Thermo fusion tribrid mass spectrometer

Immunoprecipitation was performed using Thermo Scientific™ Pierce™ MS-Compatible Magnetic IP Kit. Protein was incubated with EZH2 or acetylated-lysine antibody. Antibody bound proteins were eluted and run into SDS-PAGE. The excised gel lane pieces were reduced, alkylated, and digested in Trypsin Gold (Promega) digestion buffer (Thermo Fischer Scientific). Peptides were extracted with 70% acetonitrile (Thermo Fischer Scientific). The concentrated peptide mix was reconstituted in a solution of 2 % ACN, 2 % formic acid for MS analysis. Peptides were eluted from the column using a Dionex Ultimate 3000 Nano LC system. Using Thermo Fusion Tribrid mass spectrometer (Thermo Scientific), eluted peptides were electrosprayed. Mass spectrometer-scanning functions and HPLC gradients were controlled by the Xcalibur data system (Thermo Fischer). Experiments were performed at least twice.

This method was modified from Martínez-Acedo et al. (2014) (link). Briefly, 12 μg of purified PKL was diluted to a final volume 18uL by adding 6uL 8M urea/50mM HEPES pH7.0. Dimedone (Sigma) was added to 1mM, and incubated at 25°C for 30 min with constant mixing at 1200rpm in thermomixer. Reaction was quenched with addition of DTT to 2mM, and incubated at 1200rpm in thermomixer. A second reaction was performed at 500X excess (5mM) of IAA (Sigma), and incubated at 1200rpm in thermomixer. The second reaction was quenched by putting samples on ice and precipitating protein with addition of 100% TCA to 20% v/v, followed by incubation on ice for 30 mi and freezing at −80°C. After thawing, samples were spun at max speed in cooled centrifuge for 30 min, and supernatant removed. The pellet was washed with ice cold 10% TCA, then ice cold acetone. The pellets were dried by vacuum centrifugation, and resuspended in 50mM ammonium bicarbonate pH 8.0/10% ACN. Samples were digested with 100ng trypsin at 37°C for 4 h, desalted using a Hypersep Retain Pep 30mg cartridge (Thermo Scientific), and analyzed by LC-MS/MS on a Thermo Fusion Tribrid Mass Spectrometer using a Precursor Reaction Monitoring (PRM) method that triggered an HCD MS2 on 610.3305 (CC), 650.8538 (IC or CI), and 691.3771 (II) m/z. Data were analyzed using Skyline v 4.2.0.19072.