Name: a new approach for the comparative analysis of multiprotein complexes based on 15n metabolic labeling and quantitative mass spectrometry
Uploaded: 2014-03-13T13:00:00
Description: 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

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;...

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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			<td height="21" style="height:21px;width:392px;">Chemicals</td>
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			<td style="width:199px;"></td>
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			<td></td>
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			<td style="width:119px;">A2300</td>
			<td></td>
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			<td height="63" style="height:63px;">Acetonitrile Optigrade f&#252;r LC-MS</td>
			<td style="width:427px;">Diagonal 
			website: https://www.diagonal.de/</td>
			<td style="width:119px;">9340</td>
			<td style="width:199px;">harmful, work with gloves 
			see protocol text for further precautions</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Ammonium chloride 15N</td>
			<td style="width:427px;">Cambridge Isotope Laboratories 
			website: http://www.isotope.com/cil/index.cfm</td>
			<td style="width:119px;">39466-62-1</td>
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			<td height="42" style="height:42px;">Ammonium chloride 14N</td>
			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A0988</td>
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			<td style="width:119px;">A3583</td>
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			webiste: http://www.applichem.com/home/</td>
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			<td>10041653</td>
			<td></td>
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			<td height="42" style="height:42px;">n-Dodecyl-&#946;-D-maltoside</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0819</td>
			<td></td>
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			<td height="42" style="height:42px;">EDTA</td>
			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A2937</td>
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			<td height="42" style="height:42px;">Formic acid&#160;&#160;</td>
			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A3858</td>
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			<td height="42" style="height:42px;">Hepes</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3724</td>
			<td></td>
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			<td height="42" style="height:42px;">Magnesium chloride</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td>A4425</td>
			<td></td>
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			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2954</td>
			<td></td>
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			<td height="42" style="height:42px;">Phosphorous acid</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0989</td>
			<td></td>
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			<td height="42" style="height:42px;">Di-Potassium hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1042</td>
			<td></td>
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			<td height="42" style="height:42px;">Potassium di-hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1043</td>
			<td></td>
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			<td height="42" style="height:42px;">Sodium hydroxide</td>
			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A1551</td>
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			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A1125</td>
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			<td height="42" style="height:42px;">Tricine</td>
			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A3954</td>
			<td></td>
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			<td height="42" style="height:42px;width:392px;">Tris</td>
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			<td style="width:119px;">A2264</td>
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			<td height="42" style="height:42px;">Trypsin (sequencing grade modified) and Trypsin buffer</td>
			<td style="width:427px;">Promega 
			website: http://www.promega.de/</td>
			<td style="width:119px;">V5111</td>
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			<td height="41" style="height:41px;">Neboulizer (BioNeb cell disruptor)</td>
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			website: http://www.glascol.com/product/subproduct/id/75</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Centrifuge tubes (14 x 89 mm)&#160; 
			for preparation of takahashi style gradients</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;">331372</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Centrifuge tubes 25 x 89 mm 
			for preparation of thylakoid isolation gradients&#160;</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;">344058</td>
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			<td height="42" style="height:42px;width:392px;">Coulter Avanti Centrifuge J-20 XP</td>
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			<td height="42" style="height:42px;">Fuchs-Rosenthal cell couting chamber</td>
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			<td style="width:119px;">449/72</td>
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			<td height="63" style="height:63px;">Homogenizer (Potter) 50 ml&#160;</td>
			<td style="width:427px;">Fisherbrand 
			website: http://www.de.fishersci.com/index.php/defisherbrand</td>
			<td style="width:119px;">10618242</td>
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			<td style="width:119px;">105252220</td>
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			<td height="42" style="height:42px;width:392px;">Ultracentrifuge (Optima XPN-80 Ultracentrifuge)</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
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			<td height="42" style="height:42px;width:392px;">Antibodies&#160;</td>
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			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

a-new-approach-for-comparative-analysis-multiprotein-complexes-based

Research

JoVE Journal

Biology

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...