Synapt hdms qtof mass spectrometer

Accurate LC-MS data of cyanobacterial extracts were recorded with a Waters Acquity I-Class UPLC system and a Waters Synapt G2 HDMS mass spectrometer. High-resolution electrospray ionization-mass spectrometry (HRESI-MS) data for synthetic compounds and cyanobacterial extracts were obtained by direct infusion of methanolic solutions on a Waters Synapt HDMS QTOF mass spectrometer (Waters Corporation, Milford, MA). HPLC analyses for synthetic intermediates were performed using a Shimadzu LC-20-AT Series separations module equipped with Shimadzu SPD-M20A PDA (photo diode array) multiple wavelength detectors (180 nm-800 nm). For indole-isonitrile compounds, UV detector was set at 310 nm with a 5 nm slit-width. The overall system, CBM-20 was controlled using LC Solutions software. Raw data was plotted using Origin® software program after exporting absorbance data as an ASCII-formatted file. Analytical separations of stereoisomers (of cis and trans) mixtures were carried out on Daicel® (normal phase) AS chiral column. A 10% isopropanol/ 90% hexanes mixture was used as elution medium with a flow rate of 1 mL/min in an isocratic mode. Individual retention times for indole-isonitriles are reported along with analytical data for each isomer.

All reagents were purchased from commercial suppliers and used as received unless otherwise noted. Anhydrous acetonitrile (MeCN), dichloromethane (CH₂Cl₂), ethanol (EtOH), dimethylformamide (DMF), tetrahydrofuran (THF), methanol (MeOH), and diethyl ether (Et₂O) were purchased from commercial sources and maintained under dry N₂ conditions. Amide couplings and reactions with acid chlorides were performed under N₂ using standard Schlenk-line techniques. Compound 1 was purchased from commercial sources and used as received. ¹H and ¹³C NMR spectra were recorded in the listed deuterated solvent with a Bruker Avance III HD 500 MHz NMR spectrometer at 298 K with chemical shifts referenced to the residual protio signal of the deuterated solvent as previously reported.^{50 (link)} Low-resolution mass data were collected on an Agilent 6470AA Triple Quadrupole LC/MS system. High-resolution mass data were collected on a Waters Synapt HDMS QTOF mass spectrometer. Following the initial synthesis and screening of compounds 2–15, compound 7 was produced on the gram-scale following the same procedures described below. Purity was analyzed by analytical HPLC and Thermo LTQ MS with electrospray ionization in the positive mode with a Waters BEH 130, 5 μm, 4.6 × 150 mm C18 column, linear gradient from 90:10 to 0:100 water/acetonitrile in 10 min at a flow rate of 1 mL/min. (Supplementary Data File 2).

A Waters Synapt HDMS QTOF mass spectrometer was used to measure the intact protein mass of PLpro with and without preincubation with inhibitors to detect covalent adduct formation. To prepare the samples, 2 μL of 20 mM inhibitor stocks in DMSO were added to 100 μL PLpro at 1 mg/mL concentration and incubated 1 h at room temperature. Previously described protocols for ultrafiltration and denaturing direct infusion^{52 (link)} were implemented as follows. Samples were processed by ultrafiltration with a Vivaspin 500 10 kDa PES membrane by diluting the sample to 0.5 mL with 10 mM LC-MS grade ammonium acetate and reducing volume to 50 μL twice, followed by the same procedure with 2.5 mM ammonium acetate. Protein concentrations were estimated by A280 with a NanoDrop 2000, and samples were diluted to 2 mg/mL in 2.5 mM ammonium acetate, and then 10 μL were further diluted into 90 μL 50:50 acetonitrile:water with 0.1% formic acid. Sample was introduced into the electrospray ionization source by syringe pump at a flow rate of 10 μL/min and MS1 spectra were collected for m/z 400–1500, 5 s/scan, for 1 min. The protein monoisotopic mass was determined from the averaged spectra using mMass 5.5.^{53 (link)}