Native MS characterization of nucleotide states of the CI and CII domains on KaiCAA. Native MS characterization of nucleotide states of the CI and CII domains on KaiCAA. (A–C) Native mass spectra of KaiCAA mediated by ATP after trypsin digestion. After 5 h of incubation at 37°C in the presence of 1 mM ATP, KaiCAA was buffer-exchanged into 150 mM aqueous ammonium acetate and digested by 0.02 mg/ml trypsin for (A) 0 min, (B), 30 min, and (C) 60 min. The reaction mixture was directly analyzed by nanoflow electrospray ionization MS. The blue circles show the ion series of the KaiCAA homohexamer containing seven ATP and five ADP molecules, whereas the black circles show the hexameric CI domain (M1–S253) containing six ATP molecules. The CII domain was hardly detected as hexamer under the condition used here. Yasuhiro Yunoki et al. LSA 2019;2:e201900368 © 2019 Yunoki et al.