Bio toolkit

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.

500 ng protein was dissolved in 20 mM Tris 7.6, 50 mM KCl, 1 mM TCEP and loaded on an Eksigent MicroLC column (ChromXP C4, 3 μm particle size, 300 Å pore diameter, dimensions 50 × 0.5 mm) using a Dionex Ultimate 3000 HPLC system (Thermo Scientific). The proteins were separated on a gradient from 5% to 90% acetonitrile in water and 0.1% formic acid for 30 min at a flow rate of 30 µL/min. Mass spectra were recorded on a Sciex TripleTOF 5600 instrument equipped with a Turbo V DuoSpray ion source in positive mode. Data was analysed in PeakView version 2.1 (Sciex) using the Bio Tool Kit to reconstruct the uncharged average protein mass. All other samples were diluted in 0.1% formic acid (FA) to a concentration of 10 ng/μL and 50 ng were loaded on an Aeris Widepore C4 column, 3.6 µm particle size, dimensions 2.1 × 150 mm (Phenomenex), using a Dionex Ultimate 3000 HPLC system (Thermo Scientific) with a working temperature of 55 °C, 0.1% FA as solvent A, 90% acetonitrile, 0.08% FA as solvent B. Proteins were separated in a 6 min gradient from 10 to 70% solvent B at a flow rate of 300 μL/min. Mass spectra were recorded on a Waters Synapt G2-Si equipped with a ZSpray ESI source. Data were analysed in MassLynx V 4.1 using the MaxEnt 1 process to reconstruct the uncharged average protein mass.