Julie Howard

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.

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.

Proteomic analysis of the EhV-86 virion

BackgroundEmiliania huxleyi virus 86 (EhV-86) is the type species of the genus Coccolithovirus within the family Phycodnaviridae. The fully sequenced 407,339 bp genome is predicted to encode 473 protein coding sequences (CDSs) and is the largest Phycodnaviridae sequenced to date. The majority of EhV-86 CDSs exhibit no similarity to proteins in the public databases.ResultsProteomic analysis by 1-DE and then LC-MS/MS determined that the virion of EhV-86 is composed of at least 28 proteins, 23 of which are predicted to be membrane proteins. Besides the major capsid protein, putative function can be assigned to 4 other components of the virion: two lectin proteins, a thioredoxin and a serine/threonine protein kinase.ConclusionThis study represents the first steps toward the identification of the protein components that make up the EhV-86 virion. Aside from the major capsid protein, whose function in the virion is well known and defined, the nature of the other proteins suggest roles involved with viral budding, caspase activation, signalling, anti-oxidation, virus adsorption and host range determination.

Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius.

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled

ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter.

Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex‐related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham‐operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down‐regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex‐related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

Chemo- and Regioselective Lysine Modification on Native Proteins

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

Stat3-mediated alterations in lysosomal membrane protein composition

Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome.

Analysis of temperature-mediated changes in the wine yeast Saccharomyces bayanus var uvarum. An oenological study of how the protein content influences wine quality.

Saccharomyces bayanus var. uvarum plays an important role in the fermentation of red wine from the D.O. Ribera del Duero. This is due to the special organoleptic taste that this yeast gives the wines and their ability to ferment at low temperature. To determine the molecular factors involved in the fermentation process at low temperature, a differential proteomic approach was performed by using 2D‐DIGE, comparing, qualitatively and quantitatively, the profiles obtained at 13 and 25°C. A total of 152 protein spots were identified. We detected proteins upregulated at 13°C that were shown to be related to temperature stress, the production of aromatic compounds involved in the metabolism of amino acids, and the production of fusel alcohols and their derivatives, each of which is directly related to the quality of the wines. To check the temperature effects, an aromatic analysis by GC–MS was performed. The proteomic and “aromatomic” results are discussed in relation to the oenological properties of S. bayanus var. uvarum.

Identification of protein biomarkers in human serum using iTRAQ and shotgun mass spectrometry.

Blood serum is one of the easiest accessible sources of biomarkers and its proteome presents a significant parcel of immune system proteins. These proteins can provide not only biological explanation but also diagnostic and drug response answers independently of the type of disease or condition in question. Shotgun mass spectrometry has profoundly contributed to proteome analysis and is presently considered as an indispensible tool in the field of biomarker discovery. In addition, the multiplexing potential of isotopic labeling techniques such as iTRAQ can increase statistical relevance and accuracy of proteomic data through the simultaneous analysis of different biological samples. Here, we describe a complete protocol using iTRAQ in a shotgun proteomics workflow along with data analysis steps, customized for the challenges associated with the serum proteome.