X500b q tof

Disulfide bonds in PfCyRPA protein were identified by LC-MS. Briefly, the peptides derived from proteolysis under native conditions by the endoproteases LysC, AspN, and trypsin were analyzed by LC-MS (X500B QTOF, ABSciex). The protein samples (10 μg of purified PfCyRPA) were digested with AspN, LysC, and trypsin overnight at 37°C, without reduction to maintain intact disulfide bonds. External calibration was performed using beta-galactosidase digest (ABSciex). The twelve most intense precursor ions from the MS spectra were selected for MS/MS analysis. The data were acquired in positive TOF-MS mode using an X500B QTOF with a Turbo V ion source (ABSciex) mass spectrometer connected to the ExionLC AD UPLC system. Peptides were separated on bioZen 2.6-μm peptide XB-C18 column (2.1 × 150 mm) by a 1–90% gradient of acetonitrile in water containing 0.1% formic acid at a flow rate of 200 μl/min over 64 min and eluted into the mass spectrometer. The raw MS and MS/MS data were analyzed using Explorer software of SCIEX OS-Q and the BioPharmaView software 3.0 (Sciex) for peptide identification using the sponsor protein sequence.

Full-length KDM3B was tested for enzymatic function using histone tail with K9 dimethylation (EpigenTek, R-1026-200). Enzyme activity was performed in 50 mM HEPES pH 7.5, 1 mM TCEP, 100 μM 2-OG, 100 μM Ascorbate, and 50 μM Iron Sulfate for 3 h at room temp. Histone demethylation reactions were monitored by mass spectrometry measuring the mass of the histone H3 K9me2 substrate. Peptide masses were analyzed on a X500B Q-TOF (SCIEX) mass spectrometer coupled to an Exion UHPLC. Reactions were lyophilized before analysis and resuspended in 0.1% formic acid before injection onto an Synergi Fusion-RP C18 column (Phenomenex). Data were analyzed using Explorer and mass reconstruction was performed in Bio ToolKit, both within the SCIEX OS software package.

Protein samples were analyzed by LC/MS using a Sciex X500B Q-ToF mass spectrometer running Sciex OS v.1.6.1, coupled to an Agilent 1,290 Infinity II HPLC. Samples were injected onto a POROS R1 reverse-phase column (2.1 × 30 mm, 20 µm particle size, 4000 Å pore size), desalted, and the amount of buffer B was manually increased stepwise until the protein eluted off the column. Buffer A contained 0.1% formic acid in water and buffer B contained 0.1% formic acid in acetonitrile. The mobile phase flow rate was 300 µL/min. The acquired mass spectra for the protein of interest were deconvoluted using BioPharmaView v. 3.0.1 software (Sciex) in order to obtain the molecular weight.