Lipid extracts were analyzed with a flow-injection system using a flowrate of 1 μl/min and 5 μl sample injection. Negative and positive ion mode spectra were acquired with a LTQ-Orbitrap XL equipped with a 1200 microLC system and a Nanomate Triversa utilizing 5-μm ID ESI-chips. In the negative mode, PI, PE, PE-O, lysophosphatidylethanolamine (LPE), PC, CerPE, phosphatidylserine (PS), and phosphatidylglycerol (PG) were identified according to their accurate mass as described earlier (Schwudke et al., 2011 (link)). For PS, the specific neutral loss of 87 Da was monitored in the linear ion trap and used for quantification. In the positive ion mode Sph 14:1, ceramides and hex-ceramides were monitored with MS3 in the linear ion trap using the long chain base-related fragment ions. All MS3 for quantifying sphingolipids were analyzed using Xcalibur software while all other analyses were performed using LipidXplorer (Herzog et al., 2011 (link)).
For single and pooled brains, absolute levels of individual lipid species were summed up to arrive at lipid class quantities. At least five biological replicates were used for the analyses. GraphPad Prism (GraphPad Software, USA) and Origin 8.1 (OriginLab, USA) were used to for graphical representation and Origin 8.1 for analysis of variance (ANOVA) coupled with post-hoc Tukey test.
For single and pooled brains, absolute levels of individual lipid species were summed up to arrive at lipid class quantities. At least five biological replicates were used for the analyses. GraphPad Prism (GraphPad Software, USA) and Origin 8.1 (OriginLab, USA) were used to for graphical representation and Origin 8.1 for analysis of variance (ANOVA) coupled with post-hoc Tukey test.
