M.H. acknowledges support from the &#8220;Deutsche Forschungsgemeinschaft&#8221; (DFG). Author contributions:&#160;M.H. designed research; K.T., J. S. and M.T. performed research and analyzed the data; K.T. and M.H. wrote the paper.

1. Culturing of Chlamydomonas
<ol>
	<li>The following C. reinhardtii strains were used in the present study: WT cc124, WT cw15-arg7 (cell-wall deficient and arginine auxotroph), a &#916;PSI mutant strain29 and a pgrl1 knock-out strain4.</li>
	<li>All strains were grown in Tris-Acetate-Phosphate (TAP)-medium30, at 25&#160;&#176;C with a continuous light intensity of 20-50 &#181;E/m2 sec&#160;and shaking at 120&#160;rpm. The culture of &#916;...

The authors declare&#160;no competing financial interest.

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...

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&#160;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 stromal side of the cytochrome b6/f complex1 or at the plastoquinone pool5 and generates ATP, but no NADPH2.

The aim of the presented protocol is to demonstrate a mass spectrometry (MS) based method for the comparative, quantitative analysis of multiprotein complexes in thylakoid membranes of Chlamydomonas reinhardtii to gain insight into the composition of these complexes under different conditions (exemplified by comparing genetically different strains). This approach was applied in a publication by Terashima et al. in 2012 showing a Ca2+-dependent regulation of CEF in C. reinhardtii mediated by a multiprotein complex including the proteins CAS, ANR1, and PGRL16. The procedure will be explained by comparatively analyzing the composition of the CEF-supercomplex in two genetically different strains, thereby taking advantage of labeling one of the two strains with heavy nitrogen (15N). Briefly, the protocol includes the preparation of thylakoid membranes, followed by detergent solubilization and fractionation of photosynthetic complexes in a sucrose density gradient. After fractionation of the gradient, selected fractions of two strains are mixed based on equal volume, separated by SDS-PAGE followed by in-gel digestion and subsequent quantitative MS analysis.

As mentioned above, CEF is induced under different environmental conditions and a publication from 2010 demonstrates the isolation of a functional CEF-supercomplex from state&#160;2 locked cells of C. reinhardtii7, which was performed by separating solubilized thylakoid membranes on a sucrose density gradient during ultracentrifugation. Different from Iwai et al.7, the presented protocol describes the isolation of the CEF-supercomplex from anaerobic grown C. reinhardtii cultures by following an alternative procedure. This comprises changes in the thylakoid isolation protocol as well as differences concerning the solubilization step and the separation of protein complexes by ultracentrifugation. In the present protocol, thylakoid membranes are isolated by applying the procedure published by Chua and Bennoun8, while the buffers used for thylakoid preparation by Iwai et al. contained 25 mM Mes, 0.33 M sucrose, 5 mM MgCl2, 1.5 mM NaCl (pH 6.5) as described9. The solubilization was performed with 0.7-0.8% detergent (n-tridecyl-&#946;-D-maltoside) for 30&#160;min on ice in the case of Iwai and coworkers, while the solubilization method described here relies on the use of 0.9% detergent (n-Dodecyl-&#946;-D-maltoside (&#946;-DM)) and is performed for only 20 min on ice. Both groups used 0.8 mg of chlorophyll per ml for the solubilization with the respective detergent. For the separation of photosynthetic complexes from solubilized thylakoid membranes Iwai et al. applied sucrose concentrations between 0.1-1.3&#160;M, whereas the authors of this protocol used concentrations ranging from 0.4-1.3 M. The last difference is the centrifugation speed, which is lower compared to the earlier publication.

Solubilization of thylakoid membranes with nonionic detergents followed by sucrose density gradient fractionation has already been applied in numerous studies ranging from the 1980s until today7, 9-14 and also the application of metabolic labeling of proteins is a widespread method in the proteomics field. The described approach applies the 15N metabolic labeling for one of the two compared strains by culturing it in the presence of heavy nitrogen as sole nitrogen source in the form of 15N NH4Cl, which is incorporated into all amino acids leading to a mass shift depending on the amino acid sequence of the peptide. When analyzing a mixture of 14N and 15N within one MS run, this mass shift can be used to determine the sample origin for each peptide and relative peptide abundances can be calculated representing relative abundances for the corresponding protein15.

Numerous quantitative proteomics studies on C. reinhardtii are available, which compare a defined amount of protein to analyze changes in the proteome between experimental conditions (e.g. changes in the proteome due to nutrient16-19 or light stress20,21). Compared to those studies, in the currently presented approach equal volumes of samples are combined and analyzed. This setup allows to study the migration behavior of proteins within the gradient and moreover to analyze the composition of different complexes with respect to the investigated strains.
This method will be explained by mainly concentrating on three proteins: The first candidate is the chloroplast-localized calcium sensor protein CAS, which was shown to be involved in photo-acclimation in C. reinhardtii22. Calcium is considered to be an important signaling ion for pathways that are activated due to different biotic and abiotic stresses finally leading to changes in gene expression and cell physiology23 and it was proposed that chloroplasts might contribute to cellular Ca2+ signaling via the CAS protein22,24,25. The second protein is ANR1 (anaerobic response 1 6), a protein that was shown to be induced under anoxic growing conditions in C. reinhardtii26. Notably, CAS as well as ANR1 were identified as subunits of the CEF-supercomplex and moreover, by using reverse genetic approaches, it was demonstrated that both proteins contribute functionally to CEF in vivo6, supporting their role as functional subunits of this protein complex. The third protein is the thylakoid protein PGR5-Like&#160;1 (PGRL1), which was shown to be involved in CEF in Chlamydomonas4,27 &#160;as well as in Arabidopsis5,28 &#160;and was also identified in the work of Iwai et al.7&#160;

This approach will be presented by showing the results of two different experiments: wildtype (WT) versus (vs.) a &#916;PSI29 strain, exhibiting a deletion of the psab gene, coding for an essential photosystem I subunit, which is also part of the CEF-supercomplex and WT vs. a pgrl1 knock-out strain4. For each of those experiments the quantitative composition of the CEF-supercomplex between a 15N- and a 14N-labeled strain has been compared.

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a new approach for the comparative analysis of multiprotein complexes based on 15n metabolic labeling and quantitative mass spectrometry

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.

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...

Watch this Scientific Journal Video about A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry at JoVE.com

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

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;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.

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

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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12.1K Views.  University of Münster. 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.