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PubMed2005

Functional proteomics: a promising approach to find novel components of the circadian system.

In the postgenome era, the analysis of entire subproteomes in correlation with their function has emerged due to high throughput technologies. Early approaches have been initiated to identify novel components of the circadian system. For example, in the marine dinoflagellate Lingulodinium polyedra, a chronobiological proteome assay was performed, which resulted in the identification of already known circadian expressed proteins as well as novel temporal controlled proteins involved in metabolic pathways. In the green alga Chlamydomonas reinhardtii, two circadian expressed proteins (a protein disulfide isomerase and a tetratricopeptide repeat protein) were identified by functional proteomics. Also, the first hints of temporal control within chloroplast proteins of Arabidopsis thaliana were identified by proteome analysis.

Algal Proteins↗
PubMed2004

Expression and functional proteomics studies in colorectal cancer.

Cell dysfunction results from multiple rather than from single gene interactions in the majority of colorectal cancers (CRC). Proteins, not mRNA, are the functional molecules in the cell, and the relationship between gene expression measured at the mRNA level and the corresponding protein level is not linear. Current proteomics tools allow for the determination of post-translational modifications, and hence the presence of protein isoforms--some of them being disease-relevant. Thus, proteomics approaches are a welcome complement to traditional genetic approaches. In CRC, expression proteomics studies were carried out with colorectal cell lines, whole tissue biopsies, and purified epithelial cells. For CRC, two-dimensional electrophoresis reference maps, protein, and membrane protein databases are available on the internet. Functional proteomics studies have been performed to better understand signaling pathways, to characterize the molecular targets of novel drugs, and to identify tumor-associated antigens in CRC. The increasing use of proteomics technologies, when addressing clinical problems, will accelerate the evolution towards personalized medicine in CRC.

Adenocarcinoma↗
PubMed2000

PROfusion: genetically tagged proteins for functional proteomics and beyond.

As a new enabling technology in functional genomics, Phylos, Inc. has pioneered PROfusiontrade mark technology, whereby proteins are covalently tagged with their own genetic information (mRNA). Using this technology, both synthetic and natural libraries (representing the repertoire of proteins naturally expressed in a given cell type or tissue source) have been constructed. Due to the in vitro nature of library construction, Phylos libraries are the largest described to date, up to 10(14) in size. From a library of such molecules, one can select for a protein function of choice with the benefit that the genetic material is linked to the protein for subsequent PCR amplification, enrichment, and, ultimately, identification.

Genetic Techniques↗
PubMed2004

Identification of specific plant nucleolar phosphoproteins in a functional proteomic analysis.

The soluble fraction of nuclear proteins is a functionally significant fraction, since it has been shown that it contains ribonucleoproteins active in nuclear RNA metabolism. The aim of this work was to detect variations associated with cell proliferation, by comparing two-dimensional proteomes obtained from the soluble fractions of onion nuclei isolated from actively proliferating root meristematic cells versus nonmeristematic root cells. In particular, we have studied the physicochemical features of the major nucleolar protein NopA100, a highly phosphorylated, nucleolin-like protein. A total of 384 spots were quantified in meristematic nuclei, while only 209 were detected in nonmeristematic nuclei. The comparison of both proteomes resulted in the determination of specific spots for each proliferative state and those which were common to both cases. Furthermore, among these latter, we could discriminate quantitative differences. Interestingly, well-known nucleolar proteins, such as RNA polymerase I, B23 and the nucleolin-like protein NopA100, were significantly increased in proliferating cells. Western blots with anti-NopA100 antibody demonstrated 26 spots in the meristematic sample. All the spots detected were clustered at 100 kDa and were distributed through an isoelectric point (pI) range of 4.3-6.6. In contrast, only seven spots were found in the extract from nonmeristematic nuclei, and the pI range was shortened to 4.8-6.1. These results indicate that the state of NopA100 phosphorylation correlates with the degree of nucleolar activity, i.e. the protein is more highly phosphorylated in cycling cells. We have also analyzed the bidimensional silver staining of the nucleolar organizing region (Ag-NOR) pattern of the soluble nuclear fraction in order to identify plant cell phosphoproteins that are considered to be markers of proliferation. These experiments demonstrated that NopA100, the onion, nucleolin-like protein, is an Ag-NOR protein. In addition we found that the plant homologue of the vertebrate nucleolar phosphoprotein B23 migrated as two clusters of acidic spots, 43 and 42 kDa respectively in molecular mass. The differences between these features and those described for mammalian cells is discussed. Our results demonstrate that the use of protein fractionation procedures with functional significance and the location of candidate spots by indirect techniques are advantageous, complementary methods to random selection procedures for proteomic studies involving further mass spectrometry analysis.

Cell Division↗
PubMed2004

Technical aspects of functional proteomics in plants.

Since the completion of genome sequences of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. This analysis is achieved by separation and identification of proteins, determination of their function and functional network, and construction of an appropriate database. Many improvements in separation and identification of proteins, such as two-dimensional electrophoresis, nano-liquid chromatography and mass spectrometry, have rapidly been achieved. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. These techniques have provided the possibility of high-throughput analysis of function and functional network of proteins in plants. However, to cope with the huge information emerging from proteome analyses, more sophisticated techniques and software are essential. The development and adaptation of such techniques will ease analyses of protein profiling, identification of post-translational modifications and protein-protein interaction, which are vital for elucidation of the protein functions.

Databases, Protein↗
PubMed2003

Tackling an essential problem in functional proteomics of Saccharomyces cerevisiae.

Gene inactivation is the cornerstone of functional genetic analysis, but the analysis of essential genes requires conditional inactivation of the gene product. A new study has adapted a simple method for creating conditional alleles to allow large-scale analysis of essential genes in Saccharomyces cerevisiae and has identified a role in DNA replication for a newly identified protein complex.

DNA Replication↗
PubMed2002

Adapting chromophore-assisted laser inactivation for high throughput functional proteomics.

Recent advances in genomics and proteomics have generated a change in emphasis from hypothesis-based to discovery-based investigations. Genomic and proteomic studies based on differential expression microarrays or comparative proteomics often provide many potential candidates for functionally important roles in normal and diseased cells. High throughput technologies to address protein and gene function in situ are still necessary to exploit these emerging advances in gene and protein discovery in order to validate these identified targets. The pharmaceutical industry is particularly interested in target validation, and has identified it as the critical early step in drug discovery. An especially powerful approach to target validation is a direct protein knockdown strategy called chromophore-assisted laser inactivation (CALI) which is a means of testing the role of specific proteins in particular cellular processes. Recent developments in CALI allow for its high throughput application to address many proteins in tandem. Thus, CALI may have applications for high throughput hypothesis testing, target validation or proteome-wide screening.

Drug Industry↗
PubMed2002

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes.

The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.

Animals↗
PubMed2002

Chemical strategies for functional proteomics.

With complete genome sequences now available for several prokaryotic and eukaryotic organisms, biological researchers are charged with the task of assigning molecular and cellular functions to thousands of predicted gene products. To address this problem, the field of proteomics seeks to develop and apply methods for the global analysis of protein expression and protein function. Here we review a promising new class of proteomic strategies that utilizes synthetic chemistry to create tools and assays for the characterization of protein samples of high complexity. These approaches include the development of chemical affinity tags to measure the relative expression level and post-translational modification state of proteins in cell and tissue proteomes. Additionally, we discuss the emerging field of activity-based protein profiling, which aims to synthesize and apply small molecule probes that monitor dynamics in protein function in complex proteomes.

Affinity Labels↗
PubMed2005

Protein microarrays as tools for functional proteomics.

Protein microarrays present an innovative and versatile approach to study protein abundance and function at an unprecedented scale. Given the chemical and structural complexity of the proteome, the development of protein microarrays has been challenging. Despite these challenges there has been a marked increase in the use of protein microarrays to map interactions of proteins with various other molecules, and to identify potential disease biomarkers, especially in the area of cancer biology. In this review, we discuss some of the promising advances made in the development and use of protein microarrays.

Protein Array Analysis↗
PubMed2006

Analytical platforms for activity-based protein profiling--exploiting the versatility of chemistry for functional proteomics.

The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.

Animals↗
PubMedDate not supplied

Chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases.

Determining the biological function of newly discovered gene products requires the development of novel functional approaches. To facilitate this task, recent developments in proteomics include small molecular probes that target proteolytic enzyme families including serine, threonine, and cysteine proteases. For the families of ubiquitin (Ub) and ubiquitin-like (UBL)-specific proteases, such tools were lacking until recently. Here, we review the advances made in the development of protein-based active site-directed probes that target proteases specific for ubiquitin and ubiquitin-like proteins. Such probes were applied successfully to discover and characterize novel Ub/UBL-specific proteases. Ub/UBL processing and deconjugation are performed by a diverse set of proteases belonging to several different enzyme families, including members of the ovarian tumor domain (OTU) protease family. A further definition of this family of enzymes will benefit from a directed chemical proteomics approach. Some of the Ub/UBL-specific proteases react with multiple Ub/UBLs and members of the same protease family can recognize multiple Ub/UBLs, underscoring the need for tools that appropriately address enzyme specificity.

B-Lymphocytes↗
PubMed2001

Integrating molecular medicine with functional proteomics: realities and expectations.

We analyze key proteomic issues and cutting-edge technologies that will spearhead inroads into functional interpretations of human diseases and their therapeutic rectification, following the availability of the predicted human proteome. We contrast the distinctions between high quality data that are low throughput, (e.g., 3-D proteomic reconstructions in embryogenic and nervous system contexts, and multigenerational transgenic studies), versus automated data harvesting that is more distant from human disease phenotypes and currently fulfills a diagnostic role, (e.g., molecular portraits of human diseases via transcriptomic analyses). We examine the extent to which these approaches impinge upon a realistic understanding of human diseases, namely how close they come to revealing the causal events involved in the initiation of disease. While tissue sources from human embryogenesis, foetal development and the brain remain the absolute priority, the pragmatic approaches utilize judicious data integration from selected proteomic studies of model organisms. The role of genome-wide disease-related screens, "humanized" transgenic analyses, multigenerational gene interference methods, and analyses of post-translational modifications in epigenetic contexts from Drosophila will be crucial, since these avenues are far too slow and transgenically cumbersome in mammals. Finally, the implementation of multi compartment electrolyzers (MCE) and multi photon detection (MPD) systems will be pivotal for the proteomic profiling of human tissue samples.

Animals↗
PubMed2002

Functional proteomics using chromophore-assisted laser inactivation.

Proteins are the molecules that fulfil most cellular functions and represent over 90% of drug targets in the market. Chromophore-assisted laser inactivation (CALI) provides a timely and locally restricted protein inactivation and has proven to specifically destroy protein function using dye-coupled ligands and laser irradiation. CALI involves the generation of short-lived radicals thus limiting the radius of covalent modifications to spatially restricted sites on the target molecule. A transient functional inactivation occurs if the radicals modify amino acids of the target protein that are responsible for function. Here we show specific inactivation of several protein targets, that are members of relevant signal transduction pathways. For each of these targets, simple and high throughput screening-scaleable assays have been developed, making it possible to quantify the observed inactivation. Activities of target proteins have been addressed in cell-free as well as cell-based assays employing human primary and tumor-derived cell lines. In all cases, at least 50% inactivation was achieved. The data presented here demonstrate that CALI is a highly versatile tool for validating disease relevant targets at the protein level. This approach also takes into account post-translational modifications like phosphorylation, glycosylation or acylation, thereby enlarging its applicability for many different types of targets.

Apoptosis↗
PubMed2006

Functional proteomic profiling of glycan-processing enzymes.

Glycoconjugates play critical roles in regulating cellular and organismal functions. Consequently, defining the relative levels of these glycoconjugates under varied physiological conditions is important. Thus identifying and understanding the regulation of the enzymes that process these glycoconjugates are essential steps in understanding the role of this "glycocode" in development and disease. Activity-based affinity reagents are useful tools for probing these enzymes and should facilitate the unraveling of proteomes. One advantage of activity-based affinity probes is that they can simultaneously reveal multiple enzymes having similar activities. These probes can also be used to enrich proteomes of interest, thereby facilitating identification and cloning of new carbohydrate-processing enzymes. Here we review the current state of activity-based affinity probes for profiling carbohydrate-processing enzymes, focusing on successes and limitations, general design features, and a specific example describing profiling of exoglycosidases from cell lysates.

Affinity Labels↗
PubMed2006

Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate.

We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.

Animals↗
PubMed2002

Functional proteomics using microchannel plate detectors.

We describe the development of a novel detection system used for the functional imaging of proteins separated on electrophoretic gels. A microchannel plate detector is used here for real-time imaging of low levels of tritiated protein separated by two-dimensional (2-D) electrophoresis. The system employs radioisotope-free, low noise microchannel plates originally developed for photon counting in X-ray astronomy. Using the detector configuration described here, proteins were resolved on mini gels by either one or two-dimensional electrophoresis, transferred onto polyvinylidene difluoride membranes and directly imaged. Tritiated diisopropylfluorophosphate (DFP) was used as a selective label for the serine hydrolase class of enzymes and their distribution in the central nervous system was examined. This survey revealed approximately 24 protein spots by 2-D electrophoresis. We also investigated the relative sensitivity of these proteins towards DFP and found the peptidase, acylpeptide hydrolase to be the most sensitive brain protein towards this reagent. Using a number of different tritiated standards, it was found that the system can image as little as 0.1 Bq/mm(2) of tritium corresponding to 320 attomol of DFP labelled protein/mm(2). Moreover, the system has a wide dynamic range (>10(6)) allowing samples of high and low activity to be quantified on the same gel.

Animals↗
PubMed2003

High throughput protein production for functional proteomics.

A major impact of genome projects on human health will be their contribution to the understanding of protein function. Proteins are the engines of biological systems, nearly all pharmaceuticals act on proteins and increasingly proteins themselves are used therapeutically. As biology enters the post-genomic era, researchers have begun to embrace the exciting opportunity of investigating proteins in high throughput (HT) experiments. The study of proteins includes a vast array of techniques ranging from enzyme catalysis assays to interaction and structural studies. Many of these methods depend on purified proteins. The discovery of thousands of novel protein-coding sequences and the increased availability of large cDNA collections provide the opportunity to investigate protein function in a systematic manner and at an unprecedented scale. This opportunity highlights the need for development of HT methods for protein isolation. This article describes the challenges faced and the approaches taken to develop proteome-scale protein expression systems.

Cell-Free System↗