Synapt g1 hdms system

Native nESI–MS of apo SaBPL was carried out on a Waters Synapt G1 HDMS system completed according to [25 (link)]. MS parameters were as previously reported to maintain non-covalent interactions and included; capillary voltage, 1.5–1.7 kV; cone voltage, 40–80 V; trap collision energy, 20–50 V; transfer collision energy, 15–20 V; source temperature, 50 °C; extraction cone, 2.0–5.0 V; trap gas flow, 5–8 mL/min; backing pressure, 3.95 mbar. Data analysis was performed in MassLynx V4.1 using manual peak finding.

Collision induced unfolding experiments were performed on a Synapt G1 HDMS system (Waters Corp., UK). Initially, proteins were buffer exchanged into 200 mM ammonium acetate pH = 7.6 supplemented with 40 mM trimethylamine (TEA). The latter was added in order to induce charge reduction and avoid a large number of overlapping peaks. Experiments were done in 2 series combining three proteins at once to reduce experimental variability. The first series included vinculin, metavinculin and vinculin-T12, while the other included vinculin, metavinculin and vinculin-T12A974K. Collision induced unfolding was performed as previously described^{37 (link),47 (link)}. In brief, the proteins were activated by increasing the trap collision voltage from 50 to 240 V by 5 V increments. To compensate for slow traveling times due to reduced charge, IM-MS acquisition parameters were set as follows- wave velocity of 250 m/s, variable wave height between 0 to 30 V with a ramp time of 10%. IM-MS calibration for conversion of drift times to collision cross section values was done as previously described^{46 (link)} using the instrument parameters presented above.

All ion mobility measurements were recorded on a Synapt G1 HDMS system (Waters Corp., UK) with a traveling-wave ion mobility device as described in detail elsewhere⁴⁵ . Proteins were first buffer exchanged into 200 mM ammonium acetate, pH = 7.6 (Sigma) using a Biospin 6 column (Bio-Rad) and their concentration was adjusted to a final concentration of 10 µM. Typically, protein aliquots of 2–3 µl were injected via a gold coated borosilicate capillary via a nano-ESI ion source. Acquisition parameters were as following- capillary voltage−1.25 kV, source temperature−25 °C, sampling cone−17 V, extraction cone−1.1 V, trap and transfer collision energy−5 V. For IMS wave velocity of 250 m/s and wave heights of 8–10 V were used. IMS calibration for conversion of drift times to collision cross section values was done as previously described^{46 (link)}. Experiment was repeated three times. Data presented is from a representative experiment and is an average of three wave heights−8, 9, 10 V.