Targeted LC-MS/MS Analysis of Histone Peptides

Histone peptides were resuspended in 0.1% trifluoroacetic acid prior to mass spectrometric analysis on a ThermoFisher Scientific TSQ Quantum QqQ MS (San Jose CA). Using a Dionex RSLCnano system (Sunnyvale CA), peptides were loaded onto a trapping column (3cm x 150μm) and separated on a PicoChip analytical column (10cm x 75μm), both packed with ProntoSIL C18-AQ, 3μm, 200Å pore size (New Objective, Woburn MA). Elution occurred over a chromatography gradient of buffer B from 0 to 35% at a flow rate of 0.30 μL/min over 45 minutes. Solvent A: 0.1% formic acid in water, and B: 0.1% formic acid in 95% acetonitrile. The peptides were then introduced into the QqQ MS by electrospray from an integrated emitter with 10 μm tip (New Objective) after elution from the analytical column. Targeted analysis of unmodified and various modified histone peptides was performed with the following settings: collision gas pressure of 1.5 mTorr; Q1 peak width of 0.7 (FWHM); cycle time of 3 s; skimmer offset of 10 V; electrospray voltage of 2.5 kV. Histone peptides (modified and unmodified) were selected for monitoring based on previous publications [20 (link),21 (link)]. A list of all peptides monitored in the assay can be found in S1 Table.