Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.

Proteomic analysis of uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are among the most common of bacterial infections in humans. Although a number of Gram-negative bacteria can cause UTIs, most cases are due to infection by uropathogenic E. coli (UPEC). Genomic studies have shown that UPEC encode a number of specialized activities that allow the bacteria to initiate and maintain infections in the environment of the urinary tract. Proteomic analyses have complemented the genomic data and have documented differential patterns of protein synthesis for bacteria growing ex vivo in human urine or recovered directly from the urinary tracts of infected mice. These studies provide valuable insights into the molecular basis of UPEC pathogenesis and have aided the identification of putative vaccine targets. Despite the substantial progress that has been achieved, many future challenges remain in the application of proteomics to provide a comprehensive view of bacterial pathogenesis in both acute and chronic UTIs.

Investigating pathogen biology at the level of the proteome.

Bacterial infections are a major cause of morbidity and mortality throughout the world. By extending our understanding of the process of bacterial pathogenesis at the molecular level new strategies for their treatment and prevention can be developed. Proteomic technologies, along with other methods for global gene expression analysis, play an important role in understanding the mechanism(s) of bacterial pathogenesis. This review highlights the use of proteomics to identify protein biomarkers for virulent bacterial isolates and how these biomarkers can be correlated with the outcome of bacterial infection. Biomarker identification typically looks at the proteomes of bacteria grown under laboratory conditions. It is, however, the characterisation of the bacterial proteome during in vivo infection of its host that will eventually provide the most significant insights into bacterial pathogenesis. Although this area of research has significant technical challenges, a number of complementary proteome analytical approaches are being developed to identify and characterise the bacterial genes specifically expressed in vivo. Ultimately, the development of newly targeted therapies and vaccines using specific protein targets identified through proteomic analyses will be one of the major practical benefits arising from the proteomic analysis of bacterial pathogens.

Proteomic analysis of Escherichia coli associated with urinary tract infections.

Escherichia coli is a major cause of urinary tract infections (UTIs) where the initial infection arises from bacteria originating in the bowel. However, significant differences are observed between the genomes of intestinal and urinary E. coli strains with the latter possessing many adaptations that promote growth in the urinary tract. To define further the adaptation of urinary E. coli isolates, the cellular proteomes of 41 E. coli strains, collected from cases of UTIs or random faecal samples, were compared by 2-D gel electrophoresis and principal component analysis. The data indicated that individual patients carried relatively homogenous E. coli populations, as defined by their cellular proteomes, but the populations were distinct between patients. For one patient, E. coli, isolated during two recurrent infections 3 months apart, were indistinguishable, indicating that for this patient the infections were possibly caused by the same bacterial population. To understand the basis of the discrimination of the bacteria, selected protein spots were identified by peptide fragment fingerprinting. The identified proteins were involved in a variety of metabolic and structural roles. The data obtained for these E. coli strains provide a basis from which to target key bacterial proteins for further investigation into E. coli pathogenesis.

Proteomics in the study of the molecular taxonomy and epidemiology of bacterial pathogens.

The ability to discriminate bacterial isolates is important for a number of areas of research in Medical Microbiology, particularly in defining bacterial taxonomy and monitoring transmission in epidemiological investigations. Molecular techniques capable of typing bacteria at the level of the genome and proteome are now widely used for these investigations. This review considers two electrophoretic methods for typing bacteria on the basis of their proteomes, namely 1-D SDS-PAGE and 2-DE. The application of these two techniques for bacterial typing is described with reference to two publications that appeared in Electrophoresis [Costa, Electrophoresis 1990, 11, 382-391 and Cash et al., Electrophoresis 1997, 18, 1472-1482]. Even though these methods have been used for nearly 20 years to differentiate bacterial isolates they remain key technologies in proteome-based typing methods. The developments that have arisen from the two key papers are described in order to highlight the advantages and disadvantages in typing bacteria at the level of their proteomes. Some of the difficulties associated with electrophoretic typing methods can be overcome by using non-gel proteomic methods based on MS to provide improved sensitivity and specificity. The application of proteomic methods to investigate bacterial taxonomy, epidemiology and pathogenesis in general has significant potential in furthering our understanding of infectious diseases.

Acute stress alters the rates of degradation of cardiac muscle proteins.

Stressful experiences can have detrimental effects on many aspects of health and wellbeing. The zebrafish (Danio rerio) is a widely used model for stress research and a stress phenotype can be induced by manipulating the environmental conditions and social interactions. In this study we have combined a zebrafish stress model with the measurement of degradation rates of soluble cardiac muscle proteins. The results showed that the greater the stress response in the zebrafish the lower the level of overall protein degradation. On comparing the rates of degradation for individual proteins it was found that four main pathways were altered in response to stress conditions with decreased degradation for proteins involved in glucose metabolism, gluconeogenesis, the ubiquitin-proteasome system (UPS) and peroxisomal proliferator-activated receptor (PPAR) signalling pathways. Taken together, these data indicate that under stress conditions zebrafish preserve cardiac muscle proteins required for the 'fight or flight' response together with proteins that play a role in stress mitigation. SIGNIFICANCE: This study is the first to investigate the impact of stressful experiences on the dynamics of protein turnover in cardiac muscle. Using an established zebrafish model of human stress it has been possible to map key pathways at the protein level. The results show that the rates of degradation of cardiac proteins involved in glucose metabolism, UPS activity, hypoxia and PPAR signalling are decreased in stressed zebrafish. These findings indicate that proteins involved in the 'fight or flight' response to stress are conserved by the heart together with proteins that play a role in stress mitigation. This work provides the basis for more detailed investigations aimed at understanding the molecular effects of stress, which has implications for human health and disease.

In utero exposure to low doses of environmental pollutants disrupts fetal ovarian development in sheep.

Epidemiological studies of the impact of environmental chemicals on reproductive health demonstrate consequences of exposure but establishing causative links requires animal models using 'real life' in utero exposures. We aimed to determine whether prolonged, low-dose, exposure of pregnant sheep to a mixture of environmental chemicals affects fetal ovarian development. Exposure of treated ewes (n = 7) to pollutants was maximized by surface application of processed sewage sludge to pasture. Control ewes (n = 10) were reared on pasture treated with inorganic fertilizer. Ovaries and blood were collected from fetuses (n = 15 control and n = 8 treated) on Day 110 of gestation for investigation of fetal endocrinology, ovarian follicle/oocyte numbers and ovarian proteome. Treated fetuses were 14% lighter than controls but fetal ovary weights were unchanged. Prolactin (48% lower) was the only measured hormone significantly affected by treatment. Treatment reduced numbers of growth differentiation factor (GDF9) and induced myeloid leukaemia cell differentiation protein (MCL1) positive oocytes by 25-26% and increased pro-apoptotic BAX by 65% and 42% of protein spots in the treated ovarian proteome were differently expressed compared with controls. Nineteen spots were identified and included proteins involved in gene expression/transcription, protein synthesis, phosphorylation and receptor activity. Fetal exposure to environmental chemicals, via the mother, significantly perturbs fetal ovarian development. If such effects are replicated in humans, premature menopause could be an outcome.

Reducing the complexity of the Escherichia coli proteome by chromatography on reactive dye columns.

High-resolution 2-dimensional gel electrophoresis (2DGE) is a key technology in the analysis of cellular proteomes particularly in the field of microbiology. However, the restricted resolution of 2DGE and the limited dynamic range of established staining methods limit its usefulness for characterising low abundance proteins. Consequently, methods have been developed to either enrich for low abundance proteins directly or to deplete the highly abundant proteins present in complex samples. We present a protocol for affinity chromatography on reactive dye resins for the analysis of the Escherichia coli proteome. Using a range of commercially available reactive dye resins in a traditional chromatography system we were able to enrich low abundance proteins to levels suitable for their reliable detection and, most importantly, their identification using standard peptide mass mapping and MALDI-TOF MS methods. Under the chromatography conditions employed up to 4.42% of the proteins present in the total nonfractionated E. coli cell lysates bound to the reactive dye column and were subsequently eluted by 1.5 M NaCl. Of the bound proteins approximately 50% were considered to be enriched compared to the nonfractionated cell lysate. The ability to detect low abundance proteins was due to a combination of the specific enrichment of the proteins themselves as well as the depletion of highly abundant cellular proteins, which otherwise obscured the low abundance proteins. There was evidence of some selectivity between the different reactive dye resins for particular proteins. However, the selection of suitable dye resins to selectively enrich for particular classes of proteins remains largely empirical at this time.

Colorectal cancer-inflammatory bowel disease nexus and felony of Escherichia coli.

Colorectal cancer (CRC) has a multifactorial etiology. Although the exact cause of CRC is still elusive, recent studies have indicated microbial involvement in its etiology. Escherichia coli has emerged as an important factor in CRC development since the bacterium can cause changes in the gut that lead to cancerous transformation. A number of studies indicate that chronic inflammation induced by microorganisms, including E. coli, during inflammatory bowel disease (IBD) predisposes an individual to CRC. The evidence that support the role of E. coli in the etiology of CRC, through IBD, is not limited only to chronic inflammation. The growth of E. coli as an intracellular pathogen during IBD and CRC enable the bacteria to modulate the host cell cycle, induce DNA damage and accumulate mutations. These are some of the contributing factors behind the etiology of CRC. The present article considers the current status of the involvement of E. coli, through IBD, in the etiology of CRC. We discuss how intracellular E. coli infection can cause changes in the gut that can eventually lead to cellular transformation. In addition, the recent management strategies that target E. coli for prevention of CRC are also discussed.

Identification of potential biomarkers of hepatotoxicity by plasma proteome analysis of arsenic-exposed carp Labeo rohita.

Arsenic (As) is a toxic environmental contaminant and potential human carcinogen. Chronic intake of arsenic-contaminated water and food leads to arsenicosis, a major public health problem in many parts of the world. Early detection of arsenic toxicity would greatly benefit patients; however, the detection of arsenicosis needs to be done early before onset of severe symptoms in which case the tools used for detection have to be both sensitive and reliable. In this context, the present study investigated plasma proteome changes in arsenic-exposed Labeo rohita, with the aim of identifying biomarkers for arsenicosis. Changes in the plasma proteome were investigated using gel-based proteomics technology. Using quantitative image analysis of the 2D proteome profiles, 14 unique spots were identified by MALDI-TOF/TOF MS and/or LC-MS/MS which included Apolipoprotein-A1 (Apo-A1) (6 spots), α-2 macroglobulin-like protein (A2ML) (2 spots), transferrin (TF) (3 spots) and warm-temperature acclimation related 65kDa protein (Wap65). The proteome data are available via ProteomeXchange with identifier PXD003404. Highly abundant protein spots identified in plasma from arsenic-exposed fish i.e. Apo-A1 (>10-fold), A2ML (7-fold) and Wap65 (>2-fold) indicate liver damage. It is proposed that a combination of these proteins could serve as useful biomarkers of hepatotoxicity and chronic liver disease due to arsenic exposure.

Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein.

In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

Comparing Simplification Strategies for the Skeletal Muscle Proteome.

Skeletal muscle is a complex tissue that is dominated by the presence of a few abundant proteins. This wide dynamic range can mask the presence of lower abundance proteins, which can be a confounding factor in large-scale proteomic experiments. In this study, we have investigated a number of pre-fractionation methods, at both the protein and peptide level, for the characterization of the skeletal muscle proteome. The analyses revealed that the use of OFFGEL isoelectric focusing yielded the largest number of protein identifications (>750) compared to alternative gel-based and protein equalization strategies. Further, OFFGEL led to a substantial enrichment of a different sub-population of the proteome. Filter-aided sample preparation (FASP), coupled to peptide-level OFFGEL provided more confidence in the results due to a substantial increase in the number of peptides assigned to each protein. The findings presented here support the use of a multiplexed approach to proteome characterization of skeletal muscle, which has a recognized imbalance in the dynamic range of its protein complement.

Comparative proteomics of Helicobacter species: the discrimination of gastric and enterohepatic Helicobacter species.

Helicobacter pylori is a major human pathogen that infects the gastric mucosa and is responsible for a range of infections including gastritis and gastric carcinoma. Although other bacteria within the Helicobacter genus can also infect the gastric mucosa, there are Helicobacter species that infect alternative sites within the gastrointestinal (GI) tract. Two-dimensional gel electrophoresis was used to compare the cellular proteomes of seven non-pylori Helicobacters (H. mustelae, H. felis, H. cinaedi, H. hepaticus, H. fennelliae, H. bilis and H. cholecystus) against the more extensively characterised H. pylori. The different Helicobacter species showed distinctive 2D protein profiles, it was possible to combine them into a single dataset using Progenesis SameSpots software. Principal Component Analysis was used to search for correlations between the bacterial proteomes and their sites of infection. This approach clearly discriminated between gastric (i.e. those which infect in the gastric mucosa) and enterohepatic Helicobacter species (i.e. those bacteria that infect the small intestine and hepatobillary regions of the GI tract). Selected protein spots showing significant differences in abundance between these two groups of bacteria were identified by LC-MS. The data provide an initial insight into defining those features of the bacterial proteome that influence the sites of bacterial infection. BIOLOGICAL SIGNIFICANCE: This study demonstrated that representative members of the Helicobacter genus were readily discriminated from each other on the basis of their in vitro whole cell proteomes determined using 2D gel electrophoresis. Despite the intra-species heterogeneity observed it was possible, to demonstrate that the enterohepatic (represented by H. bilis, H. hepaticus, H. fennelliae, H. cinaedi and H. cholecystus) and gastric (represented by H. pylori, H. mustelae, and H. felis) Helicobacters formed discrete groups based on their 2D protein profiles. A provisional proteomic signature was identified that correlated with the typical sites of colonisation of these members of the Helicobacter genus. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Proteomics and protein analyses of ovine and caprine body fluids: current studies and future promises.

Our knowledge of the physiology and health of small ruminants, specifically sheep and goats, is frequently obtained by extrapolating information from other species, for example the cow. However, there are important genetic, physiological and anatomical differences between small and large ruminants that cannot be ignored. This review considers the advances that have been made in the investigation of sheep and goat physiology through the use of proteomic technologies. Proteomics is widely used to analyze clinically relevant body fluids for a number of animals to define productive traits and health status biomarkers as well as to monitor therapeutic interventions for infectious and metabolic diseases. Although the proteomes of body fluids have been described in detail for some animal species, there are few equivalent studies for sheep and goats. Nevertheless, the data now available for the proteomes of a range of body fluids in small ruminants have helped define new diagnostic and prognostic markers for these species. Moreover, these data are beneficial in studies where these small ruminants serve as models for human disease. However, despite the progress achieved to date, comprehensive data on the specific proteomes for many tissues and body fluids for sheep and goats remain scarce. The aim of this review is to describe the current status of small ruminant proteomic research and to demonstrate the potential benefits, as well as highlight the difficulties, of working with these animals.

E. coli and colon cancer: is mutY a culprit?

The recent demonstration of a role of Escherichia coli in the development of invasive carcinoma in mice ushers a new era of bacterial involvement in cancer etiology. It has been shown previously that the colonic mucosa of colorectal carcinoma (CRC) is exclusively colonized by intracellular E. coli instead of extracellular form found in normal colonic mucosa. Surprisingly, the DNA repair gene MUTYH, which is a homologue of the E. coli gene mutY, is responsible for CRC. The current paper discusses the potential role of mutY in CRC etiology and concludes that research in this area can bring together the diverse threads of the CRC etiology puzzle.

Marine n-3 fatty acids alter the proteomic response to methylmercury in Atlantic salmon kidney (ASK) cells.

Fish based diets have been linked to the amelioration of methylmercury (MeHg) induced symptoms in several epidemiological studies, particularly due to their contents of marine n-3 fatty acids. It has been suggested that n-3 fatty acids may mask the detrimental effects of MeHg due to their beneficial effect on the same biological functions which are negatively affected by MeHg. However, in vitro studies have implied that there may be direct interactions between the marine n-3 FAs and MeHg, which ameliorates MeHg toxicity through interactions at a biological level. To understand how marine n-3 FAs and MeHg interact in fish as a biological system, we wanted to investigate molecular interaction in a fish cell system. Atlantic salmon kidney (ASK) cells were pre-incubated with the marine n-3 FAs docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) before exposing them to MeHg. Modulating effects of the marine FAs on MeHg toxicity were subsequently assessed using the exploratory technique of proteomics, in a factorial design. Thirty-four differentially regulated proteins were identified. From these; twenty-seven were shown to be differentially regulated by MeHg, twelve were regulated by the fatty acids, and another eight showed interaction effects between MeHg and the FAs. Several of the proteins were concomitantly affected by MeHg- and FA-main effects, as well as interaction effects. Functional annotations and pathway analysis of the proteins revealed that marine n-3 FAs and MeHg concurrently affected the abundance of protein markers relating to such molecular mechanisms as: cell signaling, calcium homeostasis, structural integrity, apoptosis, and energy metabolism. In conclusion, both marine n-3 FAs and MeHg can differentially affect the abundances of the same proteins, indicating modulating effects of EPA and DHA on MeHg metabolism, and possibly on its toxicity.

Dietary methylmercury alters the proteome in Atlantic salmon (Salmo salar) kidney.

BACKGROUND: Methylmercury (MeHg) is an environmental contaminant most known for its severe neurotoxic effects. Although accumulation of MeHg tends to be several folds higher in kidney compared to other tissues, studies on nephrotoxic effects are almost non-existing. In this study we aim to investigate the toxicity of dietary MeHg in kidney of Atlantic salmon (Salmo salar). MATERIAL AND METHODS: Atlantic salmon were exposed to dietary MeHg for a period of 8 weeks, before the fish were euthanized and kidney was sampled for proteomic and real time RT-PCR analysis, as well as for mercury determination. Protein separation was done with 2-D PAGE, and differentially regulated spots were picked for analysis using liquid chromatography MS/MS analysis. Moreover, whole blood and liver tissue were sampled for mercury determination and real time RT-PCR (liver). RESULTS: MeHg exposed fish accumulated significantly more mercury (Hg) than control fish. The proteomic analysis revealed differential abundance of 26 spots in the kidney, and 14 of these protein spots were successfully identified. The proteins identified indicated effects of MeHg on; metabolism, inflammation, oxidative stress, protein-folding, and cell-structural components. Gene expression analysis of selected markers revealed few differentially regulated transcripts in kidney and liver in the exposed fish compared to the control fish. However, the affected transcripts indicated a disruption in the expression of two metabolic markers due to MeHg exposure in liver. CONCLUSION: This study suggests that dietary MeHg has similar effects in kidney as previously shown for other tissues in fish. The effects observed were in markers for oxidative stress, inflammation and energy metabolism. The identification of proteomic markers in this study provides a basis for a better understanding of MeHg-induced nephrotoxicity in fish.

Proteomic profiling of liver from Atlantic salmon (Salmo salar) fed genetically modified soy compared to the near-isogenic non-GM line.

The aim of this study was to investigate potential differences in liver protein expression of Atlantic salmon fed genetically modified (GM) Roundup Ready soy at a high inclusion level (25% inclusion, constituting 21% of crude protein in the diet) for 7 months or a compositionally similar non-GM diet. The liver was selected as the target organ due to its importance in the general metabolism, and 2D gel electrophoresis used as a screening tool. Samples from 12 individual fish from each diet group were evaluated. Of a total of 781 analysed protein spots, only 36 were significantly different by ANOVA (p < 0.05) in abundance between the diet groups. All these spots had low fold differences (1.2-1.6) and high false discovery rate (q = 0.44), indicating minor differences in liver protein synthesis between fish fed GM and non-GM soy. Additionally, low fold differences were observed. Four protein spots were analyzed by liquid chromatography tandem mass spectrometry and identified using a combination of online searches in NCBI and searches in an inhouse database containing salmonid expressed sequence tags and contigs. Follow-up on these proteins by real-time polymerase chain reaction did not identify differences at the transcriptional level.