Tripletof 5600 lc ms ms system

Based on the large-scale quantitative proteome study, proteins were selected for validation by targeted MS analysis using parallel reaction monitoring (PRM) on a Triple TOF 5600 + LC-MS/MS system (AB SCIEX U.K. Limited). Protein extraction and tryptic digestion were performed in the same way as for the iTRAQ experiment. ProteinPilot software was used to identify proteins, and the database search results were transferred into Skyline software for spectra library building. Target proteins for PRM validation were imported to Skyline software (Skyline Software Systems Inc.), and the peptides for protein quantification were selected according to ion signals in the spectra library. Data collection from each sample was performed using the final PRM acquisition method on the TOF mass spectrometer, where each precursor ion was selected by the quadrupole, fragmented in the collision cell, and then all fragment ions were quantified in the TOF mass analyzer. Data processing was carried out in Skyline, and the quantification results were manually inspected for each peptide of the targeted proteins. A fold change criterion (> 1.25) based on two times were reported as being significantly different.

Chloromethylimines are
products that are very sensitive to hydrolysis. All transformations
have been performed using common Schlenk techniques or in a glovebox.
Toluene, toluene-d₈, and THF have been
dried with sodium prior to use. Deuterated chloroform and liquid amines
have been distilled from CaH₂ prior to use. NMR analyses
have been performed in a Bruker AvanceIII 300 spectrometer, a Bruker
AV400 spectrometer, or a Bruker Neo500 spectrometer. NMR data have
been processed using MestReNova or TopSpin. Residual signals of deuterated
solvents have been used as internal references (CDCl₃ at
7.26 ppm in ¹H NMR and 77.16 ppm in ¹³C NMR
and toluene-d₈ at 2.08 and 20.43 ppm for
methyl group in ¹H NMR and ¹³C NMR, respectively).
IR spectra have been recorded on a Thermo Scientific Nicolet iS10
instrument and processed with Omnic. IR band frequencies have been
rounded to 1 cm^–1. GC-MS analyses have been performed
with an Agilent 5977A instrument equipped with a 30 m × 0.25
m × 0.25 μm HP-5ms ui column. HRMS (+ESI) analyses have
been performed in an AB SCIEX TripleTOF 5600 LC/MS/MS system, and
data have been processed using PeakView. Elemental analyses have been
performed in a Thermofisher Flashmart Eager 200 instrument.

Quantification of liver Cyp2c29 protein in mice after APAP and CCl₄ treatment was performed with targeted proteomics according to the previous reports^{29 (link)}. Tissue samples were lysed in a solution containing 200 μL of thiourea buffer (50 mM Tris-Cl, pH 8, 7 M urea, 2 M thiourea, and 1× protease inhibitor cocktail), 800 μL of ice-cold acetone, and 10 mM dithiothreitol. After centrifugation at 13,000 × g for 20 min at 4 °C, the precipitated pellets were washed 3 times and collected. The dried pellets were dissolved in 200 μL of thiourea buffer for further analysis. A triple TOF 5600+ LC-MS/MS system (AB SCIEX) was used for targeted MS analysis with parallel reaction monitoring. Spectrum library generation was analyzed in ProteinPilot software. The peptides were selected for quantification according to the ion signals in the spectrum library.