A Xevo Triple Quatrable (QqQ) mass spectrometer detector (Waters Corp.) was employed in the MS/MS studies and operated in positive or negative electron spray ionisation mode (ESI±). The analytes quantification was performed by employing multiple reaction monitoring (MRM) transitions. Before the sample analysis, each standard was manually tuned to achieve the optimum MRM conditions at 1 ppm concentration (Table S4 , Figure S3 ). To get the greatest signal level, 3.0 kV, the ideal tuning settings were as follows: 36 V at the cone, 150 °C at the source, 500 °C at the disolvation, 1000 L/h at the source disolvating gas flow, and 20 L/h at the gas flow. Ultra-high-purity argon was employed as a collision gas, and high-purity nitrogen gas served as the drying and nebulizing gas. Data were gathered and processed using the MassLynx program (version 4.1, Waters Co., Milford, MA, USA).
Masslynx program
Manufactured by Waters Corporation
Sourced in United States
MassLynx is a software program developed by Waters Corporation to control and operate mass spectrometry instruments. It provides a user interface for instrument control, data acquisition, and data processing. The core function of MassLynx is to enable the operation and management of mass spectrometry-based analytical workflows.
Automatically generated - may contain errors
Lab products found in correlation
Fasp protein digestion kit, by Abcam (1 mentions)
Hss t3, by Waters Corporation (1 mentions)
Xevo g2 xs qtof ms, by Waters Corporation (1 mentions)
Proteinlynx global server, by Waters Corporation (1 mentions)
Waters nano acquity system, by Waters Corporation (1 mentions)
Acquity uplc 1 class system, by Waters Corporation (1 mentions)
Acquity uplc beh c18, by Waters Corporation (1 mentions)
Targetlynx, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Q tof premier mass spectrometer, by Waters Corporation (1 mentions)
6 protocols using masslynx program
1
Optimizing Quantitative Mass Spectrometry Analysis
2
Protein Extraction and Identification
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For protein extraction, the trypsin digested whey fraction was mixed with Universal Protein Extraction (UPX) Kit (Expedeon-44101) and protease inhibitor cocktail (Thermo Sci.-87785). Samples were sonicated at 200 g and then boiled at 95 °C for 10 min. After the boiling procedure, samples were centrifuged for 10 min at 20,000 g. Peptide production was performed utilizing FASP Protein Digestion Kit (Expedeon-44250) and trypsin treatments (Pierce-90057) on the supernatant. Samples were diluted to 200 ng/μl with 0.1% formic acid. Detector and calibration settings were made with the MassLynx program (V4.1-Waters) that is specific to Xevo G2-XS Q-TOF-MS (Waters Corp, Milford, MA, USA) device where the analysis was carried out. The tryptic peptides were fractionated with an acetonitrile gradient (5–35%) in HSS T3 (Waters-186008818) column and analyzed by mass spectrometry upon electrospray ionization. Peptide data was identified in an m/z range of 50–1950. MS analysis was performed for 0.7 s and the data was collected from the entire peptide. UniProt protein database and Progenesis software were used for peptide identification.
3
LC-MS/MS Protein Identification Protocol
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Peptides were analyzed on LC–MS/MS to assign unique peptide sequences and perform protein identification—at the Proteomics and Mass Spectrometry Unit (UEMP) of the Federal University of Rio de Janeiro, Brazil. Samples were automatically injected into the Waters Nano Acquity System (Waters, Milford, MA) and peptides were desalted and separated throughout a 0%–50% ACN gradient on a C18 column. The ESI‐Q‐TOF mass spectrometry apparatus (Q‐Tof Micro‐Waters Corporation) was used for data acquisition. Instrument control was conducted in the MassLynx program (Version 4.1, Waters). All data were processed using the ProteinLynx Global server (version 2.5, Waters) where the mass/charge value of each precursor (MS) and fragment (MS/MS) were determined from the mass spectra from these chromatograms using the Q‐Tof LockSpray system (Waters, Milford, MA).
4
High-Resolution Mass Spectrometry Analysis
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Samples
were analyzed on a Waters SYNAPT G2-S high-definition mass spectrometer
connected to a Waters Acquity UPLC I-class system with an Acquity
UPLC BEH C18 (Waters, 2.1 mm × 75 mm, 1.7 μm particle size)
column. Solvent A was 0.1% formic acid in water and solvent B was
100% acetonitrile. The flow rate was 0.2 mL/min and gradient elution
was employed in the following manner (t (min), %
B): (0.01, 5), (6, 15), (21, 35), (23, 65). For each analysis, 10
μL of the sample was injected. The mass spectral data were collected
in high-resolution MSe continuum mode. A lock spray scan (function
3) was collected every 20 s for calibration, and the lock spray analyte
used was leucine-enkephalin. Parameters were 2.8 kV capillary voltage,
125 °C source temperature, 350 °C desolvation temperature,
35 V sampling cone, 50 L/h cone gas flow, 500 L/h desolvation gas
flow, and 6 L/h nebulizer gas flow. The collision energies for the
low-energy scans (function 1) were 4 and 2 V in the trap region and
the transfer region, respectively. Collision energies for the high-energy
scans (function 2) were ramped from 25 to 45 V in the trap region
and 2 V in the transfer region. Data were analyzed using MassLynx
program (Waters). The extracted ion chromatograms for the molecules
inFigure 1 are shown
inFigure 2 .
were analyzed on a Waters SYNAPT G2-S high-definition mass spectrometer
connected to a Waters Acquity UPLC I-class system with an Acquity
UPLC BEH C18 (Waters, 2.1 mm × 75 mm, 1.7 μm particle size)
column. Solvent A was 0.1% formic acid in water and solvent B was
100% acetonitrile. The flow rate was 0.2 mL/min and gradient elution
was employed in the following manner (t (min), %
B): (0.01, 5), (6, 15), (21, 35), (23, 65). For each analysis, 10
μL of the sample was injected. The mass spectral data were collected
in high-resolution MSe continuum mode. A lock spray scan (function
3) was collected every 20 s for calibration, and the lock spray analyte
used was leucine-enkephalin. Parameters were 2.8 kV capillary voltage,
125 °C source temperature, 350 °C desolvation temperature,
35 V sampling cone, 50 L/h cone gas flow, 500 L/h desolvation gas
flow, and 6 L/h nebulizer gas flow. The collision energies for the
low-energy scans (function 1) were 4 and 2 V in the trap region and
the transfer region, respectively. Collision energies for the high-energy
scans (function 2) were ramped from 25 to 45 V in the trap region
and 2 V in the transfer region. Data were analyzed using MassLynx
program (Waters). The extracted ion chromatograms for the molecules
in
in
5
Quantification of Tamatinib by UPLC-MS/MS
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The analysis was performed on Waters® Acquity H-Class UPLC® tandem triple quadrupole mass spectrometer (TQD) (Waters, Milford, USA). The H-Class UPLC® system contained Acquity sample manager and Acquity quaternary solvent manager. TQD was equipped with electrospray ionization (ESI) probe. Samples were detected and quantified in multiple reaction monitoring (MRM) mode. Chromatographic separation of tamatinib and IS was performed using Acquity TM CSH C18 (2.1 mm × 100 mm, 1.7 µm) column. Mobile phase composition was 10 mM ammonium acetate and acetonitrile (10:90). The mobile phase was adjusted at 0.25 mL/min. The MRM transitions of 471.1 > 122.0 and 441.1 > 84.0 were used for detection tamatinib and IS, respectively. The system was operated with the MassLynx program (Waters corporation, Milford, MA 01757, USA (Milford, MA 01757, USA), and the data acquisition used the TargetLynxTM software. Nitrogen (purity 99.999%) was used as a desolvation gas and argon as a collision gas. MS/MS parameters were optimized for tamatinib and IS by direct infusion of their solutions (500 ng/mL) individually at a flow rate of 5 µL/min. The values of the optimized parameters were illustrated in Table 7 .
6
Serum Metabolite Profiling via UPLC-TOFMS
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Diluted serum samples in 1-l aliquots were inserted into a Waters Ultra-Performance Liquid Chromatography-Time of Flight Mass Spectrometry(UPLC-TOFMS) machine (Milford, MA). Chemical components underwent separation at 35°C via an Acquity UPLC BEH C18 column (Waters). During a 10-minute run, the adjusted mobile-phase flow rate was 0.5 ml/min, and aqueous acetonitrile gradient contained 0.1% formic acid (0% acetonitrile for 0.5 min, 20% acetonitrile by 5 min, 95% acetonitrile by 9 min, followed by equilibration at 100% water for 1 min prior to subsequent administration). The Waters QTOF Premier mass spectrometer was adjusted to positive electrospray ionization. The capillary and cone voltages were maintained at 3 kV and 20 V, respectively. The source and desolvation temperatures were at 120°C and 350°C, respectively. Nitrogen was employed as the cone (50 l/h) and desolvation gas (600 l/h), whereas, argon was used as the collision gas. The flight mass spectrometry duration was calibrated using sodium formate solution (range m/z 100-1000), and observed in real time using intermittent administration of the lock mass sulfadimethoxine ([M + H]+ = 311.0814 m/z). Mass chromatograms and mass spectrum information were retrieved and assessed in the centroid format with the MassLynx program (Waters).
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