Destabilization and Mass Spectrometry Analysis of SOD1

Purified SOD1 was destabilized via incubation in 20 mM DTT 5 mM EDTA in 1 × PBS (pH 7.4) at 37 °C for 2 h, following which samples were buffer exchanged into 200 mM ammonium acetate using gel-filtration chromatography (Superdex 75 10/300 GL, GE USA). Fractions off the column were immediately placed on ice, pooled, and diluted to a final concentration of 10 µM SOD1 monomer (measured via BCA assay). Mass spectrometry analysis was performed using a SYNAPT G1 HDMS (Waters, UK) with parameters set according to previous work^{32 (link)}. Briefly, SOD1 samples at 10 μM in 200 mM NH₄OAc were loaded into gold-coated borosilicate capillaries (made in-house) and subjected to nano-electrospray ionization. Results are representative of 3 separate unfolding experiments. All spectra were externally calibrated using 10 mg/ml caesium-iodide in 50% n-propanol, and were processed using Masslynx 4.1. For determination of the abundances of the observed conformations the area under the peak for each charge state was determined and the values plotted as a function of charge state. Following this, MATLAB R2014b (Version 8.4) was used to fit Gaussians to the plots and area under the peak was determined. Instrument parameters can be found in Appendix A.