Sil 20ac ht

Lipids were extracted from 50 μl of liver homogenate with an internal standard mixture. The extracted free fatty acids were derivatized by amino methyl phenyl pyridium (AMPP) into FA-AMPP derivatives in order to obtain high sensitivity in MS. Measurement of lipids was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). Data processing was conducted with Xcalibur (Thermo).

The lysosomal samples were extracted from anti-HA magnetic beads with methanol and ethyl acetate with d7-cholesterol (2 μg per sample), and further derivatized with nicotinic acid to improve the mass spectrometric detection sensitivity of cholesterol. Measurement of cholesterol was performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer. Data processing was conducted with Xcalibur™ Software version 4.0 (Thermo Fisher Scientific). A quality control (QC) sample was prepared by pooling the aliquots of the study samples and was used to monitor the instrument stability. The QC was injected six times in the beginning to stabilize the instrument and was injected every four study samples to monitor the instrument performance. The data was accepted if the coefficient variance (CV) of cholesterol in QC sample was < 15%. The data was reported as the peak area ratio of cholesterol to d7-cholesterol.

Diacylglycerol (DAG) and subspecies were quantitatively and qualitatively detected by LC-MS through Washington University Mass Spec Facility. In brief, lipids were extracted from 5×10⁶ cells using a modified Bligh-Dyer method in the presence of an internal standard DG15:0-15:0 (0.5 µg per sample). Measurement of DAGs were performed with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer operated in SRM mode under ESI(+). Data processing was conducted with X calibur (Thermo). Quality control (QC) samples were prepared by pooling the aliquots of study samples and were used to monitor instrument stability. The QC was injected seven times at the beginning to stabilize the instrument, and was injected again every five study samples. Only the lipid species with CV < 15% in QC sample were reported. Relative quantification of lipids was provided, and data were reported as the peak area ratios of the analytes to the internal standard. Relative quantification data generated in same batches are appropriate to compare the change of an analyte in AKR1B10 expression or AKR1B10 silencing samples to the corresponding control.