Chemical synapses are key organelles for neurotransmission. The coordinated actions of protein networks in diverse synaptic subdomains drive the sequential molecular events of transmitter release from the presynaptic bouton, activation of transmitter receptors located in the postsynaptic density and the changes of postsynaptic potential. Plastic change of synaptic efficacy is thought to be caused by the alteration of protein constituents and their interaction in the synapse. As a first step toward the understanding of the organization of synapse, several proteomics studies have been carried out to profile the protein constituents and the post-translational modifications in various rodent excitatory chemical synaptic subdomains, including postsynaptic density, synaptic vesicle and the synaptic phosphoproteome. Quantitative proteomics have been applied to examine the changes of synaptic proteins during brain development, in knockout mice model developed for studies of synapse physiology and in rodent models of brain disorders. These analyses generate testable hypotheses of synapse function and regulation both in health and disease. 相似文献
The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal. 相似文献
In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and funciotnal homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling. 相似文献
Introduction: Calmodulin (CaM) is a highly conserved Ca2+-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca2+ concentration into the regulation of synaptic transmission dynamics.
Areas covered: We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes.
Expert commentary: Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimental distance constraints are applicable best when obtained by combining different cross-linking strategies, i.e. by using cross-linkers with different spacer length and reactivity, and by using the incorporation of unnatural photo-reactive amino acids. Insights from structural proteomics can be used to generate CaM-insensitive mutants of CaM targets for functional studies in vitro or ideally in vivo. 相似文献
Synaptic vesicles are key organelles in neurotransmission. Vesicle integral or membrane-associated proteins mediate the various functions the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the pre-synaptic plasma membrane during exo- and endocytosis. Within the past two decades, converging work from several laboratories resulted in the molecular and functional characterization of the proteinaceous inventory of the synaptic vesicle compartment. However, up until recently and due to technical difficulties, it was impossible to screen the entire organelle thoroughly. Recent advances in membrane protein identification and mass spectrometry (MS) have dramatically promoted this field. A comparison of different techniques for elucidating the proteinaceous composition of synaptic vesicles revealed numerous overlaps but also remarkable differences in the protein constituents of the synaptic vesicle compartment, indicating that several protein separation techniques in combination with differing MS approaches are required to identify and characterize the synaptic vesicle proteome. This review highlights the power of various gel separation techniques and MS analyses for the characterization of the proteome of highly purified synaptic vesicles. Furthermore, the newly detected protein assignments to synaptic vesicles, especially those proteins which are new to the inventory of the synaptic vesicle proteome, are critically discussed. 相似文献
The presynaptic proteome controls neurotransmitter release and the short and long term structural and functional dynamics of the nerve terminal. Using a monoclonal antibody against synaptic vesicle protein 2 we immunopurified a presynaptic compartment containing the active zone with synaptic vesicles docked to the presynaptic plasma membrane as well as elements of the presynaptic cytomatrix. Individual protein bands separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were subjected to nanoscale-liquid chromatography electrospray ionization-tandem mass spectrometry. Combining this method with 2-dimensional benzyldimethyl- n -hexadecylammonium chloride/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight and immunodetection we identified 240 proteins comprising synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the pre-synapse. Four maxi-channels, three isoforms of voltage-dependent anion channels and the tweety homolog 1 were co-isolated with the docked synaptic vesicles. As revealed by in situ hybridization, tweety homolog 1 reveals a distinct expression pattern in the rodent brain. Our results add novel information to the proteome of the presynaptic active zone and suggest that in particular proteins potentially involved in the short and long term structural modulation of the mature presynaptic compartment deserve further detailed analysis. 相似文献
Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery. 相似文献
Introduction: While selected/multiple-reaction monitoring (SRM or MRM) is considered the gold standard for quantitative protein measurement, emerging data-independent acquisition (DIA) using high-resolution scans have opened a new dimension of high-throughput, comprehensive quantitative proteomics. These newer methodologies are particularly well suited for discovery of biomarker candidates from human disease samples, and for investigating and understanding human disease pathways.
Areas covered: This article reviews the current state of targeted and untargeted DIA mass spectrometry-based proteomic workflows, including SRM, parallel-reaction monitoring (PRM) and untargeted DIA (e.g., SWATH). Corresponding bioinformatics strategies, as well as application in biological and clinical studies are presented.
Expert commentary: Nascent application of highly-multiplexed untargeted DIA, such as SWATH, for accurate protein quantification from clinically relevant and disease-related samples shows great potential to comprehensively investigate biomarker candidates and understand disease. 相似文献
The postsynaptic density (PSD) is a massive multi-protein complex whose functions include positioning signalling molecules for induction of long-term potentiation (LTP) and depression (LTD) of synaptic strength. These processes are thought to underlie memory formation. To understand how the PSD coordinates bidirectional synaptic plasticity with different synaptic activation patterns, it is necessary to determine its three-dimensional structure. A structural model of the PSD is emerging from investigation of its molecular composition and connectivity, in addition to structural studies at different levels of resolution. Technical innovations including mass spectrometry of cross-linked proteins and super-resolution light microscopy can drive progress. Integrating different information relating to PSD structure is challenging since the structure is so large and complex. The reconstruction of a PSD subcomplex anchored by AKAP79 exemplifies on a small scale how integration can be achieved. With its entire molecular structure coming into focus, this is a unique opportunity to study the PSD. 相似文献
Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH‐MS is a specific variant of data‐independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH‐MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH‐MS data, a strategy based on peptide‐centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH‐MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH‐MS data using peptide‐centric scoring. Furthermore, concepts on how to improve SWATH‐MS data acquisition, potential trade‐offs of parameter settings and alternative data analysis strategies are discussed. 相似文献
Mass spectrometry-based proteomics greatly benefited from recent improvements in instrument performance and the development of bioinformatics solutions facilitating the high-throughput quantification of proteins in complex biological samples. In addition to quantification approaches using stable isotope labeling, label-free quantification has emerged as the method of choice for many laboratories. Over the last years, data-independent acquisition approaches have gained increasing popularity. The integration of ion mobility separation into commercial instruments enabled researchers to achieve deep proteome coverage from limiting sample amounts. Additionally, ion mobility provides a new dimension of separation for the quantitative assessment of complex proteomes, facilitating precise label-free quantification even of highly complex samples. The present work provides a thorough overview of the combination of ion mobility and data-independent acquisition-based label-free quantification LC-MS and its applications in biomedical research. 相似文献
Stable isotope labeling with amino acids in cell culture (SILAC) has risen as a powerful quantification technique in mass spectrometry (MS)–based proteomics in classical and modified forms. Previously, SILAC was limited to cultured cells because of the requirement of active protein synthesis; however, in recent years, it was expanded to model organisms and tissue samples. Specifically, the super-SILAC technique uses a mixture of SILAC-labeled cells as a spike-in standard for accurate quantification of unlabeled samples, thereby enabling quantification of human tissue samples. Here, we highlight the recent developments in super-SILAC and its application to the study of clinical samples, secretomes, post-translational modifications and organelle proteomes. Finally, we propose super-SILAC as a robust and accurate method that can be commercialized and applied to basic and clinical research. 相似文献
The identification of dynamic protein phosphorylation events is critical for understanding kinase/phosphatase‐regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Here we present a method to study the global kinome and phosphoproteome in tandem in a model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry‐based label‐free proteomics. A dual enrichment strategy targets intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide‐based phosphorylation enrichment. To increase depth of coverage, both data‐dependent and data‐independent (via SWATH, Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) mass spectrometric acquisitions were performed to obtain a more than 50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2250 phosphopeptides and 1314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events. 相似文献
A great deal of current biological and clinical research is directed at the interpretation of the information contained in the human genome sequence in terms of the structure, function and control of biological systems and processes. Proteomics, the systematic analysis of proteins, is becoming a critical component in this endeavor because proteomic measurements are carried out directly on proteins – the catalysts and effectors of essentially all biological functions. To detect changes in protein profiles that might provide important diagnostic or functional insights, proteomic analyses necessarily have to be quantitative. This article summarizes recent technological advances in quantitative proteomics. 相似文献