Impact 2 qtof

The proteins from decellularized tissues were initially extracted and digested as detailed in the Supplementary methods. Peptides were analyzed in an Impact II Q-TOF mass spectrometer (Bruker, Billerica, MA) using the nano-LC dedicated CaptiveSpray source, coupled to a nanoRSLC ultimate 3000 system (Thermo Fisher). The peptide separation details are also found in the Supplementary methods.
The mass spectrometer was operated in the Instant Expertise mode, consisting of a cycle of: (i) One MS spectrum in 250 msec, mass range 150–2200 Da; (ii) as many as possible MS/MS spectra in 1.75 sec, at 32–250 msec, depending on precursor intensity, mass range 150–2200 Da; and (iii) precursor exclusion after one selection. Searches against the Swissprot (2015–08) library of mammal protein sequences were performed using the Mascot 2.5 search engine (MatrixScience, Boston, MA), with a 7 ppm tolerance at the MS level and 0.01 Da tolerance at MS/MS level, and a false discovery rate fixed to a maximum value of 1%. The variable modifications were carbamidomethyl, Gln->pyro-Glu, acetyl, oxidation, and deamidation.

The optical rotations were recorded on a Rudolph Research Analytical Autopol III automatic polarimeter. NMR data were collected on a Bruker Avance II 600 MHz, high resolution 5-mm cryoprobe spectrometer operating at 600 MHz for ¹H and 150 MHz for ¹³C, using residual solvent signals (δ_H 2.50; δ_C 39.5 ppm DMSO-d₆, δ_H 2.50 ppm (CD₃)₂CO) as internal standards. The edited HSQC and HMBC experiments were optimized for ¹J_CH = 140 Hz ⁿJ_CH = 8 Hz, unless indicated otherwise (3 Hz HMBC). HRMS data were obtained using a Bruker Daltonics, Impact II QTOF with electrospray ionization (ESI). Chiral analysis was performed using an Agilent 6230 ESI-ToF and an Applied Biosystems 3200 QTRAP triple quad/linear trap. The effect on intracellular calcium was measured using a Molecular Devices Flexstation instrument. Circular Dichroism data were collected on a Chirascan™ Circular Dichroism Spectrometer (Applied Photophysics, Surrey, UK), with the Pro-Data Chirascan and Pro-Data viewer software 4.7.0.

Plasma samples were prepared by deproteinization with methanol followed by the addition of methyl tert-butyl ether (MTBE). Water was added after 1 h incubation with shaking (1970 × g at 4 °C). Samples were centrifuged and both organic and aqueous layers were transferred to fresh autosampler vials. Pellets were resuspended at 60 °C and supernatants were combined with the aqueous layer. All fractions were dried by speed vacuum. Before analysis, samples were resuspended in either IPA/MeCN/Water (2:1:1) (for the organic phase) or milli-Q water (for the aqueous layer). Equal volumes of samples were pooled for internal control and injected through the queue. Samples were placed in an auto-sampler at 4 °C. Analytes from the organic and aqueous layers were separated using Phenomenex Luna Omega Polar C18 and Amide columns, respectively, and analyzed in both positive and negative ion mode using a Bruker Impact II QTOF. Extracted ion currents were generated using XCMS. Any analyte with an RSD ≥ 0.25 or isotopic peaks was removed. Differentially regulated ions were detected using MetaboAnalyst 5.0 and inspected visually to confirm appropriate peak shape and intensity. Bile acids identified by untargeted metabolomics were confirmed using tandem mass spectrometry (MS/MS).

Leaf tissue was collected from three plants (approximately 100 mg, three 6 mm discs from three leaves) in Eppendorf tubes with 1 g ceramic beads and frozen in liquid nitrogen. Hormone extraction procedure and salicylic acid content measurement were done as in [51 ]. Briefly, frozen samples were homogenized in tubes with silica beads using a FastPrep-24 instrument (MP Biomedicals, CA, United States) with extraction reagent methanol/water/formic acid (15:4:1, v/v/v) supplemented with stable-isotope-labelled 13C-SA internal standards. Extracts were subjected to solid phase extraction using Oasis MCX cartridges (Waters Co., Milford, MA, United States) and eluted with methanol. The eluate was evaporated to dryness and dissolved in 15% acetonitrile/water (v/v) immediately before the analysis. Quantification was performed on an Ultimate 3000 high-performance liquid chromatograph (UHPLC, Dionex; Thermo Fisher Scientific, Waltham, MA, United States) coupled to a IMPACT II Q-TOF ultra-high resolution and high-mass-accuracy mass spectrometer (HRAM-MS; Bruker Daltonik, Bremen, Germany). Separation was carried out using an Acclaim RSLC 120 C18 column (2.2 m, 2.1 × 100 mm; Thermo Fisher Scientific, Waltham, MA, United States) mobile phase consisting of 0.1% formic acid (A) and methanol (B) by gradient elution. The full-scan data were recorded in negative electrospray ionization (ESI) mode.