimize any effects of residual inorganic components carried over during lipid extraction on ion suppression and/or chemical noise.
The first and third quadrupoles were used as independent mass analyzers with a mass resolution setting of 0.7 Thomson while the second quadrupole served as a collision cell for tandem mass spectrometry. Typically, a 1 to 2-min period of signal averaging in the profile mode was employed for each full MS scan. For tandem mass spectrometry, a collision gas pressure was set at 1.0 mTorr but the collision energy varied with the classes of lipids as indicated or described previously3 (link), 7 (link). For each tandem MS mass spectrum, a 2 to 5-min period of signal averaging in the profile mode was employed. All the full MS scans and tandem MS scans were automatically acquired by a customized sequence subroutine operated under Xcalibur software. Data from biological samples were normalized to the protein content and all data are presented as the mean ± SD of multiple samples from different animals.
The first and third quadrupoles were used as independent mass analyzers with a mass resolution setting of 0.7 Thomson while the second quadrupole served as a collision cell for tandem mass spectrometry. Typically, a 1 to 2-min period of signal averaging in the profile mode was employed for each full MS scan. For tandem mass spectrometry, a collision gas pressure was set at 1.0 mTorr but the collision energy varied with the classes of lipids as indicated or described previously3 (link), 7 (link). For each tandem MS mass spectrum, a 2 to 5-min period of signal averaging in the profile mode was employed. All the full MS scans and tandem MS scans were automatically acquired by a customized sequence subroutine operated under Xcalibur software. Data from biological samples were normalized to the protein content and all data are presented as the mean ± SD of multiple samples from different animals.
