A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

Summary

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by 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 (¹⁴N/¹⁵N) 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.

Introduction

Photosynthetic processes in thylakoid membranes of plants and algae can function in a linear and cyclic mode. During linear electron flow (LEF) photosystem I (PSI), photosystem II (PSII) and cytochrome b₆/f ultimately transfer electrons from water to NADP+¹, leading to the generation of NADPH and ATP². In contrast, cyclic electron flow (CEF), which is known to be induced under diverse environmental conditions like state 2 ³ and anaerobic conditions⁴, results in the re-reduction of oxidized PSI by injecting electrons back into the electron transport chain. This process can take place either at the str....

Protocol

1. Culturing of Chlamydomonas

1. The following C. reinhardtii strains were used in the present study: WT cc124, WT cw15-arg7 (cell-wall deficient and arginine auxotroph), a ΔPSI mutant strain²⁹ and a pgrl1 knock-out strain⁴.
2. All strains were grown in Tris-Acetate-Phosphate (TAP)-medium³⁰, at 25 °C with a continuous light intensity of 20-50 µE/m² sec and shaking at 120 rpm. The culture of ΔPSI should be wrapped with some tissue paper for light exposure of <5 µE/m² sec.
3. The cultures of the labeled strain....

Results

The introduced quantitative proteomics approach aims to characterize the composition of multiprotein complexes in thylakoid membranes demonstrated by the comparative analysis of CEF-supercomplex components in genetically different C. reinhardtii strains. The described method has successfully been applied by Terashima et al.⁶ and comprises the isolation of thylakoid membranes from anaerobic grown cultures, followed by detergent solubilization. Subsequently, samples are loaded onto a sucrose de.......

Discussion

Different quantitative proteomic studies using stable isotope labeling have been published in the last years. In these experiments usually two different samples are compared, of which one sample is labeled with a stable isotope. Thereafter proteins or peptides from the two samples are combined in an equal ratio and further processed together⁴⁸. Such studies often intend to compare defined isolated cellular compartments (e.g. chloroplasts, mitochondria, or thylakoid membranes) exposed to different stre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Acetic acid	AppliChem	A0662	http://www.applichem.com/home/
Acetone	AppliChem	A2300	http://www.applichem.com/home/
Acetonitrile Optigrade für LC-MS	Diagonal	9340	Harmful, work with gloves. See protocol text for further precautions. https://www.diagonal.de/
Ammonium chloride 15N	Cambridge Isotope Laboratories	39466-62-1	http://www.isotope.com/cil/index.cfm
Ammonium chloride 14N	AppliChem	A0988	http://www.applichem.com/home/
Ammonium hydrogen phosphate	AppliChem	A3583	http://www.applichem.com/home/
Ammonium sulfate	AppliChem	A3598	http://www.applichem.com/home/
Coomassie brilliant blue R-250	Fisher Scientific	10041653	http://www.de.fishersci.com/index.php/deindex
n-Dodecyl-β-D-maltoside	AppliChem	A0819	http://www.applichem.com/home/
EDTA	AppliChem	A2937	http://www.applichem.com/home/
Formic acid	AppliChem	A3858	http://www.applichem.com/home/
HEPES	AppliChem	A3724	http://www.applichem.com/home/
Magnesium chloride	AppliChem	A4425	http://www.applichem.com/home/
Methanol	AppliChem	A2954	http://www.applichem.com/home/
Phosphorous acid	AppliChem	A0989	http://www.applichem.com/home/
Dipotassium hydrogen phosphate	AppliChem	A1042	http://www.applichem.com/home/
Potassium dihydrogen phosphate	AppliChem	A1043	http://www.applichem.com/home/
Sodium hydroxide	AppliChem	A1551	http://www.applichem.com/home/
Sucrose	AppliChem	A1125	http://www.applichem.com/home/
Tricine	AppliChem	A3954	http://www.applichem.com/home/
Tris	AppliChem	A2264	http://www.applichem.com/home/
Trypsin (sequencing grade modified) and Trypsin buffer	Promega	V5111	http://www.promega.de/
Equipment
Nebulizer (BioNeb cell disruptor)	Glas-Col		http://www.glascol.com/product/subproduct/id/75
Centrifuge tubes (14 mm x 89 mm)	Beckman Coulter	331372	for preparation of Takahashi style gradients http://www.beckmancoulter.de/
Centrifuge tubes 25 mm x 89 mm	Beckman Coulter	344058	for preparation of thylakoid isolation gradients. http://www.beckmancoulter.de/
Coulter Avanti Centrifuge J-20 XP	Beckman Coulter		http://www.beckmancoulter.de/
Fuchs-Rosenthal cell couting chamber	Diagonal	449/72	https://www.diagonal.de/
Homogenizer (Potter) 50 ml	Fisherbrand	10618242	http://www.de.fishersci.com/index.php/defisherbrand
Pistil for homogenizer	Fisherbrand	105252220	http://www.de.fishersci.com/index.php/defisherbrand
Ultracentrifuge (Optima XPN-80 Ultracentrifuge)	Beckman Coulter		website: http://www.beckmancoulter.de/
Other
Antibodies	Agrisera		http://www.agrisera.com/en/index.html