Multiplexed Parallel Reaction Monitoring Targeting Histone Modifications on the QExactive Mass Spectrometer Hui Tang, † Huasheng Fang, † Eric Yin, † Allan R. Brasier, ‡ Lawrence C. Sowers, † and Kangling Zhang* ,† † Department of Pharmacology, University of Texas Medical Branch, Galveston, Texas 77555, United States ‡ Institute for Translational Sciences, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555, United States * S Supporting Information ABSTRACT: Histone acetylation and methylation play an important role in the regulation of gene expression. Irregular patterns of histone global acetylation and methylation have frequently been seen in various diseases. Quantitative analysis of these patterns is of high value for the evaluation of disease development and of outcomes from therapeutic treatment. Targeting histone acetylation and methylation by selected reaction monitoring (SRM) is one of the current quantitative methods. Here, we reported the use of the multiplexed parallel reaction monitoring (PRM) method on the QExactive mass spectrometer to target previously known lysine acetylation and methylation sites of histone H3 and H4 for the purpose of establishing precursor-product pairs for SRM. 55 modified peptides among which 29 were H3 K27/K36 modified peptides were detected from 24 targeted precursor ions included in the inclusion list. The identification was carried out directly from the trypsin digests of core histones that were separated without derivatization on a homemade capillary column packed with Waters YMC ODS-AQ reversed phase materials. Besides documenting the higher-energy c-trap dissociation (HCD) MS 2 spectra of previously known histone H3/H4 acetylated and methylated tryptic peptides, we identified novel H3 K18 methylation, H3 K27 monomethyl/acetyl duel modifications, H2B K23 acetylation, and H4 K20 acetylation in mammalian histones. The information gained from these experiments sets the foundation for quantification of histone modifications by targeted mass spectrometry methods directly from core histone samples. C ore histones including H2A, H2B, H3, and H4 form chromatin scaffolds which are wrapped by ∼147 bp DNA and bound with linker histone H 1s, transcriptional factors, and chromatin remodeling complexes to form highly ordered genome architectures, the chromosomes. These molecules in the human genome are highly modified post-translationally by acetylation, methylation, phosphorylation, ubiquitination, etc. Both histone modifications and DNA methylation comprise the epigenetic modifications that regulate gene expression. Histone acetylation is normally correlated to gene activation 1 while DNA methylation is correlated to gene repression. 2 However, regulation of gene expression by histone methylation depends on not only the sites that are methylated but also the methylation states whether they are mono-, di-, or trimethy- lated. For example, in many cases, H3 K4 trimethylation, K36 trimethylation, and K79 methylation activate gene expression while H3 K9 methylation, K27 methylation, and H4 K20 methylation deactivate gene expression. 3 Epigenetic modifica- tion does not remain unchanged throughout the whole lifetime of a cell; it is a dynamic process as histones are acetylated by histone acetyltransferases and deacetylated by histone deacety- lases and methylated by histone methyltransferases and demethylated by histone demethylases. 4 In parallel, DNA is methylated by DNA methyltransferases (DNMT1 and DNMT 3A/B) and demethylated through oxidation (with Tet enzymes, cofactors α-ketoglutarate (αKG), and Fe 2+ ) and base excision repair (BER) pathways. 5 These dynamic modification processes are believed to play critical roles in mammalian cell develop- ment. 6 In the past decade, mass spectrometry has made marked contribution to the epigenetic field by identification of numerous novel modifications of histones. (References were omitted because there too many to be cited and equally considered.) Many of those identified modifications have been shown to have significant biological functions. Although the identification of protein modifications including histone modifications has become less tedious compared to that of the past, owing to a significant advancement of mass spectrometers whose scan rate, resolution, and sensitivity are tremendously increased, quantification of histone modifications is still a major challenge to analytical chemistry because modifications are highly congested on histone N-termini. We Received: March 7, 2014 Accepted: May 13, 2014 Published: May 13, 2014 Article pubs.acs.org/ac © 2014 American Chemical Society 5526 dx.doi.org/10.1021/ac500972x | Anal. Chem. 2014, 86, 5526-5534