Effect of calibrant ion Ω on TWIMS calibration accuracy. The Ω of the main charge states of the membrane proteins AmtB (pink), AqpZ (green; charge states 11–14+) and MATE (blue) were determined using non-charge-reduced either only β-lactoglobulin (2,900 Å2), only alcohol dehydrogenase (6,900 Å2) or only pyruvate kinase (10,300 Å2) as calibrant ions. The smallest deviations from the literature values are achieved when the calibrant ions have a significantly larger Ω than the membrane proteins. Dashed lines indicate the literature Ω for the membrane proteins. Spectra were recorded using a wave velocity of 350 m/s and wave height of 13 V.