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.
Cell cycle Flow cytometry Gel electrophoresis In-gel digestion Mass spectrometry Nuclear proteome Plant nucleus Protein analysis
Coomassie Brilliant Blue
High-performance liquid chromatography
Liquid chromatography coupled to electrospray ionization mass spectrometry
Liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry
Liquid chromatography coupled to tandem mass spectrometry
Loading sample buffer
Matrix-assisted laser desorption/ionization
Tandem mass spectrometry
Sodium dodecyl sulfate
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Ultra-high-resolution quadrupole time-of-flight mass spectrometer
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This work was supported by a grant from the National Program of Sustainability I (LO1204) from the Ministry of Education, Youth and Sports of the Czech Republic.
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