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PubMed2007

Design characteristics that affect speed of information access and clarity of presentation in an electronic neuroanatomy atlas.

Functional Neuroanatomy, an interactive electronic neuroanatomical atlas, was designed for first year medical students. Medical students have much to learn in a limited time; therefore a major goal in the atlas design was that it facilitate rapid, accurate information retrieval. To assess this feature, we designed a testing scenario in which students who had never taken a neuroanatomy course were asked to complete two equivalent tests, one using the electronic atlas and one using a comparable hard copy atlas, in a limited period of time. The tests were too long to be completed in the time allotted, so test scores were measures of how quickly correct information could be retrieved from each source. Statistical analysis of the data showed that the tests were of equal difficulty and that accurate information retrieval was significantly faster using the electronic atlas when compared with the hard copy atlas (P < 0.0001). Post-test focus groups (n = 4) allowed us to infer that the following design features contributed to rapid information access: the number of structures in the database was limited to those that are relevant to a practicing physician; all of the program modules were presented in both text and image form on the index screen, which doubled as a site map; pages were layered electronically such that information was hidden until requested, structures available on each page were listed alphabetically and could be accessed by clicking on their name; and an illustrated glossary was provided and equipped with a search engine.

Humans↗

PubMed2006

Information-seeking behaviors and reflective practice.

INTRODUCTION: As they care for patients, physicians raise questions, but they pursue only a portion of them. Without the best information and evidence, care and patient safety may be compromised. Understanding when and why problems prompt physicians to look for information and integrate results into their knowledge base is critical and shapes one part of reflection about care. This study explores the role of the Internet in gathering medical information as one step in that reflective practice, the barriers to its use, and changes in utilization over time. METHODS: A questionnaire with 18 items adapted from previous studies was sent by facsimile to a randomly selected sample of U.S. physicians in all specialties and active in practice. RESULTS: Specific patient problems and latest research in a specific topic most often prompt physicians to search on the Internet. Younger physicians and female physicians were most likely to seek information on a specific patient problem. Only 9% of all respondents (n = 2,500) searched for information during a patient encounter. When unsure about diagnostic and management issues for a complex case, 41.3% chose to consult with a colleague or read from a text (22.8%). Searching most often occurred at home after work (38.2%) or during breaks in the day (35.7%). Most (68.7%) found the information they were looking for more than 51% of the time. Searching was facilitated by knowing preferred sites and access in the clinical setting. The greatest barriers to answering clinical questions included a lack of specific information and too much information to scan. DISCUSSION: Although physicians are increasingly successful and confident in their Internet searching to answer questions raised in patient care, few choose to seek medical information during a patient encounter. Internet information access may facilitate overall reflection on practice; physicians do not yet use this access in a just-in-time manner for immediately solving difficult patient problems but instead continue to rely on consultation with colleagues. Professional association Web sites and point-of-care databases are helpful. From physicians' use of the Internet, professionals in continuing medical education must learn which search engines and sites are trusted and preferred.

Attitude of Health Personnel↗

PubMed2004

An evaluation of web-based information.

OBJECTIVE: To evaluate the quality of web-based information on the treatment of eating disorders and to investigate potential indicators of content quality. METHOD: Two search engines were queried to obtain 15 commonly accessed websites about eating disorders. Two reviewers evaluated the characteristics, quality of content, and accountability of the sites. Intercorrelations between variables were calculated. RESULTS: The overall quality of the sites was poor based on the outcome measures used. All quality of content measures correlated with a measure of accountability (Silberg, W.M., Lundberg, G.D., & Mussachio, R.A., 1993). DISCUSSION: There is a lack of quality information on the treatment of eating disorders on the web. Although accountability criteria may be useful indicators of content quality, there is a need to investigate whether these can be usefully applied to other mental health areas.

Feeding and Eating Disorders↗

PubMed1997

The 2DWG meta-database of two-dimensional electrophoretic gel images on the Internet.

The 2DWG meta-database is a searchable database of two-dimensional (2-D) electrophoretic gel images found on the Internet. A meta-database contains information about locating data in other databases - but not that data itself. This database was constructed because of a need for an enriched set of World Wide Web (WWW) locations (URLs) of 2-D gel images on the Internet. These gel images are used in conjunction with the National Cancer Institute (NCI) Flicker Server to manipulate and visually compare 2-D gel images across the Internet. User's gels may also be compared with those in the database. The 2DWG is organized as a spreadsheet table with each gel image being represented by a row sorted by tissue type. Data for each gel includes tissue type, species, cell-line, image URL, database URL, gel protocol, organization URL, image properties, map URL if it exists, etc. The 2DWG may be searched to find relevant subsets of gels. Searching is done using the dbEngine - a WWW database search engine which accesses selected rows of gels from the full 2DWG table. The 2DWG meta-database is accessible on the WWW at http://www-lecb.ncifcrf.gov/2dwgDB/ and the NCI Flicker server at http://www-lecb.ncifcrf.gov/flicker/

Computer Communication Networks↗

PubMed2002

Identification and sequencing analysis of intact proteins via collision-induced dissociation and quadrupole time-of-flight mass spectrometry.

Identifying unknown proteins has become a central focal point for proteomic and biopharmaceutical development laboratories. Our laboratory investigated using quadrupole time-of-flight mass spectrometry (Qq/TOFMS) for the analysis of intact proteins for the purpose of identifying unknowns while limiting the number of sample-handling steps between protein extraction and identification. Eight standard proteins, both unmodified and disulfide-bonded and ranging in mass from 5 to 66 kDa, were analyzed using nanoelectrospray and collision-induced dissociation to generate peptide sequence tags. An MS analysis, followed by MS/MS analyses on two to five individual protein charge states, were obtained to make an identification. Peptide sequence tags were extracted from the MS/MS data and used, in conjunction with molecular mass and source origin, to obtain protein identifications using the web-based search engine ProteinInfo (www.proteometrics.com). All of the proteins were unambiguously identified from the input data, after which, all of the major product ions were identified for structural information. In most cases, N- and/or C-terminal ions, and also stretches of consecutive product ions from the protein interior, were observed. This method was applied to the analysis and identification of an unknown detected via reversed-phase high-performance liquid chromatography.

Amino Acid Sequence↗

PubMed2004

Locating and retaining research participants for follow-up studies.

Two common pitfalls of longitudinal research are loss of participants over time and inability to locate participants whose contact information has changed. This article is based on our experiences in locating and retaining a sample of caregivers of persons with Parkinson's disease 8-10 years after we last contacted them. The strategies we used resulted in locating 86% of our sample and retaining 80% of those who were eligible. These strategies included asking participants for a backup contact, asking participants if they would be willing to be contacted again for a future study, making the most of existing search engines, keeping in touch, and being flexible, patient, and professional.

Caregivers↗

PubMed2003

Proteome of Caulobacter crescentus cell cycle publicly accessible on SWICZ server.

Here we present the Swiss-Czech Proteomics Server (SWICZ), which hosts the proteomic database summarizing information about the cell cycle of the aquatic bacterium Caulobacter crescentus. The database provides a searchable tool for easy access of global protein synthesis and protein stability data as examined during the C. crescentus cell cycle. Protein synthesis data collected from five different cell cycle stages were determined for each protein spot as a relative value of the total amount of [(35)S]methionine incorporation. Protein stability of pulse-labeled extracts were measured during a chase period equivalent to one cell cycle unit. Quantitative information for individual proteins together with descriptive data such as protein identities, apparent molecular masses and isoelectric points, were combined with information on protein function, genomic context, and the cell cycle stage, and were then assembled in a relational database with a world wide web interface (http://proteom.biomed.cas.cz), which allows the database records to be searched and displays the recovered information. A total of 1250 protein spots were reproducibly detected on two-dimensional gel electropherograms, 295 of which were identified by mass spectroscopy. The database is accessible either through clickable two-dimensional gel electrophoretic maps or by means of a set of dedicated search engines. Basic characterization of the experimental procedures, data processing, and a comprehensive description of the web site are presented. In its current state, the SWICZ proteome database provides a platform for the incorporation of new data emerging from extended functional studies on the C. crescentus proteome.

Bacterial Proteins↗

PubMed2004

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Amino Acid Sequence↗

PubMed2005

Sequence tag scanning: a new explorative strategy for recognition of unexpected protein alterations by nanoelectrospray ionization-tandem mass spectrometry.

Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a2, b2, b3, y1, y2, and y3. The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms.

Acetylation↗

PubMed2006

Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible host-pathogen interaction.

Wheat leaf rust is caused by the fungus Puccinia triticina. The genetics of resistance follows the gene-for-gene hypothesis, and thus the presence or absence of a single host resistance gene renders a plant resistant or susceptible to a leaf rust race bearing the corresponding avirulence gene. To investigate some of the changes in the proteomes of both host and pathogen during disease development, a susceptible line of wheat infected with a virulent race of leaf rust were compared to mock-inoculated wheat using 2-DE (with IEF pH 4-8) and MS. Up-regulated protein spots were excised and analyzed by MALDI-QqTOF MS/MS, followed by cross-species protein identification. Where possible MS/MS spectra were matched to homologous proteins in the NCBI database or to fungal ESTs encoding putative proteins. Searching was done using the MASCOT search engine. Remaining unmatched spectra were then sequenced de novo and queried against the NCBInr database using the BLAST and MS BLAST tools. A total of 32 consistently up-regulated proteins were examined from the gels representing the 9-day post-infection proteome in susceptible plants. Of these 7 are host proteins, 22 are fungal proteins of known or hypothetical function and 3 are unknown proteins of putative fungal origin.

Amino Acid Sequence↗

PubMed2005

Further steps in standardisation. Report of the second annual Proteomics Standards Initiative Spring Workshop (Siena, Italy 17-20th April 2005).

The spring workshop of the HUPO-PSI convened in Siena to further progress the data standards which are already making an impact on data exchange and deposition in the field of proteomics. Separate work groups pushed forward existing XML standards for the exchange of Molecular Interaction data (PSI-MI, MIF) and Mass Spectrometry data (PSI-MS, mzData) whilst significant progress was made on PSI-MS' mzIdent, which will allow the capture of data from analytical tools such as peak list search engines. A new focus for PSI (GPS, gel electrophoresis) was explored; as was the need for a common representation of protein modifications by all workers in the field of proteomics and beyond. All these efforts are contextualised by the work of the General Proteomics Standards workgroup; which in addition to the MIAPE reporting guidelines, is continually evolving an object model (PSI-OM) from which will be derived the general standard XML format for exchanging data between researchers, and for submission to repositories or journals.

Mass Spectrometry↗

PubMed2006

Automated reprocessing pipeline for searching heterogeneous mass spectrometric data of the HUPO Brain Proteome Project pilot phase.

The newly available techniques for sensitive proteome analysis and the resulting amount of data require a new bioinformatics focus on automatic methods for spectrum reprocessing and peptide/protein validation. Manual validation of results in such studies is not feasible and objective enough for quality relevant interpretation. The necessity for tools enabling an automatic quality control is, therefore, important to produce reliable and comparable data in such big consortia as the Human Proteome Organization Brain Proteome Project. Standards and well-defined processing pipelines are important for these consortia. We show a way for choosing the right database model, through collecting data, processing these with a decoy database and end up with a quality controlled protein list merged from several search engines, including a known false-positive rate.

Algorithms↗

PubMed2007

MascotDatfile: an open-source library to fully parse and analyse MASCOT MS/MS search results.

MS-based protein identification is an important part of both gel-based and gel-free proteome studies. The MASCOT search engine (http://www.matrixscience.com) provides one of the most popular automated algorithms for this task. Here we present an open-source software library written in Java that parses raw MASCOT results into an easily accessible and fully functional object model (http://genesis.ugent.be/MascotDatfile). Several scripts based on this library are provided as examples, allowing direct automation of important routine tasks involved in processing peptide identifications.

Algorithms↗

PubMed2002

Local energy landscape flattening: parallel hyperbolic Monte Carlo sampling of protein folding.

Among the major difficulties in protein structure prediction is the roughness of the energy landscape that must be searched for the global energy minimum. To address this issue, we have developed a novel Monte Carlo algorithm called parallel hyperbolic sampling (PHS) that logarithmically flattens local high-energy barriers and, therefore, allows the simulation to tunnel more efficiently through energetically inaccessible regions to low-energy valleys. Here, we show the utility of this approach by applying it to the SICHO (SIde-CHain-Only) protein model. For the same CPU time, the parallel hyperbolic sampling method can identify much lower energy states and explore a larger region phase space than the commonly used replica sampling (RS) Monte Carlo method. By clustering the simulated structures obtained in the PHS implementation of the SICHO model, we can successfully predict, among a representative benchmark 65 proteins set, 50 cases in which one of the top 5 clusters have a root-mean-square deviation (RMSD) from the native structure below 6.5 A. Compared with our previous calculations that used RS as the conformational search procedure, the number of successful predictions increased by four and the CPU cost is reduced. By comparing the structure clusters produced by both PHS and RS, we find a strong correlation between the quality of predicted structures and the minimum relative RMSD (mrRMSD) of structures clusters identified by using different search engines. This mrRMSD correlation may be useful in blind prediction as an indicator of the likelihood of successful folds.

Models, Molecular↗

PubMed2006

Association of calcineurin with mitochondrial proteins.

Using mouse hearts from Swiss Webster mice, calcineurin was immunoprecipitated using commercially available anti-calcineurin antibody and the resulting complex analyzed by using sodium dodecyl sulfate-gel electrophoresis with silver staining. Distinct proteins were observed and subjected to in situ trypsin digestion followed by extraction of the resulting peptides. Peptides from each protein band were loaded onto a target for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and analyzed. The resulting peptide mass spectrum was compared with the Mascot and Protein Prospector databases and resulted in the specific identification of heart mitochondrial proteins, specifically Mn-superoxide dismutase (SOD), aconitase (ACN), and malate dehydrogenase (MDH). Each of the three mitochondrial enzymes was identified with approximately 15-25% sequence coverage and all with statistical significance (P < 0.05) according to the Mascot database search engine. Tandem mass spectrometry analysis of the peptide fragmentation spectra confirmed the identification of these protein partners and also yielded the identification of mitochondrial isocitrate dehydrogenase (ICDH) as another protein in the immunoprecipitated complex. Using antibody preparations against Mn-SOD, ACN, and ICDH showed the presence of calcineurin and each of the three proteins in the immunoprecipitated complex by Western slot blotting. The activity of ACN, but not MDH or ICDH, was enhanced after incubation with calcineurin indicating one possible regulatory function for the complex. The mitochondrial forms of Mn-SOD, ACN, MDH, and ICDH were identified as partner proteins of calcineurin with all the proteins present in a single multiprotein complex.

Aconitate Hydratase↗

PubMed2004

Proteome analysis in the bovine adrenal medulla using liquid chromatography with tandem mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS)-based proteomics has been used to identify soluble proteins in the bovine adrenal medulla. This gland is a major source of hormones, opioids, neurotransmitters, and several vital proteins. The adrenal medulla proteins were first purified using ammonium sulfate precipitation. The resulting proteins were then pre-fractionated with a C-4 high-performance liquid chromatography (HPLC) column. Each 2-min HPLC fraction was digested with trypsin, and separated further and analyzed using capillary liquid chromatography/tandem mass spectrometry (capLC/nanospray-MS/MS) to map the proteome of the adrenal medulla. The parent mass and sequence ion information thus obtained for tryptic peptides was used to search the NCBInr database using the SEQUEST search engine. A total of 195 proteins were identified, of which 71 had good scores (delta correlation value greater than 0.1, preliminary score above 200, and cross-correlation value above 2.5). The prominent proteins thus identified are secretogranin I precursor, chromogranin A, proenkephalin A precursor, myosin X, hemoglobin beta chain, hemoglobin alpha chain, heat shock protein 10 kDa, and replicase.

Adrenal Medulla↗

PubMed2006

'Information-Based-Acquisition' (IBA) technique with an ion-trap/time-of-flight mass spectrometer for high-throughput and reliable protein profiling.

Highly complex protein mixtures can be analyzed after proteolysis using liquid chromatography/mass spectrometry (LC/MS). In an LC/MS run, intense peptide ions originating from high-abundance proteins are preferentially analyzed using tandem mass spectrometry (MS(2)), so obtaining the MS(2) spectra of peptide ions from low-abundance proteins is difficult even if such ions are detected. Furthermore, the MS(2) spectra may produce insufficient information to identify the peptides or proteins. To solve these problems, we have developed a real-time optimization technique for MS(2), called the Information-Based-Acquisition (IBA) system. In a preliminary LC/MS run, a few of the most intense ions detected in every MS spectrum are selected as precursors for MS(2) and their masses, charge states and retention times are automatically registered in an internal database. In the next run, a sample similar to that used in the first run is analyzed using database searching. Then, the ions registered in the database are excluded from the precursor ion selection to avoid duplicate MS(2) analyses. Furthermore, real-time de novo sequencing is performed just after obtaining the MS(2) spectrum, and an MS(3) spectrum is obtained for accurate peptide identification when the number of interpreted amino acids in the MS(2) spectrum is less than five. We applied the IBA system to a yeast cell lysate which is a typical crude sample, using a nanoLC/ion-trap time-of flight (IT/TOF) mass spectrometer, repeating the same LC/MS run five times. The obtained MS(2) and MS(3) spectra were analyzed by applying the Mascot (Matrix Science, Boston, MA, USA) search engine to identify proteins from the sequence database. The total number of identified proteins in five LC/MS runs was three times higher than that in the first run and the ion scores for peptide identification also significantly increased, by about 70%, when the MS(3) spectra were used, combined with the MS(2) spectra, before being subjected to Mascot analysis.

Chromatography, High Pressure Liquid↗

PubMed2006

An evaluation for cross-species proteomics research by publicly available expressed sequence tag database search using tandem mass spectral data.

With 1383 tandem mass spectra derived from 120 individual protein spots separated by the two-dimensional (2-D) gel electrophoresis of protein samples from three different species, comparative analyses were performed by searching the Expressed Sequence Tag (EST) database (DB) and the NCBI non-redundant (nr) DB of green plants, respectively, which uses the Mascot search engine to establish a statistical basis. It was confirmed that the former could identify more peptides manually validated by de novo sequencing (DNS) from fewer species in more closely phylogenetic relationships than the latter in a statistically significant manner. Our data demonstrated that correct peptide identifications were given low Mascot scores (e.g. 6-14) and incorrect peptide identifications were given high Mascot scores (e.g. 68-83). Our data also showed that the current evaluation approaches to protein assignments are unsatisfactory because a few 'false-positive' proteins are recognized and several 'false-negative' proteins are rescued by manual validation.

Databases, Protein↗