Orbitrap mass analyzer

To quantify the different collagen crosslinks in bone powder samples from affected and control cows, three biological replicates were used, with each biological replicate being tested for extraction efficiency using three technical replicates. Before crosslink separation and quantification, samples of the prepared bone powder were rehydrated in 1000 µL of water and 200 µL of this was inserted into a 1.5 mL short thread vial (Thermo Scientific, Waltham, MA, USA). Separation of collagen crosslinks was performed, as previously reported, by liquid chromatography (LC) using a Dionex UltiMate^TM 3000 system with an autosampler and a LPG-3400RS Rapid Separation Quaternary Pump, Thermo Fisher Scientific, USA) with a Cogent Diamond Hydride™ HPLC column (2.2 µm, 100Å, 150 mm × 2.1 mm ID, PM Separations, Capalaba, Queensland, Australia) [17 (link)]. The LC system was coupled to a Q Exactive^TM Focus mass spectrometer equipped with a high-energy collision-induced dissociation collision cell, an Orbitrap mass analyzer, and a HESI-II ion source (Thermo Fisher Scientific, USA) for crosslink quantification. Parallel reaction monitoring using tandem mass spectrometry acquisition with an inclusion list of ions was used to detect and quantitate the relevant ions for each crosslink. Details for the chromatographic and mass spectrometry settings are listed in Supplementary Tables S1–S3.

LC-MS/MS analysis was performed using Orbitrap Mass Analyzer (Thermo Fisher Scientific), according to the manufacturer’s protocol. Briefly, each sample of digested peptides was reconstituted in 20 μL of 0.1% formic acid. Peptides were first separated by the nanoflow HPLC on Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) using C18 column (Agilent Technologies) with a flow rate of 0.4 μL/min, and were ionized after passing through the nanospray tip (New Objective, Woburn, MA, USA). LC gradient for the LC-MS/MS system ramped from 2–40% ACN in 120 min, and the system was set up for automated data-dependent acquisition, with a mode of 200–2000 m/z full scan for the maximum three most intense peaks from each Orbitrap MS scan. Peptides with +2 or +3 charge were further subjected to CID. Spectra were obtained in raw data files with Xcalibur (version 2.0 SR2). Protein identification was accomplished by TurboSEQUEST (Thermo Fisher Scientific) using the UniProt database. A protein was confirmed once three peptides with Xcorr >2.5 were matched in sequencing.

Individual, unpooled samples were measured by the Metabolon analytical system (Metabolon, Inc., Durham, NC)^{60 (link), 61 (link)}. Samples were prepared and subjected to ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) utilizing an ACQUITY ultra-performance liquid chromatography (UPLC) (Waters, Milford, MA) and a Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution (Thermo Scientific, Waltham, MA). Raw data was extracted, peak-identified and QC processed using Metabolon’s hardware and software^{60 (link), 61 (link)}. Two-way ANOVA testing was used to calculate the p-values and the metabolites that were selected for display in figures and tables had either: (1) a fold change ≥1.2 with a p-value ≤ 0.05; or (2) a fold change ≥2.0 with a p-value ≤ 0.1 in at least one of the various comparisons (e.g., Oat1KO vs WT; Oat3KO vs WT; Oat3KO + probenecid vs Oat3KO). The separability of the uremic toxins/retention solutes in the wildtype, Oat3KO, and probenecid-treated Oat3KO plasma samples was assessed by partial least squares discriminant analysis (PLS-DA) using Metaboanalyst 3.0 (http://www.metaboanalyst.ca/)^{62 (link)}.