Bio spin microcentrifuge columns

Prior to MS analysis, the protein complex was buffer exchanged into 100 mM ammonium acetate buffer pH 7.0 (Sigma-Aldrich) using Bio-Spin microcentrifuge columns (Bio-Rad Laboratories). Intact MS spectra were recorded on a Synapt G2-Si HDMS instrument (Waters Corporation) modified for high mass analysis and operated in ToF mode. Samples were introduced into the ion source using borosilicate emitters (Thermo Fisher Scientific). Optimised instrument parameters were as follows: capillary voltage 1.4 kV, sampling cone voltage 150 V, offset voltage 120 V, trap collision energy 100, transfer collision voltage 25 V, argon flow rate 8 ml/min and trap bias 5 V. Data were processed using MassLynx v.4.2 (Waters).

Prior to ESI-MS analysis, rh Bri2 BRICHOS fractions upon SEC were exchanged into 200 mM ammonium acetate pH 7.5 using BioSpin microcentrifuge columns (BioRad, US). Final protein concentrations (referred to monomeric subunit) were ~80 μM for rh Bri2 BRICHOS oligomers and 20 μM for the monomers. Spectra were recorded on a Waters Synapt G1 mass spectrometer (Waters, Milford, MA) modified for high mass analysis. Samples were introduced into the mass spectrometer using in-house produced gold-coated borosilicate capillaries. Instrument settings were: Capillary voltage 1.5 V, sample cone voltage 30 V, extraction cone voltage 4 V. The collision voltages in the trap were step-wise increased from 10 V to 180 V in 10 V increments. The transfer voltage was 10 V. The source pressure was increased to 7 mbar. Trap gas was N₂ with a flow rate of 8 mL h⁻¹. Data analysis was performed using Waters MassLynx 4.1 software.

Prior to ESI-MS analysis, rh Bri2 BRICHOS R221E monomers isolated by SEC were buffer exchanged using BioSpin microcentrifuge columns (BioRad, US) into 200 mM ammonium acetate pH 7.5. The final concentration of monomeric subunit was 20 μM. Spectra were recorded on a Waters Synapt G1 mass spectrometer (Waters, Milford, MA). Rh Bri2 BRICHOS R221E monomers were introduced using in-house produced gold-coated borosilicate capillaries. Instrument settings were: sample cone voltage 30 V, capillary voltage 1.5 V, collision trap voltage 50 V, extraction cone voltage 4 V, and transfer voltage 10 V. The source pressure was 7 mbar, trap gas was N₂ with a flow rate of 8 mL h⁻¹. Data analysis was performed using Waters MassLynx 4.1 software.