HXMS. Samples were prepared in a 1:5 dilution of A1A2A3 into Tris-buffered saline in deuterium oxide (pD = 7.4). After incubation, hydrogen exchange was quenched by low pD ≅ 2.3. Samples were injected and digested on column with pepsin. Proteolytic fragments were collected on a C₈-trap and separated on a C₁₈-column in line with the mass spectrometer. (A) Hydrogen-deuterium exchange fraction of wild type (blue area) and C1669S/C1670S A1A2A3 (red area) after 1 minute, 1 hour, and 18 hours (top to bottom). (B) One-hour exchange fraction mapped onto the crystal structures of A1 (pdb ID 5BV8),¹² A2 (pdb ID 3gxb),⁵ and A3 (pdb ID 1ao3).⁶ Black = not resolved, blue = 0, white = 0.25, and red ≥ 0.5. (C) Comparison of peptide envelopes (normalized intensity as a function of the mass shift relative to the “all H” peak) of various peptides within the A1, A2, and A3 domains after 1 hour of exchange. (See supplemental Figures 17-26 for peptide mapping, hydrogen-deuterium exchange fractions as a function of residue number, structural location, time of exchange, and representative peptide envelopes; and supplemental Table 3 for HXMS experimental parameters in the supplemental materials.)