Additional file 4.
Figure S4 - Identification of H2AR29me2 in vivo by mass spectrometry (MS) analysis. (A) Overlay of the collisionally activated dissociation (CAD) tandem MS (MS/MS) spectra (derived from the endogenous H2A, isolated from Raji cells) of the specific tryptic (18 hour digest) peptide 22AGLQFPVGR(me2)29 (black) and the corresponding synthetic peptide (supplementary methods) (red). Both spectra are virtually identical, and allow the assignment of b and y ions and the internal fragment ion PV+H+. (B) Trypsin cleavage carboxyterminal to the dimethylated R29 in the histone H2A N-terminal tail is inefficient, and requires extended incubation times. Acid-extracted and gel-separated endogenous H2A was digested with trypsin for the time points indicated. and subsequently analysed by nano liquid chromatography (LC)-MS/MS. An extracted ion chromatogram (XIC; illustrated in red) of the peptide ion 22AGLQFPVGR(me2)29 (mass:charge ratio (m/z) 486.77), which eluted between 40.5 to 40.9 minutes from the C18 reverse-phase column, was derived from the total ion chromatogram (TIC; illustrated in black). (C) The peak area under the curve for the XIC of m/z 486.77 (mass was calculated and plotted against the trypsin digestion time points as indicated. An increase of more than 10-fold in peptide ion intensity was seen in the 18 hour compared with the 1 hour trypsin digest.
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Waldmann et al. Epigenetics & Chromatin 2011 4:11 doi:10.1186/1756-8935-4-11