title: SILAC-Based Quantitative Strategies for Accurate Histone Posttranslational Modification Profiling Across Multiple Biological Samples.
abstract: Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented "shotgun" workflow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cuomo
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Soldi
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy. tiziana.bonaldi@ieo.eu.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bonaldi
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057890:Epigenomics / Q000379:methods; D006657:Histones / Q000378:metabolism*; D007553:Isotope Labeling; D011499:Protein Processing, Post-Translational
chemical_list: D006657:Histones
keywords: Epigenetics; High-resolution mass spectrometry; Histone posttranslational modifications; SILAC; SILAC spike-in; Ultrahigh-pressure liquid chromatography