6550 accurate mass quadrupole time of flight mass spectrometer

SpySwitch was dialyzed into 50 mM Tris-HCl pH 7.5 + 1 mM TCEP and diluted to 10 μM, then acidified at a final concentration of 0.9% (v/v) formic acid. Samples were loaded onto a C4 solid phase extraction cartridge and washed with 0.1% (v/v) formic acid, then eluted with 85% (v/v) acetonitrile and 0.1% (v/v) formic acid in deionized water. Samples were analyzed using an Agilent 6550 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer operated in positive ion mode and utilizing a jet-stream electrospray ion source (Mass Spectrometry Research Facility, Department of Chemistry, University of Oxford). Data were analyzed in Mass Hunter Qualitative Analysis software B.07.00 (Agilent) and protein ionization data were deconvoluted using the maximum entropy algorithm. The mass of reduced SpySwitch without N-terminal formylmethionine was predicted using ExPASy ProtParam.

A previously validated method (linearity, precision, accuracy, limits of detection, and quantification) was used to analyze RSV and its derived metabolites [36 (link)]. Briefly, the analyses were performed on an Agilent 1290 Infinity UPLC system coupled to a 6550 Accurate-Mass quadrupole-time-of-flight (QTOF) mass spectrometer (Agilent Technologies, Waldbronn, Germany) using an electrospray interface (Jet Stream Technology). Chrysin was used as an internal control of the ionization signal. Spectra were acquired in the m/z range from 100 to 1100, in negative polarity mode (m/z⁻) and an acquisition rate of 1.5 spectra/s. Data were processed using the Mass Hunter Qualitative Analysis software (version B.06.00, Agilent). A targeted screening was used to identify possible phase-II metabolites (glucuronides, sulfates, and sulfoglucuronides) after RSV metabolism. In addition, MS/MS analysis provided additional information to achieve reliable compound identification. MS/MS product ion spectra were collected at m/z 50–800 range using a retention time window of 1 min, collision energy of 20 V, and acquisition rate of 4 spectra/s.

A previously validated method for analyzing RSV and its metabolites in biological samples was used [30 (link),31 (link)]. The analyses were performed on an Agilent 1290 Infinity UPLC system coupled to a 6550 Accurate-Mass quadrupole-time-of-flight (QTOF) mass spectrometer (Agilent Technologies, Waldbronn, Germany) using an electrospray interface (Jet Stream Technology, Auburn, AL, USA).
Data were processed using the Mass Hunter Qualitative Analysis software (version B.08.00), which lists and rates possible molecular formulas consistent with the accurate mass measurement and the actual isotopic pattern. A target screening strategy was applied, searching for more than 20 possible masses of metabolites, i.e., free (unconjugated) RSV and its derived gut microbial metabolites (DHRSV and LUNU), as well as their corresponding phase-II conjugates (mainly glucuronides, sulfates, and sulfoglucuronides of RSV, DHRSV, and LUNU) that could be present in the plasma and (or) the EVs after consuming the RSV capsules [30 (link),31 (link)]. The screening was based on mass filtering at the exact mass using a narrow mass extraction window (0.01 m/z), and the quantification was performed with available authentic standards. Other values are given as the area obtained from the extracted ion chromatogram (EIC) of those metabolites with no available standards.