Xcalibur qual browser software

For the measurement of metabolites release from the MCF-7 and A375 cells, the samples were separated on a HPLC Accela (Thermo Fischer Scientific, Bremen, Germany) using an ACE Equivalence C18 column (VWR/Avantar, Alfragide, Portugal), 5 µm particle size and dimensions of 3.0 mm ID × 75 mm. The samples were eluted over a gradient of 100% solvent A (CH₃COOBH₄, 10 mM) to reach 100% Solvent B (acetonitrile with HCOOH 0.1%) during 8 min at a flow rate of 0.4 mL/min. Analyses were done on an LTQ OrbitrapTM XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0. The capillary voltage of the electrospray ionization source (ESI) was set to 3.1 kV. The capillary temperature was 275 °C. The sheath gas and auxiliary gas flow rate were 40 and 10 (arbitrary units, as provided by the software settings). The capillary voltage was 32 V, and the tube lens voltage was 55 V. An MS data handling software (Xcalibur QualBrowser software, Thermo Fischer Scientific, Waltham, MA USA) was used to search for metabolites by their m/z value.

Following detection of potential precursors from its accurate MS1 mass, the fragmentation spectrum of specific target precursor was visualized using the filter setting to MS2, obtained from specific target compound. The settings involved are:(a)

Selection of MS2 for specific m/z [M-H] shown in Table 2 at a retention time when the peak for precursor mass was observed

(b)

The fragment ions from the MS2 spectrum were visualized in Xcalibur Qual browser software (Thermo Fisher) to ensure clean signal to noise, free from interfering signal.

(c)

This way, in the same window, experimental data including the precursor along with fragment ion pattern obtained from it as well as the theoretically predicted isotope pattern using isotope simulation was visualized as shown in Supplementary Figure S1A.

(d)

To validate that the precursors and fragments are detected correctly, the untreated test sample was run alongside and the absence of matching precursor as well as matching fragment ion patterns signified that the process was precise (Supplementary Figure S1B).

HRMS data were obtained on a Thermo Orbitrap Exactive Mass Spectrometer with an Orbitrap mass analyzer. The instrument was calibrated using electrospray ionization with Pierce^™ LTQ ESI Positive Ion Calibration Solution from ThermoFisher Scientific. Samples were introduced into the instrument and ionized via an Atmospheric Solids Analysis Probe (ASAP). Data was analyzed in the Thermo Xcalibur Qual Browser software and identity was confirmed if <5 ppm error. Measurement parameters were as follows: Aux gas flow rate-8, Spray Voltage-3.50 kV, Capillary temperature-275°C, Capillary Voltage-25.00 V, Tube Lens Voltage-65.00 V, Skimmer Voltage-14.00 V, Heater Temperature-100°C.

MLODs and MLOQs of the analytes were assessed as reported in previous works [8 (link),33 (link)]. Briefly, a first estimation was done in the classical way, i.e., according to the following equations:
and

where σ is the standard deviation of the intercept and S the slope of the matrix-matched calibration graph. For MLOQ calculation, the peak area obtained by the sum of all the MRM transitions was used, whereas for MLOD calculation, the peak area obtained only by the second most intense MRM transition was considered.
After those calculations, the MLOD and MLOQ values obtained according to Equations (5) and (6), respectively, were verified. Therefore, corn meal and wheat flour samples were spiked with the six mycotoxins at levels very close to the extrapolated MLOQ values, and subject to the whole analytical procedure. For limits confirmation, the following equations were used:
and

where S/N is the signal to noise ratio manually estimated by the LC-MRM data set, since the S/N provided by Thermo Xcalibur Qual Browser software by both INCOS noise method and manual noise region selection, was unlikely high.

The proteins were subjected to an EASY-nLC 1000 interfaced via a Nanospray Flex ion source to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, USA) (nano-liquid chromatography-tandem mass spectrometry [LC-MS/MS]) for analysis. This analysis was done at the Technological Platform of Mass Spectrum Centre of Institute of Microbiology, Chinese Academy of Sciences. The protein was loaded onto a trap column (C₁₈, 3 μm particles, 100 μm inside diameter [i.d.], 3 cm length, Dr. Maisch GmbH) and separated using an analytical column (C₁₈, 1.9 μm particles, 150 μm i.d., 15 cm length, Dr. Maisch GmbH) at a flow rate of 400 nL/min. The LC gradient time was 30 min and was composed of solvent A (0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid). The gradient was first 20 to 70% B for 25 min and finally 70 to 100% B for 5 min. The precursor MS1 scan (m/z 400 to 2,000) was acquired in the Orbitrap at a resolution setting of 120,000. The molecular weight was calculated using the deconvolution technique employing the Xtract algorithm of Xcalibur Qual Browser software (Thermo Fisher Scientific).