Massign

Skp:OMP complexes were prepared by rapid dilution of the denatured OMP (400 μM in 8 M urea, 50 mM glycine-NaOH, pH 9.5) to a final concentration of 5 μM into a solution of Skp (5 μM in 50 mM glycine-NaOH, pH 9.5). The samples were then buffer exchanged into 200 mM ammonium acetate, pH 10 using Zeba spin desalting columns (Thermo Scientific, UK) immediately prior to MS analysis. nanoESI-IMS-MS spectra were acquired using a Synapt HDMS mass spectrometer (Waters Corporation, UK) using platinum/gold-plated borosilicate capillaries prepared in-house. Typical instrument parameters include: capillary voltage 1.2-1.6 kV, cone voltage 40 V, trap collision voltage 6 V, transfer collision voltage 10 V, trap DC bias 20 V, backing pressure 4.5 mBar, IMS gas pressure 0.5 mBar, travelling wave height 7 V, travelling wave velocity 250 ms^-1. Data were processed using MassLynx v4.1, Driftscope 2.5 (Waters Corporation, UK) and Massign55 (link). CCSs were estimated by a calibration approach28 (link),33 (link),56 using arrival time data for ions with known CCSs (β-lactoglobulin A, avidin, concanavilin A and yeast alcohol dehydrogenase, all Sigma Aldrich, UK). Estimated modal CCSs are shown as mean ± standard deviation of three independent experiments. Theoretical CCSs for globular proteins with a given effective gas phase density were calculated according to published methods57 (link).