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Database Searching (database + searching)
Selected AbstractsDatabase searching by flexible protein structure alignmentPROTEIN SCIENCE, Issue 7 2004Yuzhen Ye Abstract We have recently developed a flexible protein structure alignment program (FATCAT) that identifies structural similarity, at the same time accounting for flexibility of protein structures. One of the most important applications of a structure alignment method is to aid in functional annotations by identifying similar structures in large structural databases. However, none of the flexible structure alignment methods were applied in this task because of a lack of significance estimation of flexible alignments. In this paper, we developed an estimate of the statistical significance of FATCAT alignment score, allowing us to use it as a database-searching tool. The results reported here show that (1) the distribution of the similarity score of FATCAT alignment between two unrelated protein structures follows the extreme value distribution (EVD), adding one more example to the current collection of EVDs of sequence and structure similarities; (2) introducing flexibility into structure comparison only slightly influences the sensitivity and specificity of identifying similar structures; and (3) the overall performance of FATCAT as a database searching tool is comparable to that of the widely used rigid-body structure comparison programs DALI and CE. Two examples illustrating the advantages of using flexible structure alignments in database searching are also presented. The conformational flexibilities that were detected in the first example may be involved with substrate specificity, and the conformational flexibilities detected in the second example may reflect the evolution of structures by block building. [source] Speeding up tandem mass spectrometry based database searching by peptide and spectrum indexingRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2010You Li Database searching is the technique of choice for shotgun proteomics, and to date much research effort has been spent on improving its effectiveness. However, database searching faces a serious challenge of efficiency, considering the large numbers of mass spectra and the ever fast increase in peptide databases resulting from genome translations, enzymatic digestions, and post-translational modifications. In this study, we conducted systematic research on speeding up database search engines for protein identification and illustrate the key points with the specific design of the pFind 2.1 search engine as a running example. Firstly, by constructing peptide indexes, pFind achieves a speedup of two to three compared with that without peptide indexes. Secondly, by constructing indexes for observed precursor and fragment ions, pFind achieves another speedup of two. As a result, pFind compares very favorably with predominant search engines such as Mascot, SEQUEST and X!Tandem. Copyright © 2010 John Wiley & Sons, Ltd. [source] Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2001Maceij Adamczyk A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S -(2-mercaptoethyl)cysteinyl or ,-methyl- S -(2-mercaptoethyl)cysteinyl residues by ,-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of ,-casein, ,-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation. Copyright © 2001 John Wiley & Sons, Ltd. [source] A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth modeFEMS MICROBIOLOGY LETTERS, Issue 1 2002Frédéric Trémoulet Abstract Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (d -ribose-binding periplasmic protein, d -galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode. [source] What determines the management of anxiety disorders and its improvement?JOURNAL OF EVALUATION IN CLINICAL PRACTICE, Issue 2 2008Mirrian Smolders MSc Introduction, Although anxiety disorders are highly prevalent, lack of correct diagnosis and related concerns about treatment are serious clinical problems. Several factors affect, positively or negatively, management of anxiety and its improvement. A literature review and thematic analysis was executed to obtain an overview of the types of determinants of anxiety care and its improvement. Methods, Literature was identified from electronic database searching (January 1995,March 2006), contact with authors of studies, and searching of websites of organizations concerned with mental health. By using a template analysis approach, a set of strong themes relating to determinants of anxiety care and its improvement was identified. Results, The 15 eligible studies identified 43 factors that impeded or facilitated optimal anxiety care and its improvement. Individual characteristics of both patients (n = 13) and professionals (n = 6) were most frequently reported as determinants of anxiety care and its improvement. A considerable number of factors were related to the organizational context (n = 12), such as practice type and location. Some factors related to the social context (n = 4), the economic context (n = 2), or to the innovation itself (n = 6) were identified. Conclusion, The findings show that there is a multitude of barriers and facilitators to optimal anxiety care and its improvement. Some determinants are modifiable, and thus responsive to interventions. Examples are collaboration within and between organizations, financial resources and assignment of both an opinion leader and responsible staff. The quality of anxiety care can be improved by systematically designing innovation strategies which are tailored to a selection of the determinants identified in this study. [source] Proteomic Alterations of Antarctic Ice Microalga Chlamydomonas sp.JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 8 2006Under Low-Temperature Stress Abstract Antarctic ice microalga can survive and thrive in cold channels or pores in the Antarctic ice layer. In order to understand the adaptive mechanisms to low temperature, in the present study we compared two-dimensional polyacrylamide gel electrophoresis (2-DE) profiles of normal and low temperature-stressed Antarctic ice microalga Chlamydomonas sp. cells. In addition, new protein spots induced by low temperature were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE patterns of both normal and low temperature-stressed cells were acquired. A total of 626 spots was detected in control cells and 652 spots were detected in the corresponding low temperature-stressed cells. A total of 598 spots was matched between normal and stressed cells. Two newly synthesized proteins (a and b) in low temperature-stressed cells were characterized. Protein spot A (53 kDa, pI 6.0) was similar to isopropylmalate/homocitrate/citramalate synthases, which act in the transport and metabolism of amino acids. Protein spot b (25 kDa, pI 8.0) was related to glutathione S -transferase, which functions as a scavenger of active oxygen, free radicals, and noxious metabolites. The present study is valuable for the application of ice microalgae, establishing an ice microalga Chlamydomonas sp. proteome database, and screening molecular biomarkers for further studies. (Managing editor: Li-Hui Zhao) [source] Systematic determination of ion score cutoffs based on calculated false positive rates: application for identifying ubiquitinated proteins by tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2008Julian Vasilescu Abstract We report a simple approach for determining ion score cutoffs that permit the confident identification of ubiquitinated proteins by tandem mass spectrometry (MS/MS). Initial experiments involving the analysis of gel bands containing multi-Ubiquitin chains with quadrupole time-of-flight and quadrupole ion trap mass spectrometers revealed that standard ion score cutoffs used for database searching were not sufficiently stringent. We also found that false positive and false negative rates (FPR and FNR) varied significantly depending on the cutoff scores used and that appropriate cutoffs could only be determined following a systematic evaluation of false positive rates. When standard cutoff scores were used for the analysis of complex mixtures of ubiquitinated proteins, unacceptably high FPR were observed. Finally, we found that FPR for ubiquitinated proteins are affected by the size of the protein database that is searched. These observations may be applicable for the study of other post-translational modifications. Copyright © 2007 John Wiley & Sons, Ltd. [source] Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ionsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2008Jeremiah J. Bowers Abstract A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion. Copyright © 2007 John Wiley & Sons, Ltd. [source] Proteomics in Alzheimer's disease: insights into potential mechanisms of neurodegenerationJOURNAL OF NEUROCHEMISTRY, Issue 6 2003D. Allan Butterfield Abstract Proteomics involves the identification of unknown proteins following their separation, often using two-dimensional electrophoresis, digestion of particular proteins of interest by trypsin, determination of the molecular weight of the resulting peptides, and database searching to make the identification of the proteins. Application of proteomics to Alzheimer's disease (AD), the major dementing disorder of the elderly, has just begun. Differences in protein expression and post-translational modification (mostly oxidative modification) of proteins from AD brain and peripheral tissue, as well as in brain from rodent models of AD, have yielded insights into potential molecular mechanisms of neurodegeneration in this dementing disorder. This review surveys the proteomics studies relevant to AD, from which new understandings of the pathology, biochemistry, and physiology of AD are beginning to emerge. [source] Database searching by flexible protein structure alignmentPROTEIN SCIENCE, Issue 7 2004Yuzhen Ye Abstract We have recently developed a flexible protein structure alignment program (FATCAT) that identifies structural similarity, at the same time accounting for flexibility of protein structures. One of the most important applications of a structure alignment method is to aid in functional annotations by identifying similar structures in large structural databases. However, none of the flexible structure alignment methods were applied in this task because of a lack of significance estimation of flexible alignments. In this paper, we developed an estimate of the statistical significance of FATCAT alignment score, allowing us to use it as a database-searching tool. The results reported here show that (1) the distribution of the similarity score of FATCAT alignment between two unrelated protein structures follows the extreme value distribution (EVD), adding one more example to the current collection of EVDs of sequence and structure similarities; (2) introducing flexibility into structure comparison only slightly influences the sensitivity and specificity of identifying similar structures; and (3) the overall performance of FATCAT as a database searching tool is comparable to that of the widely used rigid-body structure comparison programs DALI and CE. Two examples illustrating the advantages of using flexible structure alignments in database searching are also presented. The conformational flexibilities that were detected in the first example may be involved with substrate specificity, and the conformational flexibilities detected in the second example may reflect the evolution of structures by block building. [source] Computational analysis of unassigned high-quality MS/MS spectra in proteomic data setsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2010Kang Ning Abstract In a typical shotgun proteomics experiment, a significant number of high-quality MS/MS spectra remain "unassigned." The main focus of this work is to improve our understanding of various sources of unassigned high-quality spectra. To achieve this, we designed an iterative computational approach for more efficient interrogation of MS/MS data. The method involves multiple stages of database searching with different search parameters, spectral library searching, blind searching for modified peptides, and genomic database searching. The method is applied to a large publicly available shotgun proteomic data set. [source] Prenatal alcohol exposure alters phosphorylation and glycosylation of proteins in rat offspring liverPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2010Bourlaye Fofana Abstract To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2-D PAGE with total, phospho- and glycoprotein staining and MALDI-MS/MS and database searching were conducted. The results, based on fold-change expression, revealed a down-regulation of total protein expression by prenatal alcohol exposure in 7-day-old and 3-month-old rats. There was an up-regulation of protein phosphorylation but a down-regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo-keto reductase, tumor rejection antigen gp96, fructose-1,6-bisphosphatase, glycerol-3-phosphate dehydrogenase, malate dehydrogenase, and ,-actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S-transferase, glucose regulated protein 58, ,-enolase, eukaryotic translation elongation factor 1 ,-2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3-hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications. [source] Protein probabilities in shotgun proteomics: Evaluating different estimation methods using a semi-random sampling modelPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2006Xiaofang Xue Abstract The calculation of protein probabilities is one of the most intractable problems in large-scale proteomic research. Current available estimating methods, for example, ProteinProphet, PROT_PROBE, Poisson model and two-peptide hits, employ different models trying to resolve this problem. Until now, no efficient method is used for comparative evaluation of the above methods in large-scale datasets. In order to evaluate these various methods, we developed a semi-random sampling model to simulate large-scale proteomic data. In this model, the identified peptides were sampled from the designed proteins and their cross-correlation scores were simulated according to the results from reverse database searching. The simulated result of 18 control proteins was consistent with the experimental one, demonstrating the efficiency of our model. According to the simulated results of human liver sample, ProteinProphet returned slightly higher probabilities and lower specificity than real cases. PROT_PROBE was a more efficient method with higher specificity. Predicted results from a Poisson model roughly coincide with real datasets, and the method of two-peptide hits seems solid but imprecise. However, the probabilities of identified proteins are strongly correlated with several experimental factors including spectra number, database size and protein abundance distribution. [source] Proteomic analysis of pulmonary sclerosing hemangiomaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2006Lian-Jin Jin Abstract Sclerosing hemangioma (SH) is a rare benign pulmonary tumor derived from the primitive respiratory epithelium. However, the pathogenesis of SH has not yet been clear. Surfactant protein, thyroid transcription factor-1, epithelial membrane antigen, cytokeratin, and vimentin have been identified in SH by immunohistochemistry and electron microscopy. To identify proteins specifically regulated in SH, 2-D PAGE was performed using SH and paired normal tissues. Ten selected differentially expressed protein spots were identified by PMF, MALDI-TOF-MS, and database searching. Apolipoprotein,A-1, antizyme inhibitor, heat shock 27-kDa protein,1, and antioxidant proteins, such as peroxiredoxin,II (Prx,II) and GST, were identified among the down-regulated proteins in SH. Western blot and immunohistochemistry confirmed reduced expressions of Prx,II and GST in SH versus normal lung tissue. This study is the first report on the reduced expressions of Prx,II and GST in SH. [source] OLAV: Towards high-throughput tandem mass spectrometry data identificationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2003Jacques Colinge Abstract Mass spectrometry combined with database searching has become the preferred method for identifying proteins in proteomics projects. Proteins are digested by one or several enzymes to obtain peptides, which are analyzed by mass spectrometry. We introduce a new family of scoring schemes, named OLAV, aimed at identifying peptides in a database from their tandem mass spectra. OLAV scoring schemes are based on signal detection theory, and exploit mass spectrometry information more extensively than previously existing schemes. We also introduce a new concept of structural matching that uses pattern detection methods to better separate true from false positives. We show the superiority of OLAV scoring schemes compared to MASCOT, a widely used identification program. We believe that this work introduces a new way of designing scoring schemes that are especially adapted to high-throughput projects such as GeneProt large-scale human plasma project, where it is impractical to check all identifications manually. [source] A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humansPROTEOMICS - CLINICAL APPLICATIONS, Issue 8 2009Joshua Boateng Abstract An integrated MS-based proteomic approach is described that combines MALDI-MS and LC-MS with artificial neural networks for the identification of protein and peptide biomarkers associated with recombinant human growth hormone (rhGH) administration. Serum from exercised males administered with rhGH or placebo was analysed using ELISA to determine insulin-like growth factor-I concentrations. Diluted serum from rhGH- and placebo-treated subjects was analysed for protein biomarkers by MALDI-MS, whereas LC-MS was used to analyse tryptically digested ACN-depleted serum extracts for peptide biomarkers. Ion intensities and m/z values were used as inputs to artificial neural networks to classify samples into rhGH- and placebo-treated groups. Six protein ions (MALDI-MS) correctly classified 96% of samples into their respective groups, with a sensitivity of 91% (20 of 22 rhGH treated) and specificity of 100% (24 of 24 controls). Six peptide ions (LC-MS) were also identified and correctly classified 93% of samples with a sensitivity of 90% (19 of 21 rhGH treated) and a specificity of 95% (20 of 21 controls). The peptide biomarker ion with the highest significance was sequenced using LC-MS/MS and database searching and found to be associated with leucine-rich ,-2-glycoprotein. [source] Speeding up tandem mass spectrometry based database searching by peptide and spectrum indexingRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2010You Li Database searching is the technique of choice for shotgun proteomics, and to date much research effort has been spent on improving its effectiveness. However, database searching faces a serious challenge of efficiency, considering the large numbers of mass spectra and the ever fast increase in peptide databases resulting from genome translations, enzymatic digestions, and post-translational modifications. In this study, we conducted systematic research on speeding up database search engines for protein identification and illustrate the key points with the specific design of the pFind 2.1 search engine as a running example. Firstly, by constructing peptide indexes, pFind achieves a speedup of two to three compared with that without peptide indexes. Secondly, by constructing indexes for observed precursor and fragment ions, pFind achieves another speedup of two. As a result, pFind compares very favorably with predominant search engines such as Mascot, SEQUEST and X!Tandem. Copyright © 2010 John Wiley & Sons, Ltd. [source] Highly efficient and selective enrichment of peptide subsets combining fluorous chemistry with reversed-phase chromatographyRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2009Wantao Ying The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptide's hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides. Copyright © 2009 John Wiley & Sons, Ltd. [source] Comprehensive analysis of short peptides in sera from patients with IgA nephropathyRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2009Nagayuki Kaneshiro We analyzed serum short peptides comprehensively to know whether they were useful to characterize IgA nephropathy (IgAN). Serum samples from 26 patients with untreated IgAN and 25 healthy donors were tested. Short peptides with molecular weights of ,7,kDa, purified from the serum samples by magnetic-beads-based weak cation exchange, were detected by mass spectrometry. Then the peptide peaks detected were subjected to the multivariate data analysis by SIMCA-P+® containing principal component analysis (PCA) and orthogonal partial-least-squares-discriminate analysis (OPLS-DA). A total of 92 peptide peaks were detected in the tested serum samples. The OPLS-DA analysis revealed that the profile of all the peptide peak intensities discriminated the IgAN group and the healthy group completely with a high R2 value (0.919) and a high Q2 value (0.861). Further, the profile of only five peptide peaks was found to discriminate the two groups. By tandem mass spectrometry and database searching, three of the five peptides which increased in the IgAN group were identified as fragments of fibrinogen alpha chain, and the two peptides which increased in the healthy group were identified as fragments of complement C3f and kininogen-1 light chain. Taken together, the profile of the serum short peptides would be useful to discriminate IgAN and healthy conditions. Further, the five peptides may be candidate serum markers for IgAN and may be related to pathogenesis of IgA. Copyright © 2009 John Wiley & Sons, Ltd. [source] Closely spaced external standard: a universal method of achieving 5 ppm mass accuracy over the entire MALDI plate in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2003Eugene Moskovets Close deposition of the sample and external standard was used in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to achieve mass accuracy equivalent to that obtained with an internal standard across the entire MALDI plate. In this work, the sample and external standard were deposited by continuous deposition in separate traces, each approximately 200,,m wide. The dependence of the mass accuracy on the distance between the sample and standard traces was determined across a MALDI target plate with dimensions of 57.5,mm,×,57.0,mm by varying the gap between the traces from 100,,m to 4,mm. During acquisition, two adjacent traces were alternately irradiated with a 200-Hz laser, such that the peaks in the resulting mass spectra combined the sample and external standard. Ion suppression was not observed even when the peptide concentrations in the two traces differed by more than two orders of magnitude. The five peaks from the external standard trace were used in a four-term mass calibration of the masses of the sample trace. The average accuracy across the whole plate with this method was 5,ppm when peaks of the sample trace had signal-to-noise ratios of at least 30 and the gap between the traces was approximately 100,,m. This approach was applied to determining peptide masses of a reversed-phase liquid chromatographic (LC) separation of a tryptic digest of , -galactosidase deposited as a long serpentine trace across the MALDI plate, with accuracy comparable to that obtainable using internal calibration. In addition, the eluent from reversed-phase LC separation of a strong cation-exchange fraction containing tryptic peptides from a yeast lysate along with the closely placed external standard was deposited on the MALDI plate. The data obtained in the MS and MS/MS modes on a MALDI-TOF/TOF mass spectrometer were combined and used in database searching with MASCOT. Since the significant score is a function of mass accuracy in the MS mode, database searching with high mass accuracy reduced the number of false positives and also added peptides which otherwise would have been eliminated at lower mass accuracy (false negatives). Copyright © 2003 John Wiley & Sons, Ltd. [source] High accuracy mass measurement of peptides with internal calibration using a dual electrospray ionization sprayer system for protein identificationRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2002Feng Zhou A dual-ESI-sprayer system was constructed and applied to achieve high accuracy of peptide mass measurement for protein identification by means of peptide mapping. Sample was introduced in one sprayer, and reference in the other, thus making internal calibration possible greatly enhancing the mass accuracy. Several samples were utilized to evaluate the reliability of this dual-ESI-sprayer system. The range of mass errors was 0.16,5.37,ppm. The peptide masses of tryptic digests of myoglobin (horse) were measured by the HPLC/dual-ESI-MS system, with mass deviations ranging from 0.01,7.67,ppm, and about 75% mass deviations below 5,ppm with 40% below 1,ppm. These peptide masses were utilized to perform database searching for protein identification, and compared to results obtained by external calibration. This comparison showed that the internal calibration provides a more reliable method of protein identification, with a much smaller number of required peptides for matching, and with less CPU time consumed for database searching. Copyright © 2002 John Wiley & Sons, Ltd. [source] The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomicsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2001R. Cramer Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed. Copyright © 2001 John Wiley & Sons, Ltd. [source] A Transgenic Insertional Inner Ear Mutation on Mouse Chromosome 1,THE LARYNGOSCOPE, Issue 4 2000Rick A. Friedman MD Abstract Objectives/Hypothesis To clone and characterize the integration site of an insertional inner ear mutation, produced in one of fourteen transgenic mouse lines. The insertion of the transgene led to a mutation in a gene(s) necessary for normal development of the vestibular labyrinth. Study Design Molecular genetic analysis of a transgene integration site. Methods Molecular cloning, Southern and northern blotting, DNA sequencing and genetic database searching were the methods employed. Results The integration of the transgene resulted in a dominantly inherited waltzing phenotype and in degeneration of the pars superior. During development, inner ear fluid homeostasis was disrupted. The integration consisted of the insertion of a single copy of the transgene. Flanking DNA was cloned, and mapping indicated that the genomic DNA on either side of the transgene was not contiguous in the wild-type mouse. Localization of unique markers from the two flanks indicated that both were in the proximal region of mouse chromosome 1. However, in the wild-type mouse the markers were separated by 6.3 cM, indicating a sizable rearrangement. Analysis of the mutant DNA indicated that the entire region between the markers was neither deleted nor simply inverted. Conclusions These results are consistent with a complex rearrangement, including at least four breakpoints and spanning at least 6.3 cM, resulting from the integration of the transgene. This genomic rearrangement disrupted the function of one or more genes critical to the maintenance of fluid homeostasis during development and the normal morphogenesis of the pars superior. [source] | ||||||||||||||||||||||||||||