Identification of plant nuclear proteins based on a combination of flow sorting, SDS-PAGE, and LC-MS/MS analysis

Chamrád, I., Uřinovská, J., Petrovská, B., Jeřábková, H., Lenobel, R., Vrána, J., Doležel, J., Šebela, M.
Plant Membrane Proteomics: Methods and Protocols : 57-79, 2018

Keywords: Cell cycle, Flow cytometry, Gel electrophoresis, In-gel digestion, Mass spectrometry, Nuclear proteome, Plant nucleus, Protein analysis
Abstract: In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.
DOI: 10.1007/978-1-4939-7411-5_4
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IEB authors: Jaroslav Doležel, Hana Jeřábková, Beáta Petrovská, Jan Vrána