Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry
Top-down mass spectrometry is a valuable tool for understanding gene expression through characterization of combinatorial histone post-translational modifications (i.e., histone code). In this protocol, we describe a top-down workflow that employs liquid chromatography (LC) coupled to mass spectrometry (MS), for fast global profiling of changes in histone proteoforms, and apply LCMS top-down approach for comparative analysis of a wild-type and a mutant fungal species. The proteoforms exhibiting differential abundances can be subjected to further targeted studies by other MS or orthogonal (e.g., biochemical) assays. This method can be generally adapted for screening of changes in histone modifications between samples such as wild type vs. mutant or healthy vs. diseased.
Key wordsHistone Post-translational modification Liquid chromatography Mass spectrometry Top-down Screening
The authors thank Christopher Wilkins, Jung Kap Park, and Sangtae Kim at the Pacific Northwest National Laboratory (PNNL) for developing the bioinformatics software used in this work. We appreciate the help from other PNNL colleagues: Matthew Monroe and Nikola Tolić for data analysis and Rosalie K. Chu, Rui Zhao, Anil K. Shukla, and Ron Moore for running LCMS experiments. We also thank Jonathan Galazka at the Oregon State University for preparing the fungal histone samples. The research was performed in Environmental Molecular Sciences Laboratory (EMSL), a US Department of Energy (DOE) national user facility at the Pacific Northwest National Laboratory (PNNL) in Richland, WA.
- 15.LaMarche BL, Orton DJ, Clark DA, Ryan JD, Hopkins DF, Anderson GA, Moore RJ, Smith RD LC control software for maximum flexibility and rapid automation of new mode of operation. In: 59rd ASMS conference on mass spectrometry and allied topics, Denvor, CO, 2011Google Scholar
- 18.Liu X, Sirotkin Y, Shen Y, Anderson G, Tsai YS, Ting YS, Goodlett DR, Smith RD, Bafna V, Pevzner PA (2012) Protein identification using top-down spectra. Mol Cell Proteomics 11(6). doi:10.1074/mcp.M111.008524Google Scholar
- 19.LeDuc RD, Taylor GK, Kim Y-B, Januszyk TE, Bynum LH, Sola JV, Garavelli JS, Kelleher NL (2004) ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry. Nucleic Acids Res 32(suppl 2):W340–W345. doi: 10.1093/nar/gkh447 CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Sidoli S, Lin S, Karch KR, Garcia BA (2015) Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry. Anal Chem 87(6):3129–3133. doi: 10.1021/acs.analchem.5b00072 CrossRefPubMedPubMedCentralGoogle Scholar