Protein Complex Identification and quantitative complexome by CN-PAGE.
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ABSTRACT:
SUBMITTER: Gorka M
PROVIDER: S-EPMC6687828 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The majority of cellular processes are carried out by protein complexes. Various size fractionation methods have previously been combined with mass spectrometry to identify protein complexes. However, most of these approaches lack the quantitative information which is required to understand how changes of protein complex abundance and composition affects metabolic fluxes. In this paper we present a proof of concept approach to quantitatively study the complexome in the model plant Arabidopsis thaliana at the end of the day (ED) and the end of the night (EN). We show that size-fractionation of native protein complexes by Clear-Native-PAGE (CN-PAGE), coupled with mass spectrometry can be used to establish abundance profiles along the molecular weight gradient. Furthermore, by deconvoluting complex protein abundance profiles, we were able to drastically improve the clustering of protein profiles. To identify putative interaction partners, and ultimately protein complexes, our approach calculates the Euclidian distance between protein profile pairs. Acceptable threshold values are based on a cut-off that is optimized by a receiver-operator characteristic (ROC) curve analysis. Our approach shows low technical variation and can easily be adapted to study in the complexome in any biological system.
2019-11-12 | PXD010919 | Pride
Project description:Protein complexes are a cornerstone of many biological processes and together they form various types of molecular machinery. A broad understanding of these protein complexes is crucial for revealing and building models of protein function and regulation. Pancreatic cancer is a highly lethal disease which is difficult to diagnose at early stage and even more difficult to cure. In this study, we applied a gradient clear native gel system combined with subsequent second-dimensional SDS-PAGE to separate protein complexes from cell lysates of SW1990 and PANC-1 pancreatic cancer cell lines with different degrees of differentiation. Ten heat-shock-protein- (HSP-) associated protein complexes were separated and identified, and the differentially expressed proteins related to cancers were also found, such as HSP60, protein disulfide-isomerase A4 (ERp72), and transitional endoplasmic reticulum ATPase (TER ATPase).
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Project description:Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.
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Project description:The EMBL-EBI Complex Portal is a knowledgebase of macromolecular complexes providing persistent stable identifiers. Entries are linked to literature evidence and provide details of complex membership, function, structure and complex-specific Gene Ontology annotations. Data are freely available and downloadable in HUPO-PSI community standards and missing entries can be requested for curation. In collaboration with Saccharomyces Genome Database and UniProt, the yeast complexome, a compendium of all known heteromeric assemblies from the model organism Saccharomyces cerevisiae, was curated. This expansion of knowledge and scope has led to a 50% increase in curated complexes compared to the previously published dataset, CYC2008. The yeast complexome is used as a reference resource for the analysis of complexes from large-scale experiments. Our analysis showed that genes coding for proteins in complexes tend to have more genetic interactions, are co-expressed with more genes, are more multifunctional, localize more often in the nucleus, and are more often involved in nucleic acid-related metabolic processes and processes where large machineries are the predominant functional drivers. A comparison to genetic interactions showed that about 40% of expanded co-complex pairs also have genetic interactions, suggesting strong functional links between complex members.
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Project description:Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is widely used in proteomics studies. Hundreds of proteins extracted from a biological sample can be separated and visualized on a 2D-PAGE gel. The interpretation of protein expression levels relies on the comparison of areas and intensities of the corresponding protein spots in 2D-PAGE gel images. However, determination of protein spot areas by the manual selection method is time-consuming and error-prone. The purpose of this research is to develop a highly automated program for the simultaneous detection and quantification of protein spots across a large number of 2D-PAGE protein gel images by using MATLAB image processing toolbox. This program will enhance the potential of using 2D-PAGE technique as a high throughput quantitative protein expression study tool. We developed MatGel, a simple and efficient program for protein spot area detection and intensity quantification from 2D-PAGE protein gel images. MatGel can detect and output the areas and mean intensities of corresponding protein spots across a large number of 2D-PAGE gel images simultaneously. Users also have options to adjust preferences at each step of image analysis. Basic knowledge with MATLAB programming language is required to run the program.• We developed MATLAB program MatGel to automate the determination of protein spots on 2D-PAGE protein gel images.• MatGel can analyze a large number of 2D-PAGE gel images simultaneously to minimize human errors.• MatGel is flexible and easy to use. Graphical abstract Image, graphical abstract
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Project description:Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80) and its membrane anchor protein RdhB (CbdbB3). We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory complexes in strain CBDB1 the remarkable result is not the detection of a standard ATPase but the absence of any other abundant membrane complex although an 11-subunit version of complex I (Nuo) is encoded in the genome.
| S-EPMC6005880 | biostudies-literature
Project description:Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions. Besides, complexome profiling has been utilized to study the dynamics of OXPHOS complexes, as well as the impact of an increasing number of mutations leading to mitochondrial disorders or rearrangements of the whole mitochondrial complexome. Here, we summarize the major findings obtained by this approach; emphasize its advantages and current limitations; discuss multiple examples on how this tool could be applied to further investigate pathophysiological mechanisms and comment on the latest advances and opportunity areas to keep developing this methodology.
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Project description:BackgroundProteins in organisms, rather than act alone, usually form protein complexes to perform cellular functions. We analyze the topological network structure of protein complexes and their component proteins in the budding yeast in terms of the bipartite network and its projections, where the complexes and proteins are its two distinct components. Compared to conventional protein-protein interaction networks, the networks from the protein complexes show more homogeneous structures than those of the binary protein interactions, implying the formation of complexes that cause a relatively more uniform number of interaction partners. In addition, we suggest a new optimization method to determine the abundance and function of protein complexes, based on the information of their global organization. Estimating abundance and biological functions is of great importance for many researches, by providing a quantitative description of cell behaviors, instead of just a "catalogues" of the lists of protein interactions.ResultsWith our new optimization method, we present genome-wide assignments of abundance and biological functions for complexes, as well as previously unknown abundance and functions of proteins, which can provide significant information for further investigations in proteomics. It is strongly supported by a number of biologically relevant examples, such as the relationship between the cytoskeleton proteins and signal transduction and the metabolic enzyme Eno2's involvement in the cell division process.ConclusionsWe believe that our methods and findings are applicable not only to the specific area of proteomics, but also to much broader areas of systems biology with the concept of optimization principle.
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Project description:The identification of protein tyrosine kinases (PTKs) was successfully achieved by renaturation in gels after SDS/PAGE. To this effect, samples were mixed with a PTK substrate, namely the polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr), M(r) from 30,000 to 94,000], and were simultaneously submitted to electrophoresis. Following guanidine hydrochloride denaturation, renaturation and phosphorylation with [gamma-32P]ATP, kinase activity was detected by autoradiography. When applied to cytosol from human hyperplastic prostate, eleven protein kinases were detected, among which one major (M(r) 50,000) and two minor proteins (M(r) 40,000 and 38,000) were identified as PTKs by the presence of phosphotyrosine. Incubation of the gel in hot alkali after glutaraldehyde cross-linking almost completely eliminated the detection of non-PTK enzymes. On the other hand, in the absence of poly(Glu,Tyr), no PTK activity was detected. Partial purification of cytosolic PTKs indicates that the native M(r) of the major phosphotransferase was 44,000, as estimated by gel filtration following ammonium sulphate precipitation and anion-exchange chromatography. Upon renaturation after electrophoresis, this fraction showed only one major band active on poly(Glu,Tyr) which was associated with the polypeptide of M(r) 50,000. This enzyme was also identified following two-dimensional electrophoresis and renaturation in the presence of poly(Glu,Tyr), allowing the determination of a pI in the range 7.5-7.8. Thus PTKs can be easily renatured following electrophoresis and rapidly identified on the basis of their M(r) and pI in both crude or partially purified preparations. With the crucial role played by PTKs in the activation of cell function and carcinogenesis, this procedure could be useful in the identification of such enzymes and in distinguishing them from their substrates in gels.
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Project description:BackgroundBlue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging.ResultsUsing a detergent mixture of 1% (w/V) n-dodecyl-β-D-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3-8% gel gradients for separation as methodological improvements in BN PAGE, several large photosystem (PS) I containing bands were detected. According to BN(/BN)/SDS PAGE and mass spectrometry analyses, these PSI bands proved to be PSI-NADH dehydrogenase-like megacomplexes more discernible in maize bundle sheath thylakoids, and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) more abundant in mesophyll thylakoids of lincomycin treated maize. For quantitative determination of the complexes and their comparison across taxa and physiological conditions, sample volumes applicable to the gel, correct baseline determination of the densitograms, evaluation methods to resolve complexes running together, calculation of their absolute/relative amounts and distribution among their different forms are proposed.ConclusionsHere we report our experience in Blue/Clear-Native polyacrylamide gel electrophoretic separation of thylakoid complexes, their identification, quantitative determination and comparison in different samples. The applied conditions represent a powerful methodology for the analysis of thylakoid mega- and supercomplexes.
| S-EPMC8895881 | biostudies-literature