Universität zu Köln

Lundquist, Peter K., Mantegazza, Otho, Stefanski, Anja, Stuehler, Kai and Weber, Andreas P. M. (2017). Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts. Mol. Plant., 10 (1). S. 197 - 212. CAMBRIDGE: CELL PRESS. ISSN 1752-9867

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Abstract

Blue native-PAGE (BN-PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facilitate scaled-up comparative analyses, as we demonstrate with the reticulata chloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illustrate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Lundquist, Peter K. UNSPECIFIED UNSPECIFIED UNSPECIFIED Mantegazza, Otho UNSPECIFIED UNSPECIFIED UNSPECIFIED Stefanski, Anja UNSPECIFIED UNSPECIFIED UNSPECIFIED Stuehler, Kai UNSPECIFIED UNSPECIFIED UNSPECIFIED Weber, Andreas P. M. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-242672
DOI:	10.1016/j.molp.2016.10.011
Journal or Publication Title:	Mol. Plant.
Volume:	10
Number:	1
Page Range:	S. 197 - 212
Date:	2017
Publisher:	CELL PRESS
Place of Publication:	CAMBRIDGE
ISSN:	1752-9867
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language BLUE NATIVE ELECTROPHORESIS; MEMBRANE-PROTEIN COMPLEXES; PHOTOSYSTEM-II; SACCHAROMYCES-CEREVISIAE; PROTEOMICS; KINASE; BIOGENESIS; FERREDOXIN; ENVELOPE; REPAIR Multiple languages Biochemistry & Molecular Biology; Plant Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/24267