Abstract
Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.
Highlights
The nucleosome, the basic unit of chromatin, consists of 147 base pairs of DNA wrapped around histone proteins (H2A, H2B, H3, and H4)
We evaluated the accuracy of EpiProfile by mixing different ratios of synthetic histone peptides, and tested EpiProfile by analyzing nanoflow liquid chromatography (nanoLC)-Mass spectrometry (MS)/MS data sets of the following samples: purified histones from HeLa cells, a synthetic histone peptide library, and histone peptides labeled during cell growth with 13C-labeled glucose media or stable isotope labeling by amino acids in cell culture (SILAC) [22]
We found that manual quantification is obviously timeconsuming and that Skyline cannot generate the layouts of histone peptides and cannot discriminate four or six-component isobaric peptides, a common occurrence in histone data
Summary
The nucleosome, the basic unit of chromatin, consists of 147 base pairs of DNA wrapped around histone proteins (H2A, H2B, H3, and H4). Quantification of histone peptides is challenging because of presence of isobaric peptides, near isobaric PTMs such as tri-methylation (42.047 Da) and acetylation (42.011 Da), and low abundant species. When using relative retention time information to assign peak identities, reproducible nanoLC is crucial, especially because some isobaric peptides co-elute. In this case, the MS acquisition method must perform targeted MS2 for the co-eluting isobaric peptides at the specific time that they elute. The MS acquisition method must perform targeted MS2 for the co-eluting isobaric peptides at the specific time that they elute These species can be discriminated and quantified based on the intensity of fragment ions unique to each species. The co-eluting isobaric peptides could not be quantified separately based on the MS1 signal, but the unique fragment ions present in MS2 spectra allow them to be quantified individually
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