HDX-MS and data analyses were conducted as previously described (Du et al, 2019; Qu et al, 2021a). Briefly, the quenched samples underwent online digestion by passage through an immobilized pepsin column (2.1 × 30 mm; Life Technologies, Carlsbad, CA, USA). The digested peptide fragments were collected on a C18 VanGuard trap column (1.7 mm × 30 mm; Waters, Milford, MA, USA), followed by ultra-pressure liquid chromatography using an ACQUITY UPLC C18 column (1.7 mm, 1.0 mm × 100 mm; Waters). All settings and conditions for the system, such as voltage, temperature, collision energy, and lockspray, were as previously reported (Du et al, 2019; Qu et al, 2021a). Peptic peptides from nondeuterated samples were identified using ProteinLynx Global Server 2.4 (Waters). To process HDX-MS data, the amount of deuterium in each peptide was determined by measuring the centroid of the isotopic distribution using DynamX 3.0 (Waters).
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