Tandem Mass Tag Proteomics of PBMCs

PBMC samples were transferred to a BSL-2 freezer and stored at − 80 °C until processed by iFASP [15 (link)]. Briefly, 30 µg of each inactivated PBMC lysate was added to 200 µL 8 M Urea/0.1 M Tris-HCl pH 8.5 and filtered through a Microcon-30 kDa Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore; Cat# MRCF0R030) at 14,000×g for 15 min. Filters were washed 3× with 100 mM Tris pH 8.0, and proteins alkylated with 55 mM iodoacetamide followed by digestion with 4 µg Trypsin/Lys-C (Promega, Cat# V5071) overnight at 37 °C. TMT six-Plex labeling (Thermo Fisher Scientific; Cat# 90061) was performed directly on the FASP filters per the manufacturer’s instructions. TMT- labeled digests were then purified by C18 spin column, dried to completion by speed-vac and stored at − 20 °C until analyzed by LC–MS/MS.