Hdx system

Hydrogen deuterium exchange mass spectrometry (HDX‐MS) was performed largely as described 32 on a Waters HDX system with nanoAcquity UPLC (Waters, Milford, MA, USA) and Micromass Q‐ToF Premier mass spectrometer. Samples were measured in tandem using the same buffers to minimize the difference in back exchange. Samples at 1.5 mg mL⁻¹ of human plasma purified PK and PKa supplied by Enzyme Research Laboratories (termed HPK and HPKa) were diluted 1 : 7 (v : v) into labeling buffer (10 mm phosphate, 99.9% D₂O, pD 7.0) for 10 to 10 000 s at 20 °C by automated LEAP robot pipetting. The 0‐s time point was represented by the dilution into the H₂O‐based labeling buffer. The HDX was quenched 1 : 1 (v : v) with precooled buffer containing 100 mm phosphate, 0.5 m TCEP, 0.8% formic acid, 2% acetonitrile, pH 2.5 for 180 s at 1 °C and digested on a Waters Enzymate BEH Pepsin Column (2.1 × 30 mm) at 20 μL min⁻¹. Fragments were separated on a Waters Nano ACQUITY UPLC BEH C18 column (1.7 μm, 1.0 × 100 mm) at 40 μL min⁻¹ with a gradient of 40% to 90% acetonitrile. Mass spectrometry was performed using electrospray ionization in positive ion mode. Peptides with no exchange were sequenced via Protein Lynx Global Software V3.0.2, followed by amide deuterium uptake analysis done manually via Dynamx 3.0 software. Fragments with >0.2‐Da mass deviations were removed.

Hydrogen-deuterium exchange was carried out using the commercial Waters HDX system. Briefly: samples were mixed and injected in a labeling time-randomized order by a PAL3 Autosampler followed by UPLC separation and mass spectrometry analysis using a Waters M-Class ACQUITY UPLC with peptides detected on a Waters Select Series Cyclic IMS Mass Spectrometer. Labeling times of 1, 10, and 60 min at RT were used, followed by quenching (7.5 M guanidine hydrochloride and 0.5M TCEP) at 0°C and digestion using an Enzymate BEH Pepsin column. The peptides were reverse-phase separated using an ACQUITY UPLC BEH C18 column. Peptide identification was carried out using HDMSe with CID fragmentation in the transfer cell and ProteinLynx Global Server (PLGS), followed by HDX analysis using DynamX. Structures were visualized using PyMOL 2.5.