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The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectrometry methods, liquid chromatography analysis, exoglycosidase digestions, glycan microarray methods, and nuclear magnetic resonance methods. Here, we present the first version of reporting guidelines for glyco(proteo)mics analysis by capillary electrophoresis (CE) for standardized and high-quality reporting of experimental conditions in the scientific literature. The guidelines cover all aspects of a glyco(proteo)mics CE experiment including sample preparation, CE operation mode (CZE, CGE, CEC, MEKC, cIEF, cITP), instrument configuration, capillary separation conditions, detection, data analysis, and experimental descriptors. These guidelines are linked to other MIRAGE guidelines and are freely available through the project website https://www.beilstein-institut.de/en/projects/mirage/guidelines/#ce_analysis (doi:10.3762/mirage.7).

EnzymeML is an XML-based data exchange format that supports the comprehensive documentation of enzymatic data by describing reaction conditions, time courses of substrate and product concentrations, the kinetic model, and the estimated kinetic constants. EnzymeML is based on the Systems Biology Markup Language, which was extended by implementing the STRENDA Guidelines. An EnzymeML document serves as a container to transfer data between experimental platforms, modeling tools, and databases. EnzymeML supports the scientific community by introducing a standardized data exchange format to make enzymatic data findable, accessible, interoperable, and reusable according to the FAIR data principles. An application programming interface in Python supports the integration of software tools for data acquisition, data analysis, and publication. The feasibility of a seamless data flow using EnzymeML is demonstrated by creating an EnzymeML document from a structured spreadsheet or from a STRENDA DB database entry, by kinetic modeling using the modeling platform COPASI, and by uploading to the enzymatic reaction kinetics database SABIO-RK.

The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to design guidelines that improve the reporting and reproducibility of glycoanalytical methods. Previously, the MIRAGE Commission has published guidelines for describing sample preparation methods and the reporting of glycan array and mass spectrometry techniques and data collections. Here, we present the first version of guidelines that aim to improve the quality of the reporting of liquid chromatography (LC) glycan data in the scientific literature. These guidelines cover all aspects of instrument setup and modality of data handling and manipulation and is cross-linked with other MIRAGE recommendations. The most recent version of the MIRAGE-LC guidelines is freely available at the MIRAGE project website doi:10.3762/mirage.4.

The minimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for sample preparation. The sample preparation guidelines include all aspects of sample generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE sample preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets.

Glycomics targets released glycans from proteins, lipids and proteoglycans. High throughput glycomics is based on mass spectrometry (MS) that increasingly depends on exchange of data with databases and the use of software. This requires an agreed format for accurately recording of experiments, developing consistent storage modules and granting public access to glycomic MS data. The introduction of the MIRAGE (Mimimum Requirement for A Glycomics Experiment) reporting standards for glycomics was the first step towards automating glycomic data recording. This report describes a glycomic e-infrastructure utilizing a well established glycomics recording format (GlycoWorkbench), and a dedicated web tool for submitting MIRAGE-compatible MS information into a public experimental repository, UniCarb-DR. The submission of data to UniCarb-DR should be a part of the submission process for publications with glycomics MSn that conform to the MIRAGE guidelines. The structure of this pipeline allows submission of most MS workflows used in glycomics.

A key component of enzyme function experiments is reporting of considerable metadata, to allow other researchers to replicate, interpret properly or use fully the results. This paper evaluates the completeness of enzyme function data reporting for reproducibility. We present a detailed examination of 11 recent papers (and their supplementary material) from two leading journals. We found that in every paper we were not able to collect some critical information necessary to reproduce the enzyme function findings. Study of 100 papers used by the SABIO-RK database confirmed some of the most common omissions: concentration of enzyme or its substrates, identity of counter-ions in buffers. A computer system should be better at preventing such omissions, helping secure the scientific record. Many of the omissions found would be trapped by the currently available version of STRENDA DB.

STRENDA DB, freely available at http://www.strenda-db.org, is an online validation and storage system for functional enzyme data that aims at being integrated into the publication practices of the scientific community and into the publication processes of journals. It provides a simple-to-use web submission tool and searchable database allowing the sharing, comparison and accurate reporting of enzyme kinetics data. The submission tool incorporates the STandards for Reporting ENzymology DAta (STRENDA), Guidelines which specify minimum information requested in the reporting of enzyme function data, including kinetic parameter values and full experimental conditions under which they were acquired. STRENDA DB checks the manuscript data entered by the author for compliance with the STRENDA Guidelines. If data is submitted prior to or during the publication process, the submission tool aids the author of a manuscript in the submission of kinetic parameters, ensuring that all required data and metadata are supplied. Data sets compliant with the Guidelines are assigned a STRENDA Registry Number and registered a Direct Object Identifier (DOI), which provides a perennial and resolvable identifier for each dataset. The data will normally be publicly available in STRENDA DB only after the corresponding article has been peer-reviewed and published in a journal. Data can also be submitted after publication. By promoting the practice of simultaneously submitting articles to journals and kinetics data to STRENDA DB, reviewers of journal articles as well as authors and consumers of data will benefit from the availability of standardised data in multiple ways.

The Annual 2014 Spring Workshop of the Proteomics Standards Initiative (PSI) of the Human Proteome Organization (HUPO) was held this year jointly with the metabolomics COordination of Standards in MetabOlomicS (COSMOS) group. The range of existing MS standards (mzML, mzIdentML, mzQuantML, mzTab, TraML) was reviewed and updated in the light of new methodologies and advances in technologies. Adaptations to meet the needs of the metabolomics community were incorporated and a new data format for NMR, nmrML, was presented. The molecular interactions workgroup began work on a new version of the existing XML data interchange format. PSI-MI XML3.0 will enable the capture of more abstract data types such as protein complex topology derived from experimental data, allosteric binding, and dynamic interactions. Further information about the work of the HUPO-PSI can be found at http://www.psidev.info.

International audience

The MIRAGE (minimum information required for a glycomics experiment) initiative was founded in Seattle, WA, in November 2011 in order to develop guidelines for reporting the qualitative and quantitative results obtained by diverse types of glycomics analyses, including the conditions and techniques that were applied to prepare the glycans for analysis and generate the primary data along with the tools and parameters that were used to process and annotate this data. These guidelines must address a broad range of issues, as glycomics data are inherently complex and are generated using diverse methods, including mass spectrometry (MS), chromatography, glycan array-binding assays, nuclear magnetic resonance (NMR) and other rapidly developing technologies. The acceptance of these guidelines by scientists conducting research on biological systems in which glycans have a significant role will facilitate the evaluation and reproduction of glycomics experiments and data that is reported in scientific journals and uploaded to glycomics databases. As a first step, MIRAGE guidelines for glycan analysis by MS have been recently published (Kolarich D, Rapp E, Struwe WB, Haslam SM, Zaia J., et al. 2013. The minimum information required for a glycomics experiment (MIRAGE) project – Improving the standards for reporting mass spectrometry-based glycoanalytic data. Mol. Cell Proteomics. 12:991–995), allowing them to be implemented and evaluated in the context of real-world glycobiology research. In this paper, we set out the historical context, organization structure and overarching objectives of the MIRAGE initiative.

Scientific publishing is changing; Open Access allows for published information to be freely shared, and Open Data repositories [1] are improving our ability to share and (re-)use data. These and initiatives such as RDA, FORCE11, and FAIR, [2, 3, 4] are changing the way we perceive the notion of publishing science. In the laboratory, equipment is becoming interconnected and new technologies are producing vast amounts of data; transformative change is underway. New publication practices are required that address the needs of the data producers and the data users. Many community-based grassroots projects and initiatives have already successfully set up infrastructures for storing and sharing data.

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme-kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme-kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113-7D grown in aerobic glucose-limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h(-1). The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo-like medium containing 300 mM potassium, 50 mM phosphate, 245 mM glutamate, 20 mM sodium, 2 mM free magnesium and 0.5 mM calcium, at a pH of 6.8. The V(max) values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme-specific, optimal conditions.

The activity of the vacuolar H(+)-ATPase has been characterized in isolated vacuoles of the yeast Saccharomyces cerevisiae by means of the patch-clamp technique. With cytosolic calcium at virtually zero (

Since the mid-1980s, use of the budding yeast, Saccharomyces cerevisiae, for expression of heterologous (foreign) genes and proteins has burgeoned for several major purposes, including facile genetic manipulation, large-scale production of specific proteins, and preliminary functional analysis. Expression of heterologous membrane proteins in yeast has not kept pace with expression of cytoplasmic proteins for two principal reasons: (1) although plant and fungal proteins express and function easily in yeast membranes, animal proteins do not, at least yet; and (2) the yeast plasma membrane is generally regarded as a difficult system to which to apply the standard electrophysiological techniques for detailed functional analysis of membrane proteins. Especially now, since completion of the genome-sequencing project for Saccharomyces, yeast membranes themselves can be seen as an ample source of diverse membrane proteins - including ion channels, pumps, and cotransporters - which lend themselves to electrophysiological analysis, and specifically to patch-clamping. Using some of these native proteins for assay, we report systematic methods to prepare both the yeast plasma membrane and the yeast vacuolar membrane (tonoplast) for patch-clamp experiments. We also describe optimized ambient conditions - such as electrode preparation, buffer solutions, and time regimens - which facilitate efficient patch recording from Saccharomyces membranes. There are two main keys to successful patch-clamping with Saccharomyces. The first is patience; the second is scrupulous cleanliness. Large cells, such as provided by polyploid strains, are also useful in yeast patch recording, especially while the skill required for gigaseal formation is being learned. Cleanliness is aided by (1) osmotic extrusion of protoplasts, after minimal digestion of yeast walls; (2) use of a rather spare suspension of protoplasts in the recording chamber; (3) maintenance of continuous chamber perfusion prior to formation of gigaseals; (4) preparation (pulling and filling) of patch pipettes immediately before use; (5) application of a modest pressure head to the pipette-filling solution before the tip enters the recording bath; (6) optical control for debris at the pipette tip; and (7) discarding of any pipette that does not "work" on the first try at gigaseal formation. Other useful tricks toward gigaseal formation include the making of protoplasts from cells grown aerobically, rather than anaerobically; use of sustained but gentle suction, rather than hard suction; and manipulation of bath temperature and/or osmotic strength. Yeast plasma membranes form gigaseals with difficulty, but these tend to be very stable and allow for long-term cell-attached or whole-cell recording. Yeast tonoplasts form gigaseals with ease, but these tend to be unstable and rarely allow recording for more than 15 min. The difference of stability accrues mainly because of the fact that yeast protoplasts adhere only lightly to the recording chamber and can therefore be lifted away on the patch pipette, whereas yeast vacuoles adhere firmly to the chamber bottom and are subsequently stressed by very slight relative movements of the pipette. With plasma membranes, conversion from cell-attached recording geometry to isolated ISO patch (inside-out) geometry is accomplished by blowing a fine stream of air bubbles across the pipette tip; to whole-cell recording geometry, by combining suction and one high-voltage pulse; and from whole-cell to OSO patch (outside-out) geometry, by sudden acceleration of the bath perfusion stream. With tonoplasts, conversion from the vacuole-attached recording geometry to whole-vacuole geometry is accomplished by application of a large brief voltage pulse; and further conversion to the OSO patch geometry is carried out conventionally, by slow withdrawal of the patch pipette from the vacuole, which usually remains attached to the chamber bottom.

The major voltage-dependent ion channel in the plasma membrane of Saccharomyces cerevisiae, a conspicuous outwardly rectifying K+ channel, was first dubbed YPK1 and later renamed according to its registered gene names (DUK1, TOK1). It has proven novel in both structure and function. Whole-cell patch-clamp studies of the channel directly on yeast protoplasts now extend our earlier description obtained from isolated patches of yeast membrane (Bertl & Slayman, 1992; Bertl et al., 1993), and provide new data both on the contributions of channel properties to yeast physiology and on possible contributions of molecular structure of channel properties. Three recording tactics produce completely equivalent results and thereby allow great flexibility in the design of experiments: whole-cell voltage clamp with sustained voltage steps (approximately 2.5 sec), whole-cell voltage clamp with slow voltage ramps (5 sec, -40 to +100 mV), and time-averaging of single-channel currents. Activation of Duk1 channels under steady-state conditions is dependent upon ATP in the cytoplasmic solution, and the absence of ATP results in channel "rundown"--decreasing numbers of activable channels--over periods of 10 min to 1 hr from the start of patch recording. Several putative serine- and threonine-phosphorylation sites, as well as a variant ATP-binding fold, exist in the molecule as potential mediators of the ATP effects. The channel runs down similarly following cytoplasmic acidification, but is almost completely insensitive to extracellular pH changes (8.0 to 5.5 tested). This remarkable asymmetry may depend on the protein's strongly asymmetric distribution of histidine residues, with 10 out of 12 predicted to lie close to the membrane-cytoplasm interface. Further data confirm the well-recognized observation that changes of K+ concentration, intracellular or extracellular, can shift the gating voltage of Duk1p in the direction of EK. Among the other alkali-metal cations tested, extracellular Rb+ and Cs(+)--but not Na(+)--substitute almost completely for K+. Extracellular TEA+ inhibits whole-cell K+ currents through Duk1p with a KI of 2.8 mM, and does so probably by reducing the single-channel current.

Glycomics is an emerging discipline that seeks to identify and quantify the complex carbohydrates in an organism, tissue, or cell type in order to understand their functions in diverse biological contexts. Recent advances in the analytical technology used for this purpose have dramatically increased the amount of glycomics data that is being generated, interpreted, and published. However, the intrinsic challenges of identifying individual glycans in complex biological samples almost always make it necessary for the glycoanalyst to make fundamental assumptions about the structures present in the sample. The Minimum Information Required for A Glycomics Experiment (MIRAGE) guidelines aim to enhance the value of glycomics data by encouraging analysts to report the experimental conditions under which their data were generated and the assumptions they made when interpreting those data. This chapter discusses some of the major challenges of glycoanalysis and the efforts of the MIRAGE consortium to help glycoscientists and publishers cope with these challenges.

The MIRAGE guidelines are being developed in response to a critical need in the glycobiology community to clarify glycoanalytic results so that they are more readily evaluated (in terms of their scope and depth) and to facilitate the reproduction of important results in the laboratory. The molecular and biological complexity of the glycosylation process makes thorough reporting of the results of a glycomics experiment a highly challenging endeavor. The resulting data specify the identity and quantity of complex structures, the precise molecular features of which are sometimes inferred using prior knowledge, such as familiarity with a particular biosynthetic mechanism. Specifying the exact methods and assumptions that were used to assign and quantify reported structures allows the interested scientist to appreciate the scope and depth of the analysis. Mass spectrometry (MS) is the most widely used tool for glycomics experiments. The interpretation and reproducibility of MS-based glycomics data depend on comprehensive meta-data describing the instrumentation, instrument setup, and data acquisition protocols. The MIRAGE guidelines for MS-based glycomics have been designed to facilitate the collection and sharing of this critical information in order to assist the glycoanalyst in generating data sets with maximum information content and biological relevance.

The rate at which data is acquired frequently outstrips the capacity of the human mind to house it. Instead, we mine it. The ability to electronically cull the majority of mankind's knowledge of the functioning of a particular biomolecule at the push of a button would be an acutely effective, efficient research tool. Consider the benefits of crossing such information against single nucleotide polymorphism databases to identify the biochemical lesions associated with disease-linked mutations or associate the functional consequences of mutations with changes in the structures housed in the Protein Data Bank. Additionally, as systems biologists strive to integrate large swaths of metabolism, ready access to initial-rate equilibria and regulatory data will prove immensely useful. Perhaps the greatest value of such a database lies in the myriad ways in which it would integrate into the daily activities of individuals, worldwide. One cannot help but wonder what fraction of the protein-function literature is obscured or even lost to the researcher by imprecise search engines and retrieval strategies.

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme-kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme-kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113-7D grown in aerobic glucose-limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h(-1). The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo-like medium containing 300 mM potassium, 50 mM phosphate, 245 mM glutamate, 20 mM sodium, 2 mM free magnesium and 0.5 mM calcium, at a pH of 6.8. The V(max) values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme-specific, optimal conditions.

The extent of linkage disequilibrium (LD) is an important factor when designing experiments for mapping disease or trait loci using LD mapping methods. It depends on the population history and hence is a characteristic of each population. Here, we have assessed the extent of LD in a sub-isolate of the general Sardinian population (775 members of one village) using 22 polymorphic markers on chromosome 19. We found high levels of disequilibrium that extended to 8 cM, when based on D', and 11 cM when based on the significance level of the allelic association. The fact that conclusions based on both methods are similar suggests that the estimates are quite robust. We have also shown, through a simple resampling technique, that small sample sizes can overestimate both the mean value of D' and its variance up to a factor of about 2 and 16, respectively, when the number of diplotypes (the pair of haplotypes that compose the genotype) decreased from 186 to 26. We evaluated the effect on D' of the depth of the pedigree available when using phased founders, and compared the estimates with those obtained when using unphased founders, and also the effect of grouping alleles on the value of D' and the significance level. Owing to the high sampling variance of LD, we recommend the use of at least 200 unrelated individuals when characterizing the extent of LD

The worldwide DNA-sequencing efforts annually deposit ∼70 billion base-pairs of sequence information into the public databases – the equivalent of one Escherichia coli genome every 30 s – and the rate of deposition is doubling every 10–14 months. As biologists attempt to realize even a small fraction of the potential of this information by assembling accurate models of metabolic processes, they are thwarted by a surprising lack of uniformity in the acquisition and reporting of protein-function data – the data cannot be integrated into the models because the standards needed to link protein-function datasets to one another do not yet exist.

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP). These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from -80 to +80 mV), thus measuring the reversal voltage (ER) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed. This ratio was found to depend strongly upon the pH difference (DeltapH) across the vacuolar membrane, being approximately 2H+/ATP at high DeltapH (4 pH units) and increasing to4H+/ATP for small or zero DeltapH. That result is in quantitative agreement with previous determinations on plant vacuoles. Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit--which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.

This report summarizes the proceedings of the second workshop of the ‘Minimum Information for Biological and Biomedical Investigations’ (MIBBI) consortium held on Dec 1–2, 2010 in Rudesheim, Germany through the sponsorship of the Beilstein-Institute. MIBBI is an umbrella organization uniting communities developing Minimum Information (MI) checklists to standardize the description of data sets, the workflows by which they were generated and the scientific context for the work. This workshop brought together representatives of more than twenty communities to present the status of their MI checklists and plans for future development. Shared challenges and solutions were identified and the role of MIBBI in MI checklist development was discussed. The meeting featured some thirty presentations, wide-ranging discussions and breakout groups. The top outcomes of the two-day workshop as defined by the participants were: 1) the chance to share best practices and to identify areas of synergy; 2) defining a series of tasks for updating the MIBBI Portal; 3) reemphasizing the need to maintain independent MI checklists for various communities while leveraging common terms and workflow elements contained in multiple checklists; and 4) revision of the concept of the MIBBI Foundry to focus on the creation of a core set of MIBBI modules intended for reuse by individual MI checklist projects while maintaining the integrity of each MI project. Further information about MIBBI and its range of activities can be found at http://mibbi.org/ .

International audience; Data on enzyme activities and kinetics have often been reported with insufficient experimental detail to allow their repetition. This paper discusses the objectives and recommendations of the Standards for Reporting Enzyme Data (STRENDA) project to define minimal experimental standards for the reporting enzyme functional data.

Systems level investigation of genomic and proteomic scale information requires incomparably higher demands for data quality than in previous decades. Truly integrated databases that deal with heterogeneous data need to be developed to be able to retrieve properties of genes, for kinetics of enzymes, for behaviour of complex networks and for the analysis and modelling of complex biological processes. Despite the fast paced global efforts in biological systems research, the current analyses are limited by the lack of available systematic collections of comparable functional enzyme data. Besides its reliability, these data have to provide defined minimum experimental information, they must be available from the literature along with their accepted enzyme names, and must be as comprehensive as possible. However, the reality reveals a different picture: the quality of experimental data of enzymes is insufficient for the needs of systems level investigations. A 2003 founded working group, called STRENDA, recently published suggestions which intend both to improve the quality of reporting functional enzyme data and to support the comparability of inter alia enzyme kinetics for their application in the in silico investigation of biological systems.