Native Mass Spectrometry Analysis of Proteins

Purified proteins (and complexes) were buffer exchanged into 200 mM ammonium acetate solution at pH 8.5 using 10 kDa cut-off Amicon Ultra 0.5 ml centrifugal spin filters (Merck Millipore). Samples were centrifuged six times at 12,000 rpm for 5 min at room temperature. Protein concentrations were determined using a Qubit protein assay (ThermoFisher Scientific), and samples were diluted to a concentration of 3.2 μM for native mass spectrometry analysis. Protein samples were introduced into a first-generation Synapt QToF mass spectrometer (Waters Corp) using a nanoelectrospray gold-coated borosilicate glass capillary (prepared in house). Instrument parameters were as follows: capillary voltage 1.2 kV, sampling cone voltage 55 V, source offset 2 V, backing pressure 3.2 mbar, or 5.9 mbar, trap collision energy 8 eV, transfer collision energy 6 eV, and bias 12 V. CID was performed using a trap collision energy of 28 eV and a transfer collision energy of 26 eV. Native mass spectrometry data were processed using MassLynx 4.2 (Waters Corp), UniDec and in house software Amphitrite (38 (link), 39 ).