Q tof mass spectrometer

UV-Vis-NIR absorption spectra were recorded on a Cary 5000. The mass spectrum was recorded on an ABI4800plus ESI-TOF-MS and Waters Q-TOF mass spectrometer. Intensity data of compounds 1 and 2 were collected on an Oxford Gemini S Ultra system (Cu K). Absorption corrections were applied by using the program CrysAlis (multi-scan). The structure was solved by direct methods, and C, O, S, F, Ag, Au atoms were refined anisotropically by least-squares on F² using the SHELXTL program. SbF₆^– could not be located due to high symmetry, but the formula reported was confirmed by mass spectrometry (Fig. S1†). The diffuse electron densities resulting from the residual solvent molecules were removed from the data set using the SQUEEZE routine of PLATON.

The purified CCPH was analysed further by LC-MS/ESI-TOF. The analysis was carried out using Acquity UPLC model system from Waters equipped with auto-sampler gradient pump and Dual Wavelength Detector (DAD). The column used was Accucore C18 with a length of 50 mm having inner diameter of 4.6 mm and particle size of 2.6 μm. 10 μl of CCPH fraction was injected into the column having water, acetonitrile and 0.1% formic acid gradient as mobile phase. A constant flow rate of 1 ml/min was maintained for a run time of 10 min. UPLC system was coupled with Q-TOF mass spectrometer of XevoG2-XS model from Waters. The monoisotopic mass were calculated and the data was processed by using Mascot database search engine. The mascot search parameters were set as; 1% of the false-positive discovery rate (FDR) of peptide identification, 1 missed cleavage site, trypsin enzyme, the peptide mass tolerance of ±100 ppm, oxidation as variable modifications. The peptide with maximum Mascot scores of at least 20 was considered (Tu et al., 2020 (link)).

¹H NMR was performed on a Bruker Avance 400 MHz. The mass spectrum was recorded on a Waters Q-Tof mass spectrometer. UV absorption spectra were recorded on an Evolution 201 UV-Vis spectrophotometer using quartz cells of 1.0 cm path length. A Cary Eclipse fluorescence spectrophotometer was used to record the fluorescence spectra. FT-IR experiments were performed by using a SHIMADZU Infrared spectrophotometer.

Protocol for MOV10 purification was followed (above). A band at approximately 75 kDa was excised from the gel and sent for analysis. Samples were cleaned up using Perfect Focus (G-Biosciences, St Louis, MO, USA) and digested using Trypsin (Proteomics Grade, G-Biosciences) at a ratio of 1:50 w/w in 25 mM Ammonium bicarbonate buffer in a CEM (Matthews, NC, USA) Discover Focused Microwave digestor for 30 min at 55 C (50 Watts max power). The digested peptides were lyophilized and suspended in 5% acetonitrile + 0.1% formic acid. Nano-ESI LC/MS was performed in a system consists of a Waters (Milford, MA, USA) NanoAquity UPLC connected to a Waters Q-ToF mass spectrometer. For chromatography, the column used was Waters Atlantis C-18 3 μm 75 μm × 150 mm. The flow rate was set at 300 nanoliters using a gradient of water + 0.1% formic acid to 60% acetonitrile + 0.1% formic acid in 60 min. Mass spec results were filtered and sorted by Waters ProteinLynx Global Server to PKL format and further analyzed using Mascot (Matrix Sciences, London, UK) and searched against NCBI NR Human database.