Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis

  • Ivo ChamrádEmail author
  • Jana Uřinovská
  • Beáta Petrovská
  • Hana Jeřábková
  • René Lenobel
  • Jan Vrána
  • Jaroslav Doležel
  • Marek Šebela
Part of the Methods in Molecular Biology book series (MIMB, volume 1696)


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.

Key words

Cell cycle Flow cytometry Gel electrophoresis In-gel digestion Mass spectrometry Nuclear proteome Plant nucleus Protein analysis 





Ammonium bicarbonate


Ammonium persulfate


Coomassie Brilliant Blue


α-Cyano-4-hydroxycinnamic acid








Ethylenediaminetetraacetic acid


Electrospray ionization


Formic acid


Forward-scattered light


Gene ontology


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


Mass spectrometry


Tandem mass spectrometry




Sodium dodecyl sulfate


Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Stop-and-go Tip


Trifluoroacetic acid


Ultra-high-resolution quadrupole time-of-flight mass spectrometer



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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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Ivo Chamrád
    • 1
    Email author
  • Jana Uřinovská
    • 1
  • Beáta Petrovská
    • 2
  • Hana Jeřábková
    • 2
  • René Lenobel
    • 1
  • Jan Vrána
    • 2
  • Jaroslav Doležel
    • 2
  • Marek Šebela
    • 1
  1. 1.Department of Protein Biochemistry and ProteomicsCentre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký UniversityOlomoucCzech Republic
  2. 2.Centre of Plant Structural and Functional Genomics, Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental Botany AS CROlomoucCzech Republic

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