David Stead

A systematic approach to modeling, capturing, and disseminating proteomics experimental data

Both the generation and the analysis of proteome data are becoming increasingly widespread, and the field of proteomics is moving incrementally toward high-throughput approaches. Techniques are also increasing in complexity as the relevant technologies evolve. A standard representation of both the methods used and the data generated in proteomics experiments, analogous to that of the MIAME (minimum information about a microarray experiment) guidelines for transcriptomics, and the associated MAGE (microarray gene expression) object model and XML (extensible markup language) implementation, has yet to emerge. This hinders the handling, exchange, and dissemination of proteomics data. Here, we present a UML (unified modeling language) approach to proteomics experimental data, describe XML and SQL (structured query language) implementations of that model, and discuss capture, storage, and dissemination strategies. These make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of a proteome repository.

Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence

Population genetics of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) show that isolates are highly related and globally homogenous, data that are consistent with the recent epidemic spread of a previously endemic organism. Highly related isolates are predicted to be functionally similar due to low levels of heritable genetic diversity. To test this hypothesis, we took a global panel of Bd isolates and measured (i) the genetic relatedness among isolates, (ii) proteomic profiles of isolates, (iii) the susceptibility of isolates to the antifungal drug caspofungin, (iv) the variation among isolates in growth and phenotypic characteristics, and (v) the virulence of isolates against the European common toad Bufo bufo. Our results show (i) genotypic differentiation among isolates, (ii) proteomic differentiation among isolates, (iii) no significant differences in susceptibility to caspofungin, (iv) differentiation in growth and phenotypic/morphological characters, and (v) differential virulence in B. bufo. Specifically, our data show that Bd isolates can be profiled by their genotypic and proteomic characteristics, as well as by the size of their sporangia. Bd genotypic and phenotypic distance matrices are significantly correlated, showing that less‐related isolates are more biologically unique. Mass spectrometry has identified a set of candidate genes associated with inter‐isolate variation. Our data show that, despite its rapid global emergence, Bd isolates are not identical and differ in several important characters that are linked to virulence. We argue that future studies need to clarify the mechanism(s) and rate at which Bd is evolving, and the impact that such variation has on the host–pathogen dynamic.

PEDRo: A database for storing, searching and disseminating experimental proteomics data

BackgroundProteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets.ResultsThis paper describes the design, implementation and use of a P roteome E xperimental D ata R epo sitory (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available.ConclusionsThe PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation.

MNL1 Regulates Weak Acid-induced Stress Responses of the Fungal Pathogen Candida albicans

MNL1, the Candida albicans homologue of an orphan Msn2-like gene (YER130c in Saccharomyces cerevisiae) has no known function. Here we report that MNL1 regulates weak acid stress responses. Deletion of MNL1 prevents the long-term adaptation of C. albicans cells to weak acid stresses and compromises their global transcriptional response under these conditions. The promoters of Mnl1-dependent genes contain a novel STRE-like element (SLE) that imposes Mnl1-dependent, weak acid stress-induced transcription upon a lacZ reporter in C. albicans. The SLE (HHYYCCCCTTYTY) is related to the Nrg1 response element (NRE) element recognized by the transcriptional repressor Nrg1. Deletion of NRG1 partially restores the ability of C. albicans mnl1 cells to adapt to weak acid stress, indicating that Mnl1 and Nrg1 act antagonistically to regulate this response. Molecular, microarray, and proteomic analyses revealed that Mnl1-dependent adaptation does not occur in cells exposed to proapoptotic or pronecrotic doses of weak acid, suggesting that Ras-pathway activation might suppress the Mnl1-dependent weak acid response in dying cells. Our work defines a role for this YER130c orthologue in stress adaptation and cell death.

Guidelines for reporting the use of gel electrophoresis in proteomics

Gibson, Frank Anderson, Leigh Babnigg, Gyorgy Baker, Mark Berth, Matthias Binz, Pierre-Alain Borthwick, Andy Cash, Phil Day, Billy W. Friedman, David B. Garland, Donita Gutstein, Howard B. Hoogland, Christine Jones, Neil A. Khan, Alamgir Klose, Joachim Lamond, Angus I. Lemkin, Peter F. Lilley, Kathryn S. Minden, Jonathan Morris, Nicholas J. Paton, Norman W. Pisano, Michael R. Prime, John E. Rabilloud, Thierry Stead, David A. Taylor, Chris F. Voshol, Hans Wipat, Anil Jones, Andrew R. 2 NATURE PUBLISHING GROUP NEW YORK 335WX

The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans

ABSTRACT Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed in C. albicans, S. cerevisiae Icl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that like S. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation. C. albicans Icl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed in C. albicans ubi4 cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes from S. cerevisiae but absent from their C. albicans homologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure, C. albicans retains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients. IMPORTANCE Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available. Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.

A proteomic analysis of the salt, cadmium and peroxide stress responses in Candida albicans and the role of the Hog1 stress-activated MAPK in regulating the stress-induced proteome.

Stress responses are important for the virulence of the major fungal pathogen of humans, Candida albicans. In this study we employed a 2‐DE approach to examine the impact of exposure to peroxide (5 mM H2O2), salt (300 mM NaCl) or cadmium stress (0.5 mM Cd2+) upon the C. albicans proteome. Highly reproducible changes in the C. albicans proteome were observed in response to each stress condition. Significantly more proteins were up‐regulated in response to cadmium (77) than to the salt (35) or peroxide stresses (35). These proteomic changes displayed minimal overlap with those observed in the transcriptome under equivalent conditions and, importantly, revealed functional categories that respond to stress at the protein level but not the transcript level. Six proteins were up‐regulated by all three conditions: Adh1, Atp2, Cip1, Eft2, Ssa1 and Ssb1, which is consistent with the concept that a core stress response exists in C. albicans. This is the first time that a fungal core stress response has been defined at the proteomic level. We have also shown that the Hog1 stress‐activated mitogen‐activated protein kinase, which is activated in response to the stresses examined in this study, makes a major contribution to the C. albicans stress proteome.

Proteomic analysis of the pH response in the fungal pathogen Candida glabrata

Micro‐organisms must adapt to environmental change to survive, and this is particularly true for fungal pathogens such as Candida glabrata. C. glabrata is found both in the environment and in diverse niches in its human host. The ambient pH of these niches varies considerably, and therefore we have examined the response of C. glabrata to changes in ambient pH using a proteomic approach. Proteins expressed in C. glabrata cells growing at pH 4.0, 7.4 or 8.0 were compared by 2‐DE, and 174 spots displaying reproducible and statistically significant changes in expression level were identified by peptide mass fingerprinting, thereby extending our 2‐DE map of the C. glabrata proteome to a total of 272 identified spots. Proteins involved in glucose metabolism, the TCA cycle, respiration and protein synthesis were expressed at lower levels during growth at pH 7.4 and/or 8.0, whereas proteins involved in stress responses and protein catabolism were expressed at higher levels under these alkaline conditions. Our data suggest that C. glabrata perceives low pH as less stressful than higher pH. This contrasts with another opportunistic fungal pathogen of humans, Candida albicans

A comprehensive evaluation of colonic mucosal isolates of Sutterella wadsworthensis from inflammatory bowel disease.

Inflammatory bowel disease (IBD) arises in genetically susceptible individuals as a result of an unidentified environmental trigger, possibly a hitherto unknown bacterial pathogen. Twenty-six clinical isolates of Sutterella wadsworthensis were obtained from 134 adults and 61 pediatric patients undergoing colonoscopy, of whom 69 and 29 respectively had IBD. S. wadsworthensis was initially more frequently isolated from IBD subjects, hence this comprehensive study was undertaken to elucidate its role in IBD. Utilizing these samples, a newly designed PCR was developed, to study the prevalence of this bacterium in adult patients with ulcerative colitis (UC). Sutterella wadsworthensis was detected in 83.8% of adult patients with UC as opposed to 86.1% of control subjects (p = 0.64). Selected strains from IBD cases and controls were studied to elicit morphological, proteomic, genotypic and pathogenic differences. This study reports Scanning Electron Microscopy (SEM) appearances and characteristic MALDI-TOF MS protein profiles of S. wadsworthensis for the very first time. SEM showed that the bacterium is pleomorphic, existing in predominantly two morphological forms, long rods and coccobacilli. No differences were noted in the MALDI-TOF mass spectrometry proteomic analysis. There was no distinct clustering of strains identified from cases and controls on sequence analysis. Cytokine response after monocyte challenge with strains from patients with IBD and controls did not yield any significant differences. Our studies indicate that S. wadsworthensis is unlikely to play a role in the pathogenesis of IBD. Strains from cases of IBD could not be distinguished from those identified from controls.

Universal Metrics for Quality Assessment of Protein Identifications by Mass Spectrometry

Increasing numbers of large proteomic datasets are becoming available. As attempts are made to interpret these datasets and integrate them with other forms of genomic data, researchers are becoming more aware of the importance of data quality with respect to protein identification. We present three simple and universal metrics that describe different aspects of the quality of protein identifications by peptide mass fingerprinting. Hit ratio gives an indication of the signal-to-noise ratio in a mass spectrum, mass coverage measures the amount of protein sequence matched, and excess of limit-digested peptides reflects the completeness of the digestion that precedes the peptide mass fingerprinting. Receiver-operating characteristic plots show that the novel metric, excess of limit-digested peptides, can discriminate between correct and random matches more accurately than search score when validating the results from a state-of-the-art protein identification software system (Mascot) especially when combined with the two other metrics, hit ratio and mass coverage. Recommendations are made regarding the use of the metrics when reporting protein identification experiments.