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Front Plant Sci. 2014 Oct 29;5:557. doi: 10.3389/fpls.2014.00557. eCollection 2014.

A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons.

Author information

1
Lehrstuhl für Pflanzenphysiologie, Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena Jena, Germany.
2
Lehrstuhl für Pflanzenphysiologie, Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena Jena, Germany ; KWS SAAT AG Einbeck, Germany.
3
Lehrstuhl für Pflanzenphysiologie, Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena Jena, Germany ; Department of General Botany, Institute of General Botany and Plant Physiology, Friedrich Schiller University Jena Jena, Germany.
4
Lehrstuhl für Pflanzenphysiologie, Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena Jena, Germany ; University of Grenoble-Alpes Grenoble, France ; CNRS, UMR5168 Grenoble, France ; Commissariat a L'energie Atomique (CEA), iRTSV, Laboratoire de Physiologie Cellulaire & Végétale Grenoble, France ; INRA, USC1359 Grenoble, France.

Abstract

Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.

KEYWORDS:

Sinapis alba; chloroplast; cotyledon; mass spectrometry; nucleic acids binding protein; post-translational modification

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