Samples underwent S-trap cleanup to remove detergents and other impurities with S-trap mini columns (PROTIFI Cat# MSPPC02-MINI-80) placed in 2 ml Eppendorfs. The protein mixtures were added to columns and centrifuged briefly (1000 g / 1 minute / RT). Columns were washed with 400 µl S-trap buffer 4 times, centrifuging after each wash at 1000 g / 1 minute / RT. Columns were placed in fresh 2 ml Eppendorfs and 100 µl of 5 µg Trypsin/Lys-C freshly dissolved in 50 mM TEAB, pH 8.5 was added. Columns were centrifuged briefly (200 g / 1 minute / RT) and Trypsin/Lys-C mixture was pipetted back onto the column. 100 µl 50 mM TEAB, pH 8.5 was added directly to the 2 ml Eppendorfs to cover any digested peptides remaining in the tube. The S-trap columns in 2 ml Eppendorfs were incubated at 47°C without shaking for 1.5 hours, then at RT overnight. 80 µl 50 mM TEAB was added to S-trap columns, which were centrifuged, and eluates were collected in new 1.5 ml Eppendorf tubes. 80 µl 0.2% (v/v) formic acid was added to columns, which were centrifuged, and second eluates were pooled with first eluates. 80 µl 50% (v/v) acetonitrile diluted in 0.2% (v/v) formic acid was added to columns, which were centrifuged, and third eluates were pooled with previous eluates. 500 ng digested peptides were set aside to vacuum dry separately to verify that digestion efficiency by calculating the zero and single missed cleavages was >98%. The remaining peptides were divided in half (50 µg peptides each tube) and vacuum dried and stored in -80°C prior to continuation with tandem mass tag (TMT) labeling.