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PubMed2000

The use of recombinant antibodies in proteomics.

Recombinant antibodies are becoming increasingly important in the field of proteomics. Recent advances include the development of large phage-antibody libraries that contain high-affinity binders to almost any target protein, and new methods for high-throughput selection of antibody-antigen interactions. Coupled with a range of new screening technologies that use high-density antibody arrays to identify differentially expressed proteins, these antibody libraries can be applied to whole proteome analysis.

Antibodies, Monoclonal↗
PubMedDate not supplied

A potential biomarker of kidney damage identified by proteomics: preliminary findings.

4-Aminophenol (4-AP) and D-serine are established rodent nephrotoxins that selectively damage renal proximal tubules. In an attempt to understand the mechanism of action of these toxicants in greater detail, a high throughput proteomics approach was used to profile protein changes in the plasma of animals treated with these compounds. Male Fischer 344 and Alderley Park rats were treated with increasing doses of 4-AP or D-serine and plasma samples were collected over time. Control groups received either saline or the non-toxic enantiomer, L-serine. Using high throughput two-dimensional gel analysis, a number of plasma proteins showing dose- and time-dependent regulation were identified. One toxicity-associated plasma protein was identified as the cellular enzyme fumarylacetoacetate hydrolase (FAH), which is known to be required for tyrosine metabolism. The FAH gene is mutated in the human genetic disorder type I tyrosinaemia, which is associated with liver and kidney abnormalities and neurological disorders. FAH was elevated in the plasma of animals treated with 4-AP and D-serine at early time points and returned to baseline levels after 3 weeks. The protein was not elevated in the plasma of control animals or those treated with L-serine. The presence of FAH in plasma is intriguing as it is normally a cellular enzyme with no known function in plasma. It is possible that 4-AP and D-serine may work through a previously unknown mechanism in the kidney via regulation of tyrosine metabolism or FAH activity. Therefore, FAH may function in a fashion analogous to the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes that are used to measure liver injury. The link between kidney toxicants and inherited tyrosinaemia also raises the possibility that FAH may be a marker of kidney toxicity in humans. These observations highlight the value of proteomics in identifying new biomarkers and providing new unprecedented insights into complex biological mechanisms.

Aminophenols↗
PubMed2006

Comparative proteomics of symbiotic and aposymbiotic juvenile soft corals.

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral-algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.

Algal Proteins↗
PubMed2004

Enhanced functional information from predicted protein networks.

Experimentally derived genome-wide protein interaction networks have been useful in the elucidation of functional information that is not evident from examining individual proteins but determination of these networks is complex and time consuming. To address this problem, several computational methods for predicting protein networks in novel genomes have been developed. A recent publication by Date and Marcotte describes the use of phylogenetic profiling for elucidating novel pathways in proteomes that have not been experimentally characterized. This method, in combination with other computational methods for generating protein-interaction networks, might help identify novel functional pathways and enhance functional annotation of individual proteins.

Energy Metabolism↗
PubMed2006

Comparative proteomic analysis reveals a function of the novel death receptor-associated protein BRE in the regulation of prohibitin and p53 expression and proliferation.

The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress-modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)-mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up-regulated Akt-3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down-regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up-regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up-regulated after BRE over-expression. The majority of these proteins can target or crosstalk with NF-kappaB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress-response machinery and provide an explanation for the multifunctional nature of BRE.

Animals↗
PubMed2004

Proteomic signature corresponding to alpha fetoprotein expression in liver cancer cells.

Alpha fetoprotein (AFP) has been implicated in the development of hepatocellular carcinoma and is considered to be a diagnostic and prognostic tumor marker. Because elevated expression of AFP is associated with many characteristics of hepatocellular carcinoma tissues, we hypothesized that multiple proteins may function in a coordinated manner with AFP. To identify such proteins, we performed global protein expression analysis, namely a proteomic study. The protein expression profiles of 9 AFP-producing liver cancer cell lines (JHH-5, HuH-1, PLC/PRL/5, Hep3B, HT-17, JHH-7, HuH-7, HepG2, Li-7) and 7 nonproducing liver cancer cell lines (HLE, JHH-6, Sk-Hep-1, JHH-4, HLF, RBE, SSP-25) were generated by fluorescence 2-dimensional difference gel electrophoresis. In fluorescence 2-dimensional difference gel electrophoresis, proteins are labeled with fluorescent dyes before electrophoresis for more accurate quantitative expression analysis. We identified 11 protein spots that distinguished AFP-producing cell lines from nonproducing cell lines by multivariate studies. The spots showed consistent alterations in amount in AFP-producing cell lines (6 up-regulated and 5 down-regulated). An additional 5 liver cancer cell lines (KIM-1, KYN-2, KYN-3, PH5-CH, PH5-T) also were correctly grouped with respect to their AFP production on the basis of the intensity of the 11 protein spots. The proteins corresponding to the 11 selected spots were identified by mass spectrometry and were categorized into 4 groups based on their known role in apoptosis, glucose metabolism, cytoskeletal organization, or translation. In conclusion, we found a novel association of AFP with other proteins. Their interaction should provide insight into the biology of AFP-producing hepatocellular carcinoma cells.

Cell Line, Tumor↗
PubMed2026

Integrated multi-omics analysis reveals a pH-driven metabolic and translational switch in Ureaplasma parvum.

Human ureaplasmas are minimal-genome bacteria and pathobionts of the urogenital tract. They must adapt to fluctuating pH conditions despite the absence of canonical transcriptional regulatory systems. However, the mechanisms underlying these responses remain unclear. This study aimed to construct a system-level model of pH adaptation in this minimal pathogen. We used an integrated multi-omics platform combining proteomics, metabolomics, and RNA modification profiling to construct a system-level model of pH adaptation. The results revealed a bifurcated strategy governed by the differential activation of preexisting, co-regulated functional modules. Under neutral pH conditions (pH 7), Ureaplasma parvum activated energy metabolism and upregulated ATP synthesis while forming a stress-counteracting proteostasis pathway. This may suggest a biological energy state under high stress conditions. Conversely, under acidic stress (pH 5), it activated biosynthesis/translation, showing significant upregulation of ribosomal proteins and accumulation of translation precursors and the polyamine spermidine. This may represent a state of expanded translational capacity. This adaptive switch is accompanied by dynamic reorganization of the epitranscriptome, highlighting the importance of post-transcriptional regulation. This study suggests mechanisms by which minimal organisms achieve adaptive plasticity through sophisticated post-transcriptional and metabolic control, providing a new framework for understanding Ureaplasma physiology and the biology of genome-reduced organisms.IMPORTANCEMinimal bacteria challenge canonical views of cellular regulation. In organisms with radically reduced genomes and sparse transcription factors, how adaptive plasticity is achieved remains a core question. Our study proposes a model in which a simple physicochemical cue-extracellular pH-selects among prewired cellular programs, while post-transcriptional and epitranscriptomic layers fine-tune execution. The findings of this study suggest a multi-omics scheme for how organisms adapt to environmental changes and ensure survival without inducing new circuits or complex transcriptional regulation. Conceptually, it proposes regulation via RNA modifications in processes, such as metabolism, proteostasis, and translation. This framework may be generalizable to other genome-reduced microorganisms. Beyond microbiology, it provides design principles for synthetic biology and offers a mechanistic interpretation of phenotypic tolerance to stress factors. It may encourage the use of pH-linked epitranscriptome signals as measurable indicators of cellular state.

Hydrogen-Ion Concentration↗
PubMed2006

Arabidopsis thaliana proteomics: from proteome to genome.

Proteomics has become an important approach for investigating cellular processes and network functions. Significant improvements have been made during the last few years in technologies for high-throughput proteomics, both at the level of data analysis software and mass spectrometry hardware. As proteomics technologies advance and become more widely accessible, efforts of cataloguing and quantifying full proteomes are underway to complement other genomics approaches, such as RNA and metabolite profiling. Of particular interest is the application of proteome data to improve genome annotation and to include information on post-translational protein modifications with the annotation of the corresponding gene. This type of analysis requires a paradigm shift because amino acid sequences must be assigned to peptides without relying on existing protein databases. In this review, advances and current limitations of full proteome analysis are briefly highlighted using the model plant Arabidopsis thaliana as an example. Strategies to identify peptides are also discussed on the basis of MS/MS data in a protein database-independent approach.

Arabidopsis↗
PubMedDate not supplied

Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery.

The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n>250) sequenced in these profiles came from a surprisingly small number of proteins (n approximately 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.

Biomarkers↗
PubMed2004

Lysophosphatidic acid production and action: validated targets in cancer?

The completion of the human genome project, the evolution of transcriptional profiling and the emergence of proteomics have focused attention on these areas in the pathophysiology and therapy of cancer. The role of lysophospholipids as potential mediators in cancer pathophysiology, screening and management has taken a major leap forward with the recent cloning of several enzymes involved in the metabolism of lysophospholipids. Lysophospholipids, although small molecules, contain a high "informational" content. Differences include the nature of the phosphate head group, the regiochemistry of the fatty acyl chain on the glyceryl backbone, the presence of ether versus ester linkages to the backbone, and the length and saturation of the fatty acyl or alkyl chain. This informational content is sufficient to result in a marked structure function activity relationship at their cognate receptors. Thus the emerging discipline of "functional lipidomics" is likely to prove as important as genomics and proteomics in terms of early diagnosis, prognosis, and therapy. Lysophospholipid levels are elevated in vivo in a number of pathophysiological states including ascitic fluid from ovarian cancer patients indicating a role in the pathophysiology of this devastating disease. Although controversial, levels of specific lysophospholipids may be altered in the blood of cancer patients providing a potential mechanism for early diagnosis. Several of the enzymes involved in the metabolism of lysophospholipids are aberrant in ovarian and other cancers. Further, the enzymes are active in the interstitial space, rendering them readily accessible to the effects of inhibitors including antibodies, proteins, and small molecules. In support of a role for lysophospholipids in the pathophysiology of cancer, expression of receptors for lysophospholipids is also aberrant in cancer cells from multiple different lineages. All of the cell surface receptors for lysophospholipids belong to the G protein coupled receptor family. As over 40% of all drugs in current use target this family of receptors, lysophospholipid receptors are highly "druggable." Indeed, a number of highly specific agonists and antagonists of lysophospholipid receptors have been identified. A number are in preclinical evaluation as therapeutics. We look forward to the next several years when the role of lysophospholipids in physiology and the pathophysiology and management of cancer and other diseases are fully elucidated.

Animals↗
PubMed2006

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

Animals↗
PubMed2003

The challenge of multidrug resistance: actual strategies in the development of novel antibacterials.

Bacterial resistance against established antibiotics is becoming an increasingly important global healthcare problem. Despite enormous efforts, the number of therapeutically useful compounds that emerge from chemical derivatisation programs, which aim at circumventing mechanisms of resistance, is continuously decreasing and no truly novel class of compound has been introduced into therapy for nearly four decades. Hopes are now set on a thorough elucidation of bacterial cell functions to identify new bacterial target sites, and on the development of novel compounds with alternative modes of action. The pursuit of these strategies is rendered possible by employment of biotechnologically based methods such as in vivo modification of biosynthetic routes in antibiotic-producing organisms, large-scale screening assays with isolated bacterial targets, the molecular profiling of bacterial genomes and proteomes, and the development and clinical use of biochips as diagnostic tools for rapid identification and characterization of pathogenic strains. As one of the most promising class of compounds known to date with unique modes of action that escape most known mechanisms of resistance, peptic agents have recently came under the focus of anti-infective research, just as extracellular signalling molecules (autoinducer) have emerged as new bacterial target sites.

Anti-Bacterial Agents↗
PubMed2006

Nutrigenomics: from molecular nutrition to prevention of disease.

Until recently, nutrition research concentrated on nutrient deficiencies and impairment of health. The advent of genomics-interpreted broadly as a suite of high throughput technologies for the generation, processing, and application of scientific information about the composition and functions of genomes-has created unprecedented opportunities for increasing our understanding of how nutrients modulate gene and protein expression and ultimately influence cellular and organismal metabolism. Nutritional genomics (nutrigenomics), the junction between health, diet, and genomics, can be seen as the combination of molecular nutrition and genomics. The diverse tissue and organ-specific effects of bioactive dietary components include gene-expression patterns (transcriptome); organization of the chromatin (epigenome); protein-expression patterns, including posttranslational modifications (proteome); as well as metabolite profiles (metabolome). Nutrigenomics will promote an increased understanding of how nutrition influences metabolic pathways and homeostatic control, how this regulation is disturbed in the early phases of diet-related disease, and the extent to which individual sensitizing genotypes contribute to such diseases. Eventually, nutrigenomics will lead to evidence-based dietary intervention strategies for restoring health and fitness and for preventing diet-related disease. In this review, we provide a brief overview of nutrigenomics from our point of view by describing current strategies, future opportunities, and challenges.

Chronic Disease↗
PubMed2006

Quantitative analysis of complex peptide mixtures using FTMS and differential mass spectrometry.

Label-free LC-MS profiling is a powerful quantitative proteomic method to study relative peptide abundances between two or more biological samples. Here we demonstrate the use of a previously described comparative LC-MS method, differential mass spectrometry (dMS), to analyze high-resolution Fourier transform mass spectrometry (FTMS) data for detection and quantification of known peptide differences between two sets of complex mixtures. Six standard peptides were spiked into a processed plasma background at fixed ratios from 1.25:1 to 4:1 to make two sets of samples. The resulting mixtures were analyzed by microcapillary LC-FTMS and dMS. dMS successfully identified five out of the six peptides as statistically significant differences (p <or= 0.005). In this experiment, the smallest fold change reliably detected by our method was 1.5:1, and the errors of estimated ratios of concentrations were less than 20% for peptides spiked at 1.5:1 to 4:1. We conclude that LC-FTMS coupled with dMS is a useful label-free quantitative MS method that can be used to detect subtle yet statistically significant peptide differences in complex protein mixtures, including plasma samples.

Amino Acid Sequence↗
PubMed2005

How can toxicogenomics inform risk assessment?

Technologies that generate information about the genome are being used to explore changes in gene expression and related proteins following exposure to chemicals. Conceptually, this information allows a greater understanding of genomic level mRNA expression (transcriptomics), cell and tissue protein expression (proteomics) and information about metabolite profiles (metabonomics). Having a greater understanding of this information alongside, empirical toxicological reference data provide for the continued evolution in our ability to understand toxicological modes of action, thereby providing a more scientific basis for extrapolation of toxicological information from animals to humans. Toxicogenomics also provides specific opportunities for improvements at different stages of the risk assessment process such as the development of new predictive models for identifying human health hazards and the identification of more precise molecular biomarkers of exposure. One possibility is that molecular fingerprinting in vivo or in vitro can be used to categorize chemicals and mixtures of chemicals into different mode of action groups. As there is indication that molecular signals differ at dose levels, it is hoped that toxicogenomic information can also contribute to the understanding and interpretation of effects seen with low dose exposure. Several gene polymorphisms have already been identified which play a role in the differing intra-species response to chemicals thus providing explanation for the observed differences in effects. It is therefore likely that a better understanding of genomic expression will enable a greater insight into the factors behind the observed variability in susceptibility to chemical exposure that can be seen in human populations.

Genomics↗
PubMed2006

An approach to the validation of biomarkers of harm for use in a tobacco context.

There is both a call and a need for biomarkers of harm that are validated for use in a tobacco context. Currently, there are no validated biomarkers and there is no consensus about which ones may be suitable for this purpose. To advance the science in this area a working definition of biomarkers of harm and a shortlist of candidate biomarkers are proposed. A framework for the validation of biomarkers of harm using of a series of epidemiological studies culminating in a targeted prospective study is outlined. The candidate biomarkers have advanced to preliminary testing although this does not imply that any on the shortlist will become validated. This framework could also be used for the evaluation of proteomic, genomic, transcriptosomic or metabonomic profiles, which may turn out to be the preferred biomarkers for use in harm prediction. Biomarker studies would complement data that are generated from specific in vitro tests and from animal studies to evaluate tobacco products.

Animals↗
PubMed2001

Gaucher disease: understanding the molecular pathogenesis of sphingolipidoses.

Gaucher disease is a typical lysosomal storage disease, resulting from an inborn deficiency of glucocerebrosidase. This leads to the accumulation of glycolipids in macrophages, particularly those in the liver, bone marrow, spleen and lung. In addition, disease of the nervous system can arise as a result of the accumulation of endogenous glycosphingolipid metabolites in brain tissue. About 150 mutations of the glucocerebrosidase gene have been identified in patients with Gaucher disease, some of which are predictive of phenotype. However, even patients and siblings with the same mutation, including monozygotic twins, may exhibit marked variability in disease expression and severity, illustrating our lack of understanding of the phenotype-genotype relationship in the sphingolipidoses. Massive organomegaly, particularly of the spleen, is a frequent feature of the disease. Although the liver and spleen may increase greatly in size, the amount of pathological lipid stored in the affected macrophages (Gaucher cells) accounts for less than 2% of the additional tissue mass. It is therefore clear that an inflammatory response occurs in affected individuals and that the clinical phenotype is due to an effect of macrophage storage beyond the physical presence of the Gaucher cells. Factors released by Gaucher cells, including pro-inflammatory cytokines and perhaps cathepsins, provide a mechanistic link between lysosomal storage and the diverse clinical manifestations of Gaucher disease. Emerging proteomic technology and gene expression profiling should not only improve our understanding of pathogenesis but also offer the prospect of identifying novel biomarkers that can be used as surrogate measures of disease activity and responses to treatment.

Gaucher Disease↗
PubMed2003

Printing chemical libraries on microarrays for fluid phase nanoliter reactions.

Chemical compounds within individual nanoliter droplets of glycerol were microarrayed onto glass slides at 400 spots/cm2. Using aerosol deposition, subsequent reagents and water were metered into each reaction center to rapidly assemble diverse multicomponent reactions without crosscontamination or the need for surface linkage. This proteomics technique allowed the kinetic profiling of protease mixtures, protease-substrate interactions, and high-throughput screening reactions. An inhibitor of caspases 2, 4, and 6 was identified by using a 352-compound combinatorial library microarrayed in quadruplicates on 100 slides and screened against caspases 2, 4, and 6, as well as thrombin and chymotrypsin. From one printing run that consumes <1 nanomole of each compound, large combinatorial libraries can be subjected to numerous separation-free homogeneous assays at volumes 103-104 smaller than current high-throughput methods.

Animals↗