Plasma samples (6 time points/animal) were first processed in BSL-3 or BSL-4 containment by adding 25 µL SDS-PAGE solubilizing/reducing buffer to 75 µL sample and heating to 95 °C for 10 min. Samples were then removed from containment and stored at − 80 °C until processed by the iFASP method [28 (link)]. Briefly, 5 µL of each inactivated plasma sample was added to 200 µL 8 M Urea/100 mM Tris–HCL pH 8.5 (Solution UT8) and filtered through a Microcon-30 kDa Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore, MRCF0R030) at 14,000 × G for 15 min. Following several washing steps with 100 mM Tris pH 8.0, proteins were alkylated with 55 mM Iodoacetamide and digested with 4 µg Trypsin/Lys-C (Promega, V5071) overnight at 37 °C. TMT 6-Plex labeling (Thermo Fisher, 90061) was performed directly on the FASP filters per the manufacturer’s instructions. All 6 single labelled samples were then combined at an equal volume, purified by C18 spin column, dried to completion by speed-vac and stored at − 20 °C until analyzed by LC MS/MS.
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