Pierce ms grade trypsin

Samples were dissolved with chaotropic lysis buffer containing 8.4 M urea (USB Corporation, Cleveland, Ohio, USA), 2.4 M thiourea (Sigma-Aldrich), 2.5% SDS (Sigma-Aldrich), 5 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich) and a protease inhibitor cocktail (Sigma-Aldrich), and incubated for 15 min on ice. Homogenisation of the pellet was achieved by ultrasonication for 5 min on ultrasonic bath Branson 2510 (Marshall Scientific, New Hampshire, USA). The homogenate was centrifuged at 20 000×g for 10 min at 4°C, and the supernatant containing the solubilised proteins was used for further analysis. Then, 20 µg of protein was precipitated by methanol/chloroform method and resuspended in 20 µL of multichaotropic sample solution UTT buffer (7 M urea, 2 M thiourea, 100 mM TEAB (Sigma-Aldrich).
The resuspended sample was reduced with 2 µL of 50 mM TCEP, pH 8.0, at 37°C for 60 min, followed by addition of 1 µL of 200 mM cysteine-blocking reagent MMTS (SCIEX, Foster City, California, USA) for 10 min at room temperature. Sample was diluted to 140 µL to reduce the urea concentration with 25 mM triethylamonium bicarbonate (TEAB). Finally, digestion was initiated by adding 1 µg of Pierce MS-grade trypsin (Thermo-Fisher Scientific) to each sample in a ratio 1:20 (w/w), and then incubated at 37°C overnight on a shaker. Sample digestion was evaporated to dryness in a vacuum concentrator.

The 0.5 mM Δ16-TBEVC protein solution was treated by 0.5-, 1-; 2-, 4-, and 8-fold molar excess of bis(2,5-dioxopyrrolidin-1-yl)2,2'-(carbonyl bis(azanediyl))diacetate (C2; Urea crosslinker-C2-arm, NHS ester; CF Plus Chemicals), respectively, for 30 min to find the molar excess that generates the sufficient amount of covalently linked dimer and thus also crosslinked peptides. The reaction was quenched by the addition of 50-fold molar excess of 1 M Tris–HCl of the concentration of C2. Subsequently, the crosslinking products were visualized by Tris–glycine SDS-PAGE. Because of the absence of cysteine residues in the Δ16-TBEVC sequence, the crosslinked samples were not treated by any reduction and alkylation agent, and they were immediately digested in solution at 37 °C for 16 h by Pierce MS-grade trypsin (Thermo Fisher Scientific), using the trypsin-to-substrate ratio of 1:100 (w/w). The digestion was terminated by the addition of TFA to the final concentration of 0.5% (v/v). The obtained peptide mixture was then desalted by Pierce C18 Spin Columns (Thermo Fisher Scientific) according to the manufacturer’s instructions.