Sciex 5800 maldi tof tof mass spectrometer

GSLs were extracted from HCC tissue as described⁵⁴ . Briefly, frozen tissue (approximately 25 mg) was homogenized and extracted with methanol and chloroform and bath-sonicated for 30 min. After centrifugation, the supernatant was collected, and the pellet was extracted three times with the same solvent. The supernatants containing neutral and acidic GSLs were pooled. Neutral and acidic GSLs were further separated by anion-exchange chromatography. The purified GSLs were subjected to permethylation according to NaOH/DMSO method^{55 (link)}. MS analyses were carried out on an SCIEX 5800 MALDI-TOF/TOF mass spectrometer using 2,5-dihydroxybenzoic acid as matrix. MS spectra were acquired in positive-ion mode and accumulated in 4000 laser shots with random sampling to increase the spectral reproducibility.

The oxidation of DJ-1 was performed by incubating DJ-1 with different concentrations of hydrogen peroxide (H₂O₂) as described earlier with slight modifications^{24 (link)}. In brief, DJ-1 (400 μM) was incubated with 180 mM H₂O₂ at 37 °C in 20 mM phosphate buffer (pH 7.4) and 100 mM NaCl for 30 min for generation of complete oxidation species (Cys106-SO₃⁻). Unreacted H₂O₂ was removed by multiple washes with 20 mM phosphate buffer (pH 7.4) and 100 mM NaCl in 10 kDa Amicon Ultra. For partial oxidation, DJ-1 (400 μM) was incubated with 2 molar excess of H₂O₂ at 4 °C in 20 mM phosphate buffer (pH 7.4) and 100 mM NaCl overnight. The unreacted H₂O₂ was removed as explained above. The DJ-1 was treated with 5 mM DTT to keep it in the un-oxidized state. H₂O₂ treated DJ-1(1 μg) was diluted in 500 μl of mass spectrometry grade water and desalted using C18 tip (Pierce, Thermo Scientific) following the manufacturer’s instructions. Desalted proteins were mixed with CHCA (1:1) MALDI matrix and spotted on a MALDI plate. For MALDI peptide mass fingerprinting (PMF) and MS/MS analysis, samples were processed in Sciex 5800 MALDI TOF/TOF mass spectrometer in positive ion reflector mode with ion acceleration voltage 25 KV for MS acquisition and 1 KV for MS/MS. The normalized collision energy was set to 35% for precursor ion fragmentation. The MS and MS/MS spectrum were analysed in MASCOT (version 2.3).

CTX used for the protein array analysis was chemically synthesized using Fmoc chemistry with 2-chlorotrityl resin and oxidized using 0.1 M ammonium bicarbonate pH 8 and 1 mM reduced glutathione, with stirring overnight at room temperature. The peptide was biotinylated using a 20-fold excess of EZ-Link® Sulfo-NHS-LC-biotin (ThermoFisher Scientific), according to the manufacturer’s instructions. The biotinylated sample was analyzed utilizing an SCIEX 5800 MALDI TOF/TOF mass spectrometer (SCIEX, Foster city, CA, USA), followed by purification of labeled CTX (to remove excess non-reacted and hydrolyzed biotin reagent from the solution) via HPLC. The purified sample was dried and resuspended in 100 μl of 1.0% PBS (pH 7.2) to give a final concentration of 10 μg. Protein-protein interactions among biotinylated CTX and more than 9,000 human proteins were then investigated employing a ProtoArray® Human Protein Microarray v5.0 Protein-Protein Interaction (PPI) kit (Invitrogen™) and significant interactions detected employing a GenePix® microarray scanner (Molecular Devices), according to the manufacturer’s instructions.