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PubMed2006

Activity-based proteomics: enzymatic activity profiling in complex proteomes.

In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine proteases, tyrosine phosphatases, glycosidases, and others. These probes are usually detected by their fluorescent, radioactive or affinity tags and their protein targets are analyzed using established proteomics techniques. Recent developments, such as the design of probes for in vivo analysis of proteomes, as well as microarray technologies for higher throughput screenings of protein specificity and the application of activity-based probes for drug screening are highlighted. We focus on biological applications of activity-based probes for target and inhibitor discovery and discuss challenges for future development of this field.

Animals↗
PubMed2026

Glycolysis-dependent reactive oxygen species mediate desmopressin acetate-induced rescue of platelet dysfunction caused by antiplatelet therapy.

Antiplatelet therapy is extensively used in the prevention and treatment of cardiovascular and cerebrovascular diseases; however, life-threatening hemorrhage requires urgent reversal of platelet dysfunction. Desmopressin acetate has been proposed as a rescue strategy, yet its efficacy and underlying mechanisms remain incompletely understood, particularly regarding redox regulation. A mouse carotid artery blood flow injury model was employed to evaluate the effects of desmopressin acetate on platelet and coagulation dysfunction induced by antiplatelet therapy. Proteomic analyses were performed in both patients and mice to identify differentially expressed proteins. Genetic knockout and pharmacological inhibition approaches were used to investigate the mechanistic pathways involved. Desmopressin acetate effectively restored platelet function and coagulation capacity in antiplatelet-treated mice. Proteomic profiling identified peroxiredoxin-5, a key antioxidant enzyme, as significantly upregulated following antiplatelet therapy but markedly downregulated after desmopressin acetate administration; these findings were validated in plasma samples from 10 patients who received dual antiplatelet therapy for unruptured intracranial aneurysms. Functional studies demonstrated that proteomic profiling identified peroxiredoxin-5 supplementation impaired platelet function, whereas proteomic profiling identified peroxiredoxin-5 knockout or inhibition significantly improved platelet activity. Notably, desmopressin acetate primarily suppressed liver-derived proteomic profiling identified peroxiredoxin-5 expression. Mechanistically, desmopressin acetate enhanced platelet glycolysis via phosphofructokinase-2/fructose-2,6-bisphosphatase 3 activation, leading to increased intracellular reactive oxygen species levels. Inhibition of phosphofructokinase-2/fructose-2,6-bisphosphatase 3 attenuated glycolysis, reduced reactive oxygen species generation, and restored proteomic profiling identified peroxiredoxin-5 expression, thereby abolishing the platelet-rescuing effects of desmopressin acetate. Desmopressin acetate rescued platelet dysfunction induced by antiplatelet therapy through a glycolysis-reactive oxygen species-proteomic profiling identified peroxiredoxin-5 axis, in which glycolysis-driven reactive oxygen species generation plays a central regulatory role. These findings indicate redox modulation as a critical mechanism underlying desmopressin acetate-mediated platelet rescue and suggest a potential therapeutic strategy for managing severe bleeding associated with antiplatelet therapy.

Animals↗
PubMed2006

Exploring glycopeptide-resistance in Staphylococcus aureus: a combined proteomics and transcriptomics approach for the identification of resistance-related markers.

BACKGROUND: To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. RESULTS: In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. CONCLUSION: Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.

Anti-Bacterial Agents↗
PubMed2005

[Strategy for the protein identification of human proteome expression profile: selection of searching database].

Widely used method of protein identification for high-throughout proteome expression profile studies was database-dependent, so the selection of databases for the protein identification was very important. Despite the deficiency of available human protein databases, the complementarity of human proteins could be got mainly from human genome but not from the protein databases of other organisms. According to the comparison of the current protein databases from different aspects, IPI was recommended for the basic identification for the studies of human proteome expression profile, and other human protein or nucleic acid databases were needed for the complementary identification and novel protein mining.

Animals↗
PubMed2004

Comprehensive proteome expression profiling of undifferentiated versus differentiated neural stem cells from adult rat hippocampus.

Adult neural stem cells can be isolated from various regions of the rat brain and seem to have multilineage differentiation potential. In this study, we investigated the hypothesis that global protein expression of adult neural stem cells isolated from rat hippocampus is changed during in vitro differentiation. After 2 days of differentiation, we separated total protein extracts by two-dimensional gel electrophoresis and used mass spectrometry and gel-matching for identification. We detected a differential expression in 367 regulated protein spots, of which 128 could be identified. Regulated proteins included participants in transcription and DNA metabolism; signal transduction and Ca2+-signaling; MAP kinase pathways; cytoskeletal rearrangement; regulation of cell cycle, proliferation, and survival; protein biosynthesis, folding, and degradation; and glycine and glutamate metabolic pathways. These results suggest a massive reorganization of the stem cell proteomic profile upon differentiation and indicate potential cellular targets mediating the differentiation of neural stem cells.

Animals↗
PubMed2006

Interest of major serum protein removal for Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) proteomic blood profiling.

BACKGROUND: Surface-Enhanced Laser Desorption/Ionization - Time Of Flight (SELDI-TOF) has been proposed as new approach for blood biomarker discovery. However, results obtained so far have been often disappointing as this technique still has difficulties to detect low-abundant plasma and serum proteins. RESULTS: We used a serum depletion scheme using chicken antibodies against various abundant proteins to realized a pre-fractionation of serum prior to SELDI-TOF profiling. Depletion of major serum proteins by immunocapture was confirmed by 1D and 2D gel electrophoresis. SELDI-TOF analysis of bound and unbound (depleted) serum fractions revealed that this approach allows the detection of new low abundant protein peaks with satisfactory reproducibility. CONCLUSION: The combination of immunocapture and SELDI-TOF analysis opens new avenues into proteomic profiling for the discovery of blood biomarkers.

Journal Article↗
PubMed1999

The Feasibility of Using Proteome Expression Profile for Genome Annotation.

By investigating into the expression data from ECO2DBASE (Edition 6),the feasibility of using proteome expression profile for genome annotation was tested. Based on our newly developed CRC (cellular role cluster) method,79 proteins extracted from ECO2DBASE were clustered into 4 CRCs. Function related proteins tend to be clustered into same CRC. Total 9 aminoacyl-tRNA synthetases were clustered into CRC2, whereas 4 heat-shock proteins into CRC3. These results indicate with enough proteome expression data and the efficient algorithm, proteome expression profile can provide very important information for genome annotation, while this kind of information is sequence-independent.

Journal Article↗
PubMedDate not supplied

Characterization of human tear proteome using multiple proteomic analysis techniques.

Tear proteome profiling may generate useful information for the understanding of the interaction between an eye and its contacting objects, such as a contact lens or a lens implant. This is important for designing improved eye-care devices and maintaining the health of an eye. Proteome profiles of tear fluids may also be used for disease diagnosis and prognosis. However, only a small volume of tear fluid (<5 microL) can be collected in a clinical laboratory under normal operational conditions, which makes proteome profiling a challenge. In this work we apply several proteomic analysis techniques, including gel-based and solution-based approaches with LC-ESI and LC-MALDI MS and MS/MS to gauge the relative merits of producing proteome profiles and to generate as broad a coverage of the tear proteome as possible from this small amount of sample. It is shown that a total of 54 proteins can be confidently identified using less than 5 microL of tear fluid. Of these, 44 proteins can be detected by LC-MALDI MS alone with a consumption of 2 microL of tear fluid. Furthermore, LC-MALDI can be used to determine post-translational modifications (PTMs), such as glycosylation and phosphorylation, without any sample enrichment or treatment. This work represents one of the most extensive proteome profiles (i.e., proteins identified and PTMs characterized) generated from tear fluids using clinically relevant amounts of sample.

Chromatography↗
PubMed2023

An N terminomics toolbox combining 2-pyridinecarboxaldehyde probes and click chemistry for profiling protease specificity.

Proteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.

Peptide Hydrolases↗
PubMed2006

Proteome-wide profiling of isoniazid targets in Mycobacterium tuberculosis.

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.

Antitubercular Agents↗
PubMed2006

[Importance of development of ecotoxicogenomics in understanding molecular mechanisms of chemicals in developing animals].

Developing organisms are sensitive to estrogenic chemicals. Exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in reproductive as well as nonreproductive organs, including persistent molecular alterations. Chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans, some of which exhibit estrogenic or anti-androgenic activity by binding to estrogen or androgen receptors. Estrogen-responsive genes and critical developmental windows of various animal species, therefore, should be identified to understand the molecular basis of estrogenic activity during embryonic development. To understand molecular mechanisms of estrogenic chemicals in developing organisms, we identified estrogen-responsive genes using cDNA microarrays and a quantitative reverse transcriptase-polymerase chain reaction, and genes related to estrogen-independent vaginal changes in mice induced by estrogens during a critical window. We also analyzed genes in Xenopus laevis embryos related to abnormalities induced by estrogenic chemicals using cDNA microarrays. Estrogen receptors and other steroid hormone receptors were cloned in various species, including the American alligator, crocodile, Florida red-bellied turtle, Silurana tropicalis, roach, Fundulus and mosquito fish. An environmental androgen, trenbolone, was found to induce persistent changes in the gonopodium and ovotestis in mosquito fish. In Daphnia magna, juvenile hormone agonists induce the production of male offspring. Tributyltin (TBT) induces imposex in rockshell through retinoid X receptor (RXR) activation. Microarray technology is a powerful tool for understanding the molecular mechanism of receptor-mediated toxicology in various animal species. Ecotoxicogenomics is a multidisciplinary field concerned with elucidating how the entire genome is involved in the biological response of wild animals exposed to environmental toxicants and stressors. Ecotoxicogenomics combines information from studies of genomic-scale mRNA profiling by microarray analysis (transcriptomics), protein profiling (proteomics), metabolite profiling (metabolonomics) and computational models to understand the roles of gene-environment interaction in the chemical toxicity. To understand the molecular mechanism of chemicals in developing wildlife, the establishment of ecotoxicogenomics is essential in the near future.

Amphibians↗
PubMed2000

100th American society for microbiology annual meeting.

The 100th ASM Annual Meeting, attended by approximately 10,000 delegates, continued the trend of concentrating on bacteria and antibacterial therapy, mixed with genomics and a diverse number of additional topics. Of the various marketable drug classes, the quinolones received attention with respect to susceptibility studies and several drug comparison studies. New marketable drugs were also of interest, especially given the reservoirs of resistance presented by several speakers. Drugs in development include the antibacterial daptomycin and protegrins and the antifungal lipodepsinonapeptides and echinocandins, to name a few. It is still unclear whether or not antibiotic treatment regimens for Chlamydia pneumonia will he necessary, as association of this bacteria with several chronic diseases, such as atherosclerosis and asthma, was discussed. The development of novel antibiotics was highlighted and the potential role that microbial genomics technology could play was a recurring theme. In fact, a number of symposia treated the increasingly popular topic of genomics in a variety of themes, including phenotyping arrays, transcriptional profiling, proteomics, expression profiling, genome sequencing, target areas or essentiality of genes via gene knockout systems, the role of genomics in pharmaceutical development and fungal genomics. Similarly, genomics plays a role in developing a deeper appreciation for classical areas of interest in microbial physiology, such as gene regulation, cell division, fatty acid biosynthesis, DNA replication and cell signalling. Even in the bio-inorganic field of study in microbial metabolite activation, genomics plays a role. The sequencing of the large gene clusters of the auxiliary proteins necessary to synthesise or activate the metallo-proteins provided insights into the mechanisms of activation of these microbial enzymes, including the genes for the nif gene cluster in Azotobacter vinelandii, the urease from Kiebsiella aerogenes and the three hydrogenases in Ralstonia eutropha.

Anti-Bacterial Agents↗
PubMed2006

Proteomic analysis of nipple aspirate fluid throughout the menstrual cycle in healthy pre-menopausal women.

A proteomic approach to nipple aspiration fluid (NAF) has been used in a number of studies comparing women with breast cancer and healthy women. However, to make useful comparisons between women with breast cancer and healthy women it is necessary to establish whether there is physiological variation in the proteomic profiles of NAF. The purpose of this study was, for the first time, to examine how the proteomic profile of NAF using surface-enhanced laser desorption ionisation time-of-flight mass spectrometry varies across the menstrual cycle in healthy pre-menopausal women. Twelve women were recruited and nipple aspiration was carried out weekly from both breasts of each subject for two menstrual cycles. Matching serum samples for luteinising hormone, follicle stimulating hormone and oestradiol were obtained at each aspiration attempt. Statistically significant peaks were found for three healthy volunteers (p < 0.05). However, the peaks that varied across the menstrual cycle were different from one healthy volunteer to another and the differences were small compared with the large variation in proteomic profiles between healthy volunteers. This study provides proof of concept that the NAF proteomic profile does not vary substantially during the menstrual cycle and that therefore it is valid to compare NAF profiles from pre-menopausal women that have been taken at different stages in the menstrual cycle.

Biomarkers↗
PubMed2005

Proteomic patterns of preinvasive bronchial lesions.

PURPOSE: A proteomics approach is warranted to further elucidate the molecular steps involved in lung tumor development. We asked whether we could classify preinvasive lesions of airway epithelium according to their proteomic profile. EXPERIMENTAL DESIGN: We obtained matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiles from 10-microm sections of fresh-frozen tissue samples: 25 normal lung, 29 normal bronchial epithelium, and 20 preinvasive and 36 invasive lung tumor tissue samples from 53 patients. Proteomic profiles were calibrated, binned, and normalized before analysis. We performed class comparison, class prediction, and supervised hierarchic cluster analysis. We tested a set of discriminatory features obtained in a previously published dataset to classify this independent set of normal, preinvasive, and invasive lung tissues. RESULTS: We found a specific proteomic profile that allows an overall predictive accuracy of over 90% of normal, preinvasive, and invasive lung tissues. The proteomic profiles of these tissues were distinct from each other within a disease continuum. We trained our prediction model in a previously published dataset and tested it in a new blinded test set to reach an overall 74% accuracy in classifying tumors from normal tissues. CONCLUSIONS: We found specific patterns of protein expression of the airway epithelium that accurately classify bronchial and alveolar tissue with normal histology from preinvasive bronchial lesions and from invasive lung cancer. Although further study is needed to validate this approach and to identify biomarkers of tumor development, this is a first step toward a new proteomic characterization of the human model of lung cancer tumorigenesis.

Aged↗
PubMed2000

A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling.

Proteomic projects are often focused on the discovery of differentially expressed proteins between control and experimental samples. Most laboratories choose the approach of running two-dimensional (2-D) gels, analyzing them and identifying the differentially expressed proteins by in-gel digestion and mass spectrometry. To date, the available stains for visualizing proteins on 2-D gels have been less than ideal for these projects because of poor detection sensitivity (Coomassie blue stain) or poor peptide recovery from in-gel digests and mass spectrometry (silver stain), unless extra destaining and washing steps are included in the protocol. In addition, the limited dynamic range of these stains has made it difficult to rigorously and reliably determine subtle differences in protein quantities. SYPRO Ruby Protein Gel Stain is a novel, ruthenium-based fluorescent dye for the detection of proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels that has properties making it well suited to high-throughput proteomics projects. The advantages of SYPRO Ruby Protein Gel Stain relative to silver stain demonstrated in this study include a broad linear dynamic range and enhanced recovery of peptides from in-gel digests for matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry.

Acrylic Resins↗
PubMed2007

2-DE profiling of GDNF overexpression-related proteome changes in differentiating ST14A rat progenitor cells.

Targeted differentiation of neural progenitor cells (NPCs) is a challenge for treatment of neurodegenerative diseases by cell replacement therapy and cell signalling manipulation. Here, we applied a proteome profiling approach to the rat striatal progenitor model cell line ST14A in order to elucidate cellular differentiation processes. Native cells and cells transfected with the glial cell line-derived neurotrophic factor (GDNF) gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. 2-DE combined with MALDI-MS was used to create a reference 2-DE-map of 652 spots of which 164 were identified and assigned to 155 unique proteins. For identification of protein expression changes during cell differentiation, spot patterns of triplicate gels were matched to the 2-DE-map. Besides proteins that display expression changes in native cells, we also noted 43 protein-spots that were differentially regulated by GDNF overexpression in more than four time points of the experiment. The expression patterns of putative differentiation markers such as annexin 5 (ANXA5), glucosidase II beta subunit (GLU2B), phosphatidylethanolamine-binding protein 1 (PEBP1), myosin regulatory light chain 2-A (MLRA), NASCENT polypeptide-associated complex alpha (NACA), elongation factor 2 (EF2), peroxiredoxin-1 (PRDX1) and proliferating cell nuclear antigen (PCNA) were verified by Western blotting. The results reflect the large rearrangements of the proteome during the differentiation process of NPCs and their strong modification by neurotrophic factors like GDNF.

Animals↗
PubMed2006

Divide and conquer: rat liver tissue proteomics based on the analysis of purified constituents.

Comparative proteome data of normal and diseased tissue samples are difficult to interpret. Proteins detected in tissues are derived from different cell types and blood constituents. Pathologic or toxicant-induced aberrations may affect the proteome profile of tissues in several ways since different cell types may respond in very different and highly specific manners. The aim of this study was to analyze the proteome profiles of purified rat liver primary cells and of blood plasma in comparison to liver whole tissue. Moreover, we investigated alterations of these profiles induced by the liver toxicant N-nitrosomorpholine (NNM) used as a model compound. Whole liver samples, pure hepatocytes and Kupffer cells as well as blood plasma were obtained from saline- or NNM-treated rats. Proteins were separated by 2-D PAGE and their amounts were estimated by fluorography. Selected proteins were identified by MS analysis of tryptic digests. Among them we identified proteins exclusively expressed in the analysed constituents. Several of these proteins were assigned in the proteome profile of whole-tissue homogenates. Furthermore, we identified several proteins that were modified, up-regulated or down-regulated due to NNM treatment in total liver homogenates. Some of these protein alterations were specifically detected in primary cells isolated from NNM-treated rats. Thus, we demonstrated the successful assignment of NNM-induced proteome alterations in rat liver to the cell type of origin. The currently applied approach may help to better understand pathologic processes at a whole-tissue level.

Animals↗
PubMed2005

Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling.

Chemical genomics aims to discover small molecules that affect biological processes through the perturbation of protein function. However, determining the protein targets of bioactive compounds remains a formidable challenge. We address this problem here through the creation of a natural product-inspired small-molecule library bearing protein-reactive elements. Cell-based screening identified a compound, MJE3, that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were observed exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compounds into chemical genomics screens offers a means to discover targets of bioactive small molecules in living systems, thereby enabling downstream mechanistic investigations.

Antineoplastic Agents↗