title: A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry.
abstract: The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling ((14)N/(15)N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.
authors.affiliation: Institute of Plant Biology and Biotechnology, University of Münster.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Trompelt
authors.affiliation: Institute of Plant Biology and Biotechnology, University of Münster.
authors.forename: Janina
authors.initials: J
authors.lastname: Steinbeck
authors.affiliation: Institute of Plant Biology and Biotechnology, University of Münster; Department of Plant Biology, Carnegie Institution for Science.
authors.forename: Mia
authors.initials: M
authors.lastname: Terashima
authors.affiliation: Institute of Plant Biology and Biotechnology, University of Münster; mhippler@uni-muenster.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Hippler
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000032:analysis* / Q000737:chemistry; D009587:Nitrogen Isotopes
chemical_list: D046912:Multiprotein Complexes; D009587:Nitrogen Isotopes