Xcalibur system

Briefly, fresh and/or frozen cerebral tissue was homogenized in ice-cold 0.1 × phosphate-buffered saline (PBS) using Precellys^® Evolution Tissue Homogenizer (Bertin, France) as previously described [18 (link)]. Protein concentration of homogenates was determined using the bicinchoninic acid protein assay (Thermo Fisher Scientific, New York, NY, USA). Lipids were extracted by a modified procedure of Bligh and Dyer extraction in the presence of internal standards, which were added based on the total protein content of each sample [57 (link),58 (link),59 (link)]. Lipids were assessed using a triple-quadrupole mass spectrometer (TSQ Altis (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Nanomate device (Advion, Ithaca, NY, USA) and Xcalibur system as previously described [60 (link),61 (link),62 (link)]. Data processing including ion peak selection, baseline correction, data transfer, peak intensity comparison, ¹³C deisotoping, and quantitation were conducted using a custom programmed Microsoft Excel macro as previously described after considering the principles of lipidomics [61 (link),62 (link)].

Multidimensional mass spectrometry-based shotgun lipidomics was performed as previously described [50 (link)]. Briefly, frozen brain, spinal cord, or sciatic nerve tissues were homogenized in ice-cold phosphate-buffered saline (PBS) using Precellys® Evolution Tissue Homogenizer (Bertin, France). The protein concentration of homogenates was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Lipids were extracted by a modified procedure of Bligh and Dyer extraction in the presence of internal standards, which were added based on the total protein content of each sample [51 (link)]. Lipids were assessed using a triple-quadrupole mass spectrometer (TSQ Altis, (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Nanomate device (Advion Ithaca, NY, USA) and Xcalibur system as previously described [52 (link)]. Data processing including ion peak selection, baseline correction, data transfer, peak intensity comparison, ¹³C deisotoping, and quantitation were conducted using a custom-programmed Microsoft Excel macro as previously described [53 (link)].