One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry
- Simone Sidoli1,5,
- Yekaterina Kori1,2,
- Mariana Lopes1,3,
- Zuo-Fei Yuan1,
- Hee Jong Kim1,2,
- Katarzyna Kulej1,
- Kevin A. Janssen1,2,
- Laura M. Agosto1,2,
- Julia Pinheiro Chagas da Cunha3,
- Andrew J. Andrews4 and
- Benjamin A. Garcia1,2
- 1Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
- 2Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
- 3Laboratório Especial de Ciclo Celular, Center of Toxins, Immune Response and Cell Signaling - CeTICS, Instituto Butantan, São Paulo, 05503-900, Brazil;
- 4Cancer Epigenetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA
Abstract
DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic “histone-like” peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.247353.118.
- Received December 11, 2018.
- Accepted May 13, 2019.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
