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Results for “Functional proteome”

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At least 55 records · Page 3Linked to original sources
PubMed2006

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum.

Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.

Bacterial Proteins↗
PubMed2002

Functional proteomics of breast cancer for signal pathway profiling and target discovery.

The near completion of human genome sequencing and the introduction of mass spectrometry combined with advanced bioinformatics for protein identification have led to the emergence of proteomics as a powerful tool for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified proteins of potential clinical interest, such as the molecular chaperone 14-3-3 sigma and the heat shock protein HSP90, and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer cells. As illustrated by fibroblast growth factor-2 and the H19 noncoding oncogenic mRNA, proteomics is a pertinent approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth and metastasis. Together with genomics, proteomics is now providing a way to define molecular processes involved in breast carcinogenesis and to identify new therapeutic targets. The next challenge will be the introduction of proteomics as a tool for the clinic, for the establishment of diagnosis, prognosis, and the monitoring of treatment; however, this ambitious goal still requires further technological progress in the field.

Breast Neoplasms↗
PubMed2025

Decoding Arginine Dimethylation Isomers via pH-Tuned Reactivity with Methylglyoxal: A Chemical Approach for Functional Proteomics.

Arginine dimethylation, encompassing asymmetric and symmetric configurations, represents a fundamental post-translational modification. Despite sharing identical chemical formulas, the two arginine dimethylation isomers exhibit different or even opposite biological effects. Therefore, it is necessary to determine their specific structure before conducting a further biological investigation. However, current methods for arginine dimethylation analysis face great challenges in efficient isomer differentiation, preventing the functional investigation of arginine dimethylation. To overcome this obstacle, herein, we introduce a novel chemical strategy leveraging pH-tuned reactivity with methylglyoxal (MGO) to decode these dimethylation isomers. By utilizing molecular dynamics simulation analysis, we revealed the different chemical reactivities of asymmetrically and symmetrically dimethylated arginine when reacted with MGO at different pH conditions. This property enabled the development of a pH-tuned chemical strategy by combining the MGO reaction with boronate affinity enrichment to simultaneously enrich and differentiate the dimethylation isomers. This strategy can effectively distinguish dimethylated arginine isomers in complex cell samples, and the good feasibility of this strategy was verified by orthogonal validation with the neutral loss. Of the obtained data set, this strategy identified sDMA at R112 of SNRPN, which is confirmed to be modified by PRMT5. Further functional analysis reveals its crucial role in maintaining protein stability and in regulating spliceosome assembly. Overall, by transforming the inherent pH sensitivity of MGO reactions into a powerful analytical tool, our work establishes the first chemical platform for functional proteomic dissection of arginine dimethylation isomers, which paves the way for further regulating mechanism investigations of protein methylation.

Pyruvaldehyde↗
PubMed2005

Functional proteomic screens in therapeutic protein drug discovery.

Biopharmaceuticals, mainly protein-based therapeutics, are rapidly being developed for several disease indications. However, most biopharmaceuticals are 'me-too' drugs or are being developed against the same handful of targets. Thus, the potential of biopharmaceuticals is not being fully exploited. The bottleneck is still the lack of validated targets and the means of implementing the appropriate target validation technology. Functional proteomic screens provide a rapid route to accelerate the discovery and development of the next generation of biopharmaceuticals.

Antibodies↗
PubMed2002

Activity-based probes for functional proteomics.

Achieving an understanding of the functional role of all proteins expressed by a complex organism will require the development of methods that enable the rapid monitoring of protein function on a global scale. Current genomics and proteomics technologies fall short of this goal since they measure only the relative abundance of transcripts and proteins, respectively. Recent efforts in several laboratories have led to the development of tagged chemical probes that selectively react with families of active enzymes based on shared mechanistic features. These activity-based probes (ABPs) permit the quantitation and comparison of multiple protein activities simultaneously in complex proteomes. In this paper, the general properties and design features of ABPs will be discussed with an emphasis on the use of ABPs for activity-based proteome analysis.

Affinity Labels↗
PubMed2003

Analysis of transglutaminase protein substrates by functional proteomics.

Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.

Affinity Labels↗
PubMed2002

Monitoring of gene expression by functional proteomics: response of human lung fibroblast cells to stimulation by endothelin-1.

Proteomic methods have been used to monitor changes in protein synthesis in the first 4 h following stimulation of human lung fibroblasts with endothelin-1. Using pulsed [(35)S]methionine labeling, about 70 proteins with altered protein synthesis could be detected, and the 35 proteins showing the largest changes were identified by mass spectrometry. The observed proteins included unexpected proteins such as Sox5, two isoforms of Rab14, Rab3A, translationally controlled tumor protein, and one protein of previously unknown function. There was a wide range of different kinetic behavior, and groups of functionally linked proteins such as Rab14, nucleophosmin,and cyclin-dependent kinase inhibitor 1B could be detected from similar kinetics. We propose that the functional proteomic methods are competitive with and have some advantages compared to expression profiling methods for monitoring gene expression.

Autoradiography↗
PubMed2002

Functional proteomics: The goalposts are moving.

Holistic understanding of protein function is a primary goal of the post-genome sequencing era. Functional genomic approaches are powerful and relatively straightforward but produce an incomplete picture at the protein level. Proteomics offers physiologically enriched insights to protein function, and ongoing advances are enabling proteome analyses to proceed with increased depth and efficiency. Exciting discoveries have emerged recently amidst growing awareness of the power of proteomics. However, while proven as a potent discovery tool, proteomics is under pressure to provide improved functional value particularly in concert with other investigative approaches. As reviewed here for ERp29, a recently discovered endoplasmic reticulum protein, the role of novel proteins can remain elusive even after substantial information has accrued. Thousands more proteins of uncertain function will be unveiled in the near future. Consequently, the goalposts are moving for proteomics both through increasing demand for high-value functional information and improving capacity to deliver.

Animals↗
PubMed2006

Interaction of zonula occludens-1 (ZO-1) with alpha-actinin-4: application of functional proteomics for identification of PDZ domain-associated proteins.

The use of recombinant "bait" proteins to capture protein-binding partners, followed by identification of protein interaction networks by mass spectrometry (MS), has gained popularity and widespread acceptance. We have developed an approach using recombinant PDZ protein interaction modules of the membrane-associated guanylate kinase (MAGUK) protein zonula occludens-1 (ZO-1) to pull-down and screen for proteins that interact with these modules via their PDZ domain binding motifs. Identification of proteins by MS of pull-down material was achieved using a vacuum-based chromatography sample preparation device designed for matrix-assisted laser desorption/ionization (MALDI) MS. MS analysis of tryptic fragments in pull-down material revealed a number of potential ZO-1 interacting candidates, including the presence of peptides corresponding to the cortical membrane scaffolding protein alpha-actinin-4. Interaction of alpha-actinin-4 with ZO-1 was confirmed by coimmunoprecipitation of these two proteins from cultured cells, as well as from brain, liver, and heart, and by immunoblot detection of alpha-actinin-4 after pull-down with the first PDZ domain of ZO-1. In contrast, the highly homologous alpha-actinin family member, alpha-actinin-1, displayed no association with ZO-1. Immunofluorescence showed colocalization of alpha-actinin-4 with ZO-1 in cultured HeLa and C6 glioma cells, as well as in a variety of tissues in vivo, including brain, heart, liver, and lung. This study demonstrates the utility of MS-based functional proteomics for identifying cellular components of the ZO-1 scaffolding network. Our finding of the interaction of ZO-1 with alpha-actinin-4 provides a mechanism for linking the known protein recruitment and signaling activities of ZO-1 with alpha-actinin-4-associated plasma membrane proteins that have regulatory activities at cell-cell and cell-extracellular matrix contacts.

Actinin↗
PubMed2003

Project management system for structural and functional proteomics: Sesame.

A computing infrastructure (Sesame) has been designed to manage and link individual steps in complex projects. Sesame is being developed to support a large-scale structural proteomics pilot project. When complete, the system is expected to manage all steps from target selection to data-bank deposition and report writing. We report here on the design criteria of the Sesame system and on results demonstrating successful achievement of the basic goals of its architecture. The Sesame software package, which follows the client/server paradigm, consists of a framework, which supports secure interactions among the three tiers of the system (the client, server, and database tiers), and application modules that carry out specific tasks. The framework utilizes industry standards. The client tier is written in Java2 and can be accessed anywhere through the Internet. All the development on the server tier is also carried out in Java2 so as to accommodate a wide variety of computer platforms. The database tier employs a commercial database management system. Each Sesame application module consists of a simple user interface in the client tier, corresponding objects in the server tier, and relevant data stored in the centralized database. For security, access to stored data is controlled by access privileges. The system facilitates both local and remote collaborations. Because users interact with the system using Java Web Start or through a web browser, access is limited only by the availability of an Internet connection. We describe several Sesame modules that have been developed to the point where they are being utilized routinely to support steps involved in structural and functional proteomics. This software is available to parties interested in using it and assisting to guide its further development.

Database Management Systems↗
PubMed2001

Nucleocytoplasmic O-glycosylation: O-GlcNAc and functional proteomics.

The molecular complexity that defines different cell types and their biological responses occurs at the level of the cell's proteome. The recent increase in availability of genomic sequence information is a valuable tool for the field of proteomics. While most proteomic studies focus on differential expression levels, post-translational modifications such as phosphorylation, glycosylation, and acetylation, provide additional levels of functional complexity to the cell's proteome. The reversible post-translational modification O-linked beta-N-acetylglucosamine (O-GlcNAc) is found on serines and threonines of nuclear and cytoplasmic proteins. It appears to be as widespread as phosphorylation. While phosphorylation is recognized as a fundamental mechanism for controlling protein function, less is known about the specific roles of O-GlcNAc modification. However, evidence is building that O-GlcNAc may compete with phosphate at some sites of attachment. Aberrant O-GlcNAc modification has been linked to several disease states, including diabetes and Alzheimer's disease. Regulated enzymes catalyzing the addition (O-GlcNAc transferase, OGT) and removal (O-GlcNAcase) of the modification have been cloned and OGT is required for life at the single cell level. Here we review the properties of O-GlcNAc that suggest it is a regulatory modification analogous to phosphorylation. We also discuss the use of comparative functional proteomics to elucidate functions for this ubiquitous intracellular carbohydrate modification.

Acetylglucosamine↗
PubMed2006

Optimizing the surface plasmon resonance/mass spectrometry interface for functional proteomics applications: how to avoid and utilize nonspecific adsorption.

A great challenge in functional or interaction proteomics is to map protein networks and establish a functional relationship between expressed proteins and their effects on cellular processes. These cellular processes can be studied by characterizing binding partners to a "bait" protein against a complex background of other molecules present in cells, tissues, or biological fluids. This so-called ligand fishing process can be performed by combining surface plasmon resonance biosensors with MS. This combination generates a unique and automated method to quantify and characterize biomolecular interactions, and identify the interaction partners. A general problem in chip-based affinity separation systems is the large surface-to-volume ratio of the fluidic system. Extreme care, therefore, is required to avoid nonspecific adsorption, resulting in losses of the target protein and carry-over during the affinity purification process, which may lead to unwanted signals in the final MS analysis and a reduction in sensitivity. In this study, carry-over of protein and low-molecular weight substances has been investigated systematically and cleaning strategies are presented. Furthermore, it is demonstrated that by the introduction of colloidal particles as a capturing and transporting agent, the recovery yield of the affinity-purified ligand could be improved nearly twofold.

Adsorption↗
PubMed2004

Global analysis of predicted proteomes: functional adaptation of physical properties.

The physical characteristics of proteins are fundamentally important in organismal function. We used the complete predicted proteomes of >100 organisms spanning the three domains of life to investigate the comparative biology and evolution of proteomes. Theoretical 2D gels were constructed with axes of protein mass and charge (pI) and converted to density estimates comparable across all types and sizes of proteome. We asked whether we could detect general patterns of proteome conservation and variation. The overall pattern of theoretical 2D gels was strongly conserved across all life forms. Nevertheless, coevolved replicons from the same organism (different chromosomes or plasmid and host chromosomes) encode proteomes more similar to each other than those from different organisms. Furthermore, there was disparity between the membrane and nonmembrane subproteomes within organisms (proteins of membrane proteomes are on the average more basic and heavier) and their variation across organisms, suggesting that membrane proteomes evolve most rapidly. Experimentally, a significant positive relationship independent of phylogeny was found between the predicted proteome and Biolog profile, a measure associated with the ecological niche. Finally, we show that, for the smallest and most alkaline proteomes, there is a negative relationship between proteome size and basicity. This relationship is not adequately explained by AT bias at the DNA sequence level. Together, these data provide evidence of functional adaptation in the properties of complete proteomes.

Animals↗
PubMed2005

Toward functional proteomics of alveolar macrophages.

Alveolar macrophages (AM) belong to a phenotype of macrophages with distinct biological functions and important pathophysiological roles in lung health and disease. The molecular details determining AM differentiation from blood monocytes and AM roles in lung homeostasis are largely unknown. With the use of different technological platforms, advances in the field of proteomics have made it possible to search for differences in protein expression between AM and their precursor monocytes. Proteome features of each cell type provide new clues into understanding mononuclear phagocyte biology. In-depth analyses using subproteomics and subcellular proteomics offer additional information by providing greater protein resolution and detection sensitivity. With the use of proteomic techniques, large-scale mapping of phosphorylation differences between the cell types have become possible. Furthermore, two-dimensional gel proteomics can detect germline protein variants and evaluate the impact of protein polymorphisms on an individual's susceptibility to disease. Finally, surface-enhanced laser desorption and ionization (SELDI) time-of-flight mass spectrometry offers an alternative method to recognizing differences in protein patterns between AM and monocytes or between AM under different pathological conditions. This review details the current status of this field and outlines future directions in functional proteomic analyses of AM and monocytes. Furthermore, this review presents viewpoints of integrating proteomics with translational topics in lung diseases to define the mechanisms of disease and to uncover new diagnostic and therapeutic targets.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Exploration of the functional proteome: lessons from lipid rafts.

Lipid rafts are liquid-ordered cholesterol and glycolipid-enriched membrane microdomains that act as sorting devices for the accumulation of acylated signaling molecules. Lipid rafts are implicated in receptor signaling, protein and membrane trafficking, cytoskeletal re-organization and the entry of infectious organisms into cells. Several recent studies have investigated the composition of the lipid raft proteome by mass spectrometry. Here, those studies and the insights they afford into raft function are reviewed. Lipid rafts contain hydrophobic proteins, posing problems of isolation, recovery and analysis. Recent advances in proteome preparation that extend the boundaries of protein detection in lipid rafts are described and their implications for the exploration of the functional proteome are discussed.

Humans↗
PubMed2001

Intramers as promising new tools in functional proteomics.

Aptamers are valuable tools for studying numerous aspects of biological processes, opening up new experimental opportunities to analyse the function of a wide range of cellular molecules. Functional RNA molecules can be rapidly selected in vitro from complex combinatorial mixtures of different sequences. Recently, it was shown that in vitro selection processes can be automated: the first generation selection robots will soon mean aptamers for several targets can be isolated in parallel within days rather than weeks. Aptamers not only exhibit highly specific molecular recognition properties but are also able to modulate the function of their cognate targets in a highly specific manner by agonistic or antagonistic mechanisms. These properties prompted the development of novel technologies to exploit the use of aptamers to modulate distinct functions of biological targets. Recent controlled expression of aptamers inside cells demonstrated their impressive potential as rapidly generated intracellular inhibitors of biomolecules. Intracellularly applied aptamers are also called 'intramers'. Here we discuss recent developments and strategies for intramer-based technologies that have the potential to greatly facilitate characterisation of unknown protein functions in the context of their natural expression status in vivo. Thus, intramer-based technologies offer many promising applications in functional genomics, proteomics and drug discovery.

Bacterial Proteins↗
PubMed2005

High-resolution functional proteomics by active-site peptide profiling.

Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.

Animals↗