Abstract
It has proven particularly difficult to purify Linker (H1) histones from the model plant Arabidopsis thaliana. This is most likely due to its low nuclear DNA content and the abundance of substances that interfere with protein isolation. These problems have hindered the use of Arabidopsis for in-depth characterization of nuclear proteins by modern techniques based on mass spectrometry (MS). Here, we describe an improved methodology for preparing pure Arabidopsis H1s and separating them by HPLC into fractions corresponding to nonallelic variants. In addition, we outline basic approaches enabling the identification of posttranslational modifications of H1 by MS and their mapping by digestion with different proteases. We also discuss the analysis and interpretation of the acquired data.
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References
Zlatanova J, Caiafa P, Van Holde K (2000) Linker histone binding and displacement: versatile mechanism for transcriptional regulation. FASEB J 14:1697–1704
McBryant SJ, Lu X, Hansen JC (2010) Multifunctionality of the linker histones: an emerging role for protein-protein interactions. Cell Res 20:519–528. doi:10.1038/cr.2010.35
Stasevich TJ, Mueller F, Brown DT, McNally JG (2010) Dissecting the binding mechanism of the linker histone in live cells: an integrated FRAP analysis. EMBO J 29:1225–1234. doi:10.1038/emboj.2010.24
Wisniewski JR, Zougman A, Krüger S, Mann M (2007) Mass spectrometric mapping of linker histone H1 variants reveals multiple acetylations, methylations, and phosphorylation as well as differences between cell culture and tissue. Mol Cell Proteomics 6:72–87. doi:10.1074/mcp.M600255-MCP200
Kotliński M, Rutowicz K, Kniżewski Ł et al (2016) Histone H1 variants in Arabidopsis are subject to numerous post-translational modifications, both conserved and previously unknown in histones, suggesting complex functions of H1 in plants. PLoS One 11:e0147908. doi:10.1371/journal.pone.0147908
Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319. doi:10.1038/nprot.2016.136
Neuhauser N, Michalski A, Cox J, Mann M (2012) Expert system for computer-assisted annotation of MS/MS spectra. Mol Cell Proteomics 11:1500–1509. doi:10.1074/mcp.M112.020271
Johns EW (1964) Studies on histones. 7. Preparative methods for histone fractions from calf thymus. Biochem J 92:55–59
Acknowledgments
We thank Prof. Michał Dadlez and Jacek Olędzki for fruitful discussions.
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Kotliński, M., Jerzmanowski, A. (2018). Histone H1 Purification and Post-Translational Modification Profiling by High–Resolution Mass Spectrometry. In: Bemer, M., Baroux, C. (eds) Plant Chromatin Dynamics. Methods in Molecular Biology, vol 1675. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7318-7_10
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DOI: https://doi.org/10.1007/978-1-4939-7318-7_10
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