Figure 5. LC-MS/MS analysis of H3K56ac from K562 cells using an LTQ-Orbitrap XL mass spectrometer.

(a) Samples (see Experimental Section) were prepared by mixing solutions of increasing concentrations (from 5 to 1000 pg/mL) of [¹²C-isoleucine]-containing K56-acetylated peptide with a fixed amount (100 pg/mL) of internal standard (IS) peptide (YQK₅₆(ac)STELLI*R), where I* is a [¹³C₆¹⁵N₁]-labeled isoleucine residue). The y-axis is the peak height abundance ratio (as determined by LC-MS) of the YQK₅₆(ac)STELLIR peptide (containing only the [¹²C] isotope) to that of the [¹³C₆¹⁵N₁]-labeled internal peptide standard (IS). (b) Precursor ion mass spectrum (from LC-MS/MS analysis of histones derived from K562 cells treated with 1 μM SAHA for 24 h) showing the detection of the native YQK₅₆(ac)STELLIR peptide along with the [¹³C₆¹⁵N₁-isoleucine]-labeled internal standard. (c) MS/MS spectrum of precursor m/z 646.86²⁺ from the analysis shown in (b). This spectrum confirms the identity of the natural peptide containing H3K56ac.