Enzymate beh pepsin column

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.

HXMS experiment was performed as described previously 53 (link). Briefly, deuterium labeling was initiated with a 20-fold dilution into D₂O buffer of a pre-equilibrated (30 min, RT) aliquot of 14-3-3ζ protein and 14-3-3ζ protein with PTA stock solution. The labeling reaction was quenched with the addition of quenching buffer (37.5% [v/v] hydrochloric acid) at indicated times (0.25, 1, 10, 20, 60 and 240 min). Subsequently, samples were injected and online digested by a Waters ENZYMATE BEH pepsin column (2.1×30 mm, 5µm). The peptides were then trapped and desalted on a VanGuard Pre-Column trap (ACQUITY UPLC BEH C18, 1.7 µm) for 3 min, eluted in the trap using 15% acetonitrile at a flow rate of 100 µL/min, and separated using an ACQUITY UPLC BEH C18 column (1.7µm, 1.0×100 mm). Relative deuterium levels of all peptides were calculated by subtracting the mass of the undeuterated control sample from that of the deuterium-labeled sample. All mass spectra were analyzed using DynamX 3.0. Deuterium levels were not corrected for back exchange and thus reported as relative.

Deuterium labeling was initiated with a 20-fold dilution in D₂O buffer (100 mM phosphate, pD 7.0) of WT, L369/H373Q mutant, or I136T/L369/H373Q mutant (each 1 mg/mL). After 0.083, 0.25, 1, 10, 30, 60 and 240 min of labeling, the reaction was quenched with the addition of quenching buffer (100 mM phosphate, 4 M GdHCl, 0.5 M TCEP, pH 2.0). Samples were then injected and online digested using a Waters ENZYMATE BEH pepsin column (2.1 × 30 mm, 5 μm). The peptides were trapped and desalted on a VanGuard Pre-Column trap (ACQUITY UPLC BEH C18, 1.7 µm), eluted with 15% aqueous acetonitrile at 100 µL/min, and then separated on an ACQUITY UPLC BEH C18 column (1.7 µm, 1.0 × 100 mm). All mass spectra were acquired on a Waters Xevo G2 mass spectrometer, and processed using DynamX 3.0 software. Peptides from an unlabeled protein were identified using ProteinLynx Global Server (PLGS) searches of a protein database including WT, L369/H373Q mutant, and I136T/L369/H373Q sequences. Relative deuterium levels for each peptide were calculated by subtracting the mass of the undeuterated control sample from that of the deuterium-labeled sample. Deuterium levels were not corrected for back exchange and were thus reported as relative^{35 (link)}.

KCC3b‐PKO and KCC3b‐PM were diluted to 3.5 mg/ml using equilibration buffer which contains 25 mM HEPES pH 7.4, 150 mM NaCl and 0.002% LMNG. 5 μl of each sample was incubated with 50 μl of D₂O buffer (25 mM HEPES pD 7.4, 150 mM NaCl, 0.002% LMNG) for a time course of 5, 15 and 60 s, then quenched by 55 μl of ice‐cold quenching solution (25 mM HEPES pH 1.9, 150 mM NaCl, 0.002% LMNG). 80 μl of quenched samples was loaded into nanoACQUITY UPLC System (Waters corp.) and online digested by Enzymate™ BEH Pepsin Column (2.1 × 30 mm, Waters corp.) at 20°C. The digested peptides were trapped onto a BEH C18 trap column (1.7 μm, 2.1 × 5 mm, Waters corp.) and separated by BEH C18 analytical column (1.7 μm, 1 × 100 mm, Waters corp.) with a linear gradient of buffer B (acetonitrile with 0.1% formic acid) from 3 to 35% at a flow rate of 40 μl/min.
Mass spectra were acquired using Synapt G2‐Si HDMS mass spectrometer (Waters Corp.) in positive mode. MS/MS spectra were acquired in MS^E mode. Peptides from un‐deuterated samples were identified by ProteinLynx Global Server 2.5.1 (Waters Corp.), and HDX data were analysed by DynamX 3.0 (Waters Corp.). Relative fractional uptake was calculated by dividing the measured deuterium uptake by the theoretically maximum deuterium uptake.

Human CAR protein was prepared at 10 µM in 100 mM ammonium acetate pH 7.25 and diluted into either equilibration buffer (10 mM phosphate buffer, pH 7.5) or deuterated reaction buffer (10 mM phosphate buffer, 150 mM NaCl, pH 7.5 in D₂O). After labeling times of 1, 10 or 30 min at 25 °C, samples were mixed with equal volumes of quench buffer (100 mM phosphate buffer, pH 2.5) at 0 °C. For protein-ligand complex samples, hCAR was mixed 1:10 with CITCO or DIE compound at a final concentration of 100 µM with 2% DMSO. HDX-MS was performed using a commercial Waters HDX system (M-class ACQUITY UPLC and Water Cyclic IMS Mass Spectrometer). Leucine enkephalin was used for mass correction, and all time points were carried out in triplicate. Samples were digested using an Enzymate BEH Pepsin column (2.1 × 30 mm, Waters) at 15 °C. Peptides were desalted on an ACQUITY UPLC BEH C18 VanGuard Pre-column (2.1 × 5 mm, Waters) and separated using an ACQUITY UPLC BEH C18 column (100 × 1 mm, Waters). ProteinLynx Global server (PLGS) and DynamX software was used for peptide identification and deuterium uptake analysis, respectively. PyMOL 2.5 was used for structural visualization.