Anatomical heterogeneity of tendon: Fascicular and interfascicular tendon compartments have distinct proteomic composition
Chavaunne T. ThorpeMandy J. PeffersDeborah M. SimpsonElizabeth HalliwellHazel R. C. ScreenPeter Clegg
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Abstract Tendon is a simple aligned fibre composite, consisting of collagen-rich fascicles surrounded by a softer interfascicular matrix (IFM). The composition and interactions between these material phases are fundamental in ensuring tissue mechanics meet functional requirements. However the IFM is poorly defined, therefore tendon structure-function relationships are incompletely understood. We hypothesised that the IFM has a more complex proteome, with faster turnover than the fascicular matrix (FM). Using laser-capture microdissection and mass spectrometry, we demonstrate that the IFM contains more proteins and that many proteins show differential abundance between matrix phases. The IFM contained more protein fragments (neopeptides), indicating greater matrix degradation in this compartment, which may act to maintain healthy tendon structure. Protein abundance did not alter with ageing, but neopeptide numbers decreased in the aged IFM, indicating decreased turnover which may contribute to age-related tendon injury. These data provide important insights into how differences in tendon composition and turnover contribute to tendon structure-function relationships and the effects of ageing.Keywords:
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Proteomics is the scientific study of proteins and their biochemical identification. Proteome of the host plant is believed to be regulated by environmental conditions as well as by relationship of plant with rhizobacteria. Some crucial factors affecting the plant proteome are disease state, insect damage, developmental stage, cell and tissue type, environmental stress and soil conditions. The plant-microbe interaction can induce an intense impact on protein constituents and proteomics. Proteomics can be stated as the indirect tool for unravelling the plant-microbe interactions. Application of proteomics can be used to study pathogenicity, pathogen-related stress and expression of antioxidant proteins produced in response to interaction of microbe with host plant. Therefore, proteome of the host plant can be studied to reveal the changes induced by a microbe inoculation in host plant. The function and the structure of protein molecules involved in plant-microbe interaction can be explored through proteomics. Several techniques including mass spectrometry and microarray technology can be used to study the proteome of the microbe. This technique is into the practice for the recent times, and knowledge of proteomics for studying plant-microbe interaction is still incomplete and insignificant. This chapter provides understanding of different techniques for the study of plant growth promotion of microbes through protein identification and quantification. The future prospective of utilising proteomics to elucidate the proteome-level fluctuations for improvisations in biofertiliser industry has been discussed.
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The dynamic range of the cellular proteome approaches seven orders of magnitude—from one copy per cell to ten million copies per cell. Since a proteome's abundance distribution represents a nearly symmetric bell-shape curve on the logarithmic copy number scale, detection of half of the expressed cellular proteome, i.e. approximately 5000 proteins, should be a relatively straightforward task with modern mass spectrometric instrumentation that exhibits four orders of magnitude of the dynamic range, while deeper proteome analysis should be progressively more difficult. Indeed, metaanalysis of 15 recent papers that claim detection of >5000 protein groups reveals that the half-proteome analyses currently requires ≈5 h of chromatographic separation, while deeper analyses yield on average ≤20 new proteins per hour of chromatographic gradient. Therefore, a typical proteomics experiment consists of a "high-content" part, with the detection rate of approximately 1000 proteins/h, and a "low-content" tail with much lower rate of discovery and respectively, lower cost efficiency. This result calls for disruptive innovation in deep proteomics analysis.
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Subcellular proteomics is a new field in proteomics, the proteome of important subcellular structures has been analyzed by many strategies and techniques. Recently,almost all of subcellular structures have been analyzed by proteomic methods and proteomic analysis of sub-organelles or protein complexes have been developed.Moreover,to study proteome dynamics on some physiological or pathological conditions,quantitative analysis or differential analysis has become new direction of subcellular proteome.However,the challenge of subcellular proteome is how to explain the localization of the identified proteins,which are contaminated or really localized in this structure?This problem is a difficult and inevitable topic of subcellular proteome.The recent progress on subcellular proteomics, the challenge and prospect of subcellular proteomics are discussed.
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Proteome analysis is usually performed by separating complex cellular protein extracts by two‐dimensional‐electrophoresis followed by protein identification using mass spectrometry. In this way proteins are compared from normal and diseased tissue in order to detect disease related protein changes. In a strict sense, however, this procedure cannot be called proteome analysis: the tools of proteomics are used just to detect some interesting proteins which are then investigated by protein chemistry as usual. Real proteome research would be studying the cellular proteome as a whole, its composition, organization and its kind of action. At present however, we have no idea how a proteome works as a whole; we have not even a theory about that. If we would know how the proteome of a cell type is arranged, we probably would alter our strategy to detect and analyze disease‐related proteins. I will present a theory of proteomics and show some results from our laboratory which support this theory. The results come from investigations of the mouse brain proteome and include mouse models for neurodegenerative diseases.
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Proteins are the chief performers for physiological functions in a cell,and often function as protein complexes or interact with nucleic acids.A proteome represents all the proteins expressed by a cell,a tissue,or an organism at a defined time point under certain circumstances.Therefore,proteomes will undergo dynamic changes with alterations in time and space.The aim of proteomics is to globally study structures,functions and expression modes of proteome as well as interactions among proteins in a proteome.Based on the contents of study,proteomics can be classified as expression proteomics,structural proteomics and functional proteomics,respectively.The study of proteomes will help understand structures of proteins,functions of cells,nature and active rules of life,and will provide scientific basis for diagnosis and treatment of diseases as well as development of novel drugs.The techniques commonly used in proteomic studies include two-dimentional polyacrylamide gel electrophoresis,mass spectrometry,yeast two-hybrid system and protein chips.This article will provide a brief overview of proteomics and techniques frequently utilized for proteomic studies.
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The use of proteome-expression databases may facilitate cancer research. Proteome-expression data derived from a sufficient number of clinical cases may help establish the molecular background of malignant tumors. Recently, we published a proteome expression database, the Genome Medicine Database of Japan (GeMDBJ) Proteomics. The GeMDBJ Proteomics includes the proteome data of surgically resected tissues and tissue-cultured cells of various malignancies, as well as the corresponding biological and clinicopathological data. The proteome data in GeMDBJ Proteomics include expression data produced using 2D PAGE with the immobilized pH gradient gel and DIGE technology, and the protein identification data by liquid chromatography tandem mass spectrometry. GeMDBJ Proteomics can be freely accessed online without registration.
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Rapidly progressing proteomics techniques have been widely adopted in most areas of biology and medicine. One of the distinct advantages of proteomic analysis, not attainable with RNA expression data, is the ability to fractionate the cell's proteins into various subpopulations. In neurology and neuroscience, many applications have been entertained in neurotoxicology and neurometabolism, and used in the determination of specific proteomic aspects of individual brain areas and body fluids in neurodegeneration to identify biomarkers. Investigation of brain protein groups in neurodegeneration, such as enzymes, cytoskeleton proteins, chaperones, synaptosomal proteins and antioxidant proteins, is in progress as phenotype related proteomics. The concomitant detection of several hundred proteins on a gel provides sufficiently comprehensive data to determine a pathophysiological protein network and its peripheral representatives. The rapid spread of proteomics technology, which principally consists of two–dimensional gel electrophoresis (2–DE) with in–gel protein digestion of protein spots and identification by mass–spectrometry, has provided an explosive amount of results. In this study, quantitative proteome analysis of AD brains was performed using two–dimensional (2–D) gels. For the higher resolution of 2–DE including the detailed analysis of hydrophobic proteins, we sequentially extracted brain protein. The identified proteins thus examined include enzymes, heat shock proteins and cell structure proteins. We also examined body fluids proteomes to determine the same proteins as in brain proteome using two–dimensional difference gel electrophoresis (2D–DIGE). In neuroscience, ‘neuroproteomics’ (proteomics in the central nervous system) is still in its infancy, with a paucity of studies in the context of the brain. There are several other analytical problems which also need to be overcome, and once solved, will allow for a more comprehensive analysis of the individual disease process.
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Localized proteomics of specific cell populations will greatly aid understanding of the complex etiology of neurodegenerative diseases. The use of proteomics offers considerable advantages over traditional protein detection techniques and is likely to be particularly useful for studying neurodegenerative diseases, as selective vulnerability of specific neuron populations is a defining characteristic of these diseases. Pathogenesis is likely to involve complex interactions between multiple proteins. Therefore, a technique that can quantify all proteins in a cell population at the same time is preferred. Methodology that enables proteomics on cells isolated from archived formalin-fixed, paraffin-embedded (FFPE) tissue is of particular benefit for future studies because of the large FFPE tissue repositories. Here, we describe our protocol for localized proteomics of neurons microdissected from archived FFPE Alzheimer's disease brain tissue. Neurons are visualized by staining with cresyl violet and isolated using laser capture microdissection (LCM). Collection of 1.5 mm2 total tissue area (approximately 12,000 neurons) provides enough material for quantitative mass spectrometry. The excised tissue is directly deparaffinized, reduced, alkylated, proteolytically digested, desalted, and analyzed using LC-MS.
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A bstract : Functional decline of tissues in aged animals is a result of cellular aging. Though any process of somatic cell aging basically depends on genomic instructions, phenotypes of aged cells are expressed in a given internal environment of each cell type that was made with translated proteins and post‐translationally modified products. Therefore, research on age‐dependent protein alterations in each cell type is very important in clarifying mechanisms of aging. The novel term “proteome” is a compound of “protein” and “genome,” which means constitutive whole proteins including post‐translationally modified products in a cell type. Proteomics is a novel strategy for analyzing proteomes. In proteomics, high resolution two‐dimensional electrophoresis is exclusively performed for isolation of proteins followed by mass spectrometry for identification of proteins and determination of modifications. Thus, proteomics is becoming appreciated as a powerful tool to find out proteins responsible for cellular aging, symptoms of senility and geriatric diseases.
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