Barnstead gen pure xcad plus water purification system

Bovine serum albumin (> 99.9%, probumin, crystallized, 810014, lot number 121, Merck KGaA, Darmstadt, Germany), chicken avidin (98%, 786-582, VWR International GmbH, Darmstadt, Germany), myoglobin from horse skeletal muscle (M0630-250MG, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), jacalin (> 95%, SRP6176-5MG, Sigma-Aldrich Chemie GmbH), bovine apotransferrin (> 95%, PSB-PRO-511, Biozol Diagnostics), and recombinant protein G (> 96%, PSB-PRO-402, Biozol Diagnostics, Munich, Germany) were used. The proteins were dissolved in pure water (Barnstead Gen-Pure xCAD plus water purification system, Thermo Scientific, Braunschweig, Germany) to an expected concentration of 1 mg/mL (gravimetrically), and 500-μL aliquots were stored at − 20 ^∘C for further analysis. Commercial birch pollen extract (Allergopharma GmbH & Co. KG, Hamburg, Germany) was provided by courtesy of I. Bellinghausen (University Clinics, Mainz, Germany). NIST reference materials (total protein standard, SRM 927e - Bovine Serum Albumin) and amino acids (SRM 2389a - Amino Acids) were used. The characterization of the test proteins is shown in the Electronic Supplementary Material: Protein Characterization.

Pure water was taken from a Barnstead™ GenPure™ xCAD plus water purification system (Thermo Scientific, Braunschweig, Germany). The water was autoclaved at 121 ${}^{\circ }$ C for 20 min and filtered three times through a sterile 0.1 µm pore diameter polyethersulfone (PES) vacuum filter unit (VWR International, Radnor, PA, USA). Recombinant timothy grass (Phleum pratense) pollen allergen 5 (Phl p 5.0101) was obtained from Biomay AG (Vienna, Austria), and a stock solution (1 mg mL⁻¹) was prepared with pure water prior to each experiment. The solution was reconstituted at room temperature for at least 30 min to ensure complete reconstitution of the protein. Sodium peroxynitrite (160–200 mM) was purchased from Merck (Darmstadt, Germany), and ammonium bicarbonate (NH₄HCO₃), acetonitrile (ACN), Tris-HCl, Glycerol, SDS, and monobasic sodium phosphate monohydrate (NaH₂PO₄· H₂O) were from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). A 150 mM NaH₂PO₄· H₂O buffer was prepared, and the pH was adjusted to 7 by the addition of sodium hydroxide (NaOH). NaOH and water with 0.1% trifluoroacetic acid (TFA) were obtained from VWR International GmbH (Darmstadt, Germany). $\mathsf{\alpha }$ -Cyano-4-hydroxycinnamic acid (HCCA, ultrapure, J67635) was purchased from Th. Geyer GmbH & Co. KG (Renningen, Germany). Trifluoroacetic acid (TFA, LC-MS grade, 85183) was obtained from Thermo Fisher Scientific (Braunschweig, Germany).

Animals were administered NMR/RTV (NMR: Medkoo Biosciences, Catalog#555985 Lot#: C22R06B23, Morrisville, NC, USA. RTV: Medkoo Biosciences, Catalog#318671, Lot#: A22M08B04) for oral dosing. Artificial CSF (TOCRIS Biotechne, #3525) and normal saline (B/BRAUN, Lot#: R5200-01) were used as described in sampling methods below. LC–MS/MS standard curves were generated using commercially obtained NMR (Cayman Chemical, Lot#:0635075, Ann Arbor, MI, USA) with a purity of > 98%. Nirmatrelvir-2H9 (2H9-PF-07321332, Lot#: NA-ALS-22-044-P3, Alsachim, Illkirch, France) was used as an internal standard for the NMR quantification. Formic acid, methanol and acetonitrile were obtained from Fischer Scientific (Waltham, MA, USA). Ultra-pure water was obtained from UNMC via a Barnstead GenPure xCAD Plus water purification system (Thermo-Fisher, Waltham, MA, USA). Frozen, non-medicated, non-immunized, pooled Sprague–Dawley rat plasma and pooled human CSF (BioIVT, Westbury, NY, USA) were used for calibration of standard curves. For oral dosing, NMR and RTV were mixed into a premade vehicle formulation similar to previous methods^{40 (link),58 (link),66 (link)}.