Tandem Mass Spectrometry Protein Digestion

The MV enriched plasma samples were thawed on ice and homogenised by vortexing. Small aliquots were mixed 1:8 with 0.5 M Urea and 2.5% methanol and the protein concentration was determined using Micro BCA from Pierce (Thermo Fisher Scientific Inc). From each sample 2 × 10 µg of protein was dissolved in a final concentration of 0.1% ProteaseMax (Thermo Fisher Scientific Inc), 0.05 M Urea, 50 mM ammonium bicarbonate and 10% methanol in a total volume of 80 µL. The resulting protein solutions were sonicated (2 cycles, each cycle 20 s with 40% amplitude) on ice using a probe (Vibra-Cell CV18, Sonics & Materials, Newtown, CT, USA) followed by incubation for 30 min at 37 °C while shaking. Samples were centrifuged and directly subjected to a tryptic digestion protocol carried out by a liquid handling robot (MultiProbe II, Perkin Elmer) as previously described^{30 (link)}. This included protein reduction in 5 mM DTT at 56 °C and alkylation in 15 mM iodacetamide for 30 min at RT in the dark. Trypsin was added in an enzyme to protein ratio of 1:30 and digestion was carried out over night at 37 °C. Samples were acidified by adding 6 µL concentrated formic acid, incubated for 30 min at RT and centrifuged for 20 min at 1,000g to remove undigested material.