Abstract
DNA replication and subsequent deposition of nucleosomes is critical for the maintenance of the genome and epigenetic inheritance. Experiments using human tissue culture cells harvested at defined stages of the cell cycle can help to elucidate the mechanism of histone deposition and chromatin assembly in detail. Here, we describe a pulsed-SILAC approach to distinguish newly synthesized and deposited histones during S-phase of the cell cycle from parental “old” histones incorporated in previous replications and to decipher posttranslational histone modifications (PTMs).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Smith PA, Jackson V, Chalkley R (1984) Two-stage maturation process for newly replicated chromatin. Biochemistry 23:1576–1581
Worcel A, Han S, Wong ML (1978) Assembly of newly replicated chromatin. Cell 15:969–977
Alabert C, Barth TK, Reveron-Gomez N et al (2015) Two distinct modes for propagation of histone PTMs across the cell cycle. Genes Dev 29:585–590
Osley MA (1991) The regulation of histone synthesis in the cell cycle. Annu Rev Biochem 60:827–861
Oda Y, Huang K, Cross FR et al (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc Natl Acad Sci U S A 96:6591–6596
Ong S, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell cluture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386
Alabert C, Bukowski-Wills JC, Lee SB et al (2014) Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat Cell Biol 16:281–293
Scharf AN, Barth TK, Imhof A (2009) Establishment of histone modifications after chromatin assembly. Nucleic Acids Res 37:5032–5040
Egertson JD, Maclean B, Johnson R et al (2015) Multiplexed peptide analysis using data-independent acquisition and skyline. Nat Protoc 10:887–903
Völker-Albert MC, Schmidt A, Forne I et al (2017) Analysis of histone modifications by mass spectrometry. Curr Protoc Protein Sci Chapter 14:Unit 14.10
Cuomo A, Sanfilippo R, Vaccari T et al (2014) Proteomics meets genetics: SILAC labeling of Drosophila melanogaster larvae and cells for in vivo functional studies. In: Warscheid B (ed) Stable isotope labeling by amino acids in cell culture (SILAC): methods and protocols. Springer, New York, pp 293–311
Kruger M, Moser M, Ussar S et al (2008) SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell 134:353–364
Rosenfeld J, Capdevielle J, Guillemot JC et al (1992) In-gel digestion of proteins for internal sequence analysis after one- or two dimensional gel electrophoresis. Anal Biochem 203:173–179
Feller C, Forne I, Imhof A et al (2015) Global and specific responses of the histone acetylome to systematic perturbation. Mol Cell 57:559–571
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906
Acknowledgments
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, CRC1064-A16).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Völker-Albert, M.C., Schmidt, A., Barth, T.K., Forne, I., Imhof, A. (2018). Detection of Histone Modification Dynamics during the Cell Cycle by MS-Based Proteomics. In: Orsi, G., Almouzni, G. (eds) Histone Variants. Methods in Molecular Biology, vol 1832. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8663-7_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8663-7_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8662-0
Online ISBN: 978-1-4939-8663-7
eBook Packages: Springer Protocols