Proteomics of Polarized T Helper Cell Subsets

In vitro polarized Th17 and Th22 cells were prepared as above. Cells were harvested on day 3, washed with PBS and pellets were snap frozen and stored at −80°C before treatment for detection. Cell profiling was performed leveraging liquid chromatography-tandem mass spectrometry (LC-MS/MS) method^{21 (link)}. A C8-positive platform, which connected a Nexera X2 U-HPLC (Shimadzu Corp) to an Exactive Plus orbitrap mass spectrometer (Thermo Fisher Scientific), was used to measure polar and non-polar plasma lipids. Internal standard peak areas were monitored for quality control and to ensure system performance throughout analyses. Pooled Th17 and Th22 reference samples were inserted every 20 samples as an additional quality control. Untargeted data were processed using Progenesis QI software (Waters, Milford, MA) and TraceFinder 3.1 (Thermo Fisher Scientific, Waltham, MA) for automated LC-MS peak integration.