Papers by Kristian Flikka
This chapter contains sections titled: Coverage and complexitySelecting a representative peptide ... more This chapter contains sections titled: Coverage and complexitySelecting a representative peptide sample – COFRADICSeparating peptides into fractionsProducing MS/MS spectraSpectrum filteringSpectrum clusteringSearching the databaseLIMSExercisesBibliographic notesCoverage and complexitySelecting a representative peptide sample – COFRADICSeparating peptides into fractionsProducing MS/MS spectraSpectrum filteringSpectrum clusteringSearching the databaseLIMSExercisesBibliographic notes
This chapter contains sections titled: Separation of intact proteinsIonization of intact proteins... more This chapter contains sections titled: Separation of intact proteinsIonization of intact proteinsResolution and accuracy requirements for charge state determination and mass calculationFragmentation of intact proteinsCharges of the fragmentsProtein identificationProtein characterization – detecting modificationsProblems with top-down approachExercisesBibliographic notesSeparation of intact proteinsIonization of intact proteinsResolution and accuracy requirements for charge state determination and mass calculationFragmentation of intact proteinsCharges of the fragmentsProtein identificationProtein characterization – detecting modificationsProblems with top-down approachExercisesBibliographic notes
BMC bioinformatics, Jan 27, 2004
Microarrays have emerged as the preferred platform for high throughput gene expression analysis. ... more Microarrays have emerged as the preferred platform for high throughput gene expression analysis. Cross-hybridization among genes with high sequence similarities can be a source of error reducing the reliability of DNA microarray results. We have developed a tool called XHM (cross hybridization on microarrays) for assessment of the reliability of hybridization signals by detecting potential cross-hybridizations on DNA microarrays. This is done by comparing the sequences of the probes against an extensive database representing the transcriptome of the organism in question. XHM is available online at http://www.bioinfo.no/tools/xhm/. Using XHM with its user-adjustable parameters will enable scientists to check their lists of differentially expressed genes from microarray experiments for potential cross-hybridizations. This provides information that may be useful in the validation of the microarray results.
ABSTRACT Probiotic bacteria provide unique opportunities to study the global responses and molecu... more ABSTRACT Probiotic bacteria provide unique opportunities to study the global responses and molecular mechanisms underlying the effects of gut-associated microorganisms in the human digestive tract. In this study, we show by comparative transcriptome analysis using DNA microarrays that the established probiotic Lactobacillus plantarum 299v specifically adapts its metabolic capacity in the human intestine for carbohydrate acquisition and expression of exopolysaccharide and proteinaceous cell surface compounds. This report constitutes the first application of global gene expression profiling of a commensal microorganism in the human gut. A core L. plantarum transcriptome expressed in the mammalian intestine was also determined through comparisons of L. plantarum 299v activities in humans to those found for L. plantarum WCFS1 in germ-free mice. These results identify the niche-specific adaptations of a dietary microorganism to the intestinal ecosystem and provide novel targets for molecular analysis of microbial-host interactions which affect human health.
Computational Methods for Mass Spectrometry Proteomics, 2007
PROTEOMICS, 2007
High-throughput proteomics experiments typically generate large amounts of peptide fragmentation ... more High-throughput proteomics experiments typically generate large amounts of peptide fragmentation mass spectra during a single experiment. There is often a substantial amount of redundant fragmentation of the same precursors among these spectra, which is usually considered a nuisance. We here discuss the potential of clustering and merging redundant spectra to turn this redundancy into a useful property of the dataset. To this end, we have created the first generalpurpose, freely available open-source software application for clustering and merging MS/MS spectra. The application also introduces a novel approach to calculating the similarity of fragmentation mass spectra that takes into account the increased precision of modern mass spectrometers, and we suggest a simple but effective improvement to single-linkage clustering. The application and the novel algorithms are applied to several real-life proteomic datasets and the results are discussed. An analysis of the influence of the different algorithms available and their parameters is given, as well as a number of important applications of the overall approach.
Computational Methods for Mass Spectrometry Proteomics, 2007
PROTEOMICS – Clinical Applications, 2007
Cerebrospinal fluid (CSF) is a perfect source to search for new biomarkers to improve early diagn... more Cerebrospinal fluid (CSF) is a perfect source to search for new biomarkers to improve early diagnosis of neurological diseases. Standardization of pre-analytical handling of the sample is, however, important to obtain acceptable analytical quality. In the present study, MALDI-TOF MS was used to examine the influence of pre-analytical sample procedures on the low molecular weight (MW) CSF proteome. Different storage conditions like temperature and duration or the addition of as little as 0.2 mL blood/mL neat CSF caused significant changes in the mass spectra. The performance of different types of MW cut-off spin cartridges from different suppliers used to enrich the low MW CSF proteome showed great variance in cut-off accuracy, stability and reproducibility. The described analytical method achieved a polypeptide discriminating limit of approximately 800 pM, two to three orders of magnitude lower than reported for plasma. Based on this study, we recommend that CSF is centrifuged immediately after sampling, prior to storage at -807C without addition of protease inhibitors. Guanidinium hydrochloride is preferred to break protein-protein interactions. A spin cartridge with cut-off limit above the intended analytical mass range is recommended. Our study contributes to the important task of developing standardized pre-analytical protocols for the proteomic study of CSF.
PROTEOMICS, 2006
In contemporary peptide-centric or non-gel proteome studies, vast amounts of peptide fragmentatio... more In contemporary peptide-centric or non-gel proteome studies, vast amounts of peptide fragmentation data are generated of which only a small part leads to peptide or protein identification. This motivates the development and use of a filtering algorithm that removes spectra that contribute little to protein identification. Removal of unidentifiable spectra reduced both the amount of computational and human time spent on analyzing spectra as well as the chances of obtaining false identifications. Thorough testing on various proteome datasets from different instruments showed that the best suggested machine-learning classifier is, on average, able to recognize half of the unidentified spectra as bad spectra. Further analyses showed that several unidentified spectra classified as good were derived from peptides carrying unanticipated amino acid modifications or contained sequence tags that allowed peptide identification using homology searches.
PROTEOMICS, 2010
MS-based proteomics produces large amounts of mass spectra that require processing, identificatio... more MS-based proteomics produces large amounts of mass spectra that require processing, identification and possibly quantification before interpretation can be undertaken. Highthroughput studies require automation of these various steps, and management of the data in association with the results obtained. We here present ms_lims (http://genesis.UGent.be/ ms_lims), a freely available, open-source system based on a central database to automate data management and processing in MS-driven proteomics analyses.
Nucleic Acids Research, 2004
This work describes the development of a program that predicts whether or not a polypeptide seque... more This work describes the development of a program that predicts whether or not a polypeptide sequence from a Gram-negative bacterium is an integral beta-barrel outer membrane protein. The program, called the beta-barrel Outer Membrane protein Predictor (BOMP), is based on two separate components to recognize integral beta-barrel proteins. The first component is a C-terminal pattern typical of many integral beta-barrel proteins. The second component calculates an integral beta-barrel score of the sequence based on the extent to which the sequence contains stretches of amino acids typical of transmembrane beta-strands. The precision of the predictions was found to be 80% with a recall of 88% when tested on the proteins with SwissProt annotated subcellular localization in Escherichia coli K 12 (788 sequences) and Salmonella typhimurium (366 sequences). When tested on the predicted proteome of E.coli, BOMP found 103 of a total of 4346 polypeptide sequences to be possible integral beta-barrel proteins. Of these, 36 were found by BLAST to lack similarity (E-value score < 1e-10) to proteins with annotated subcellular localization in SwissProt. BOMP predicted the content of integral beta-barrels per predicted proteome of 10 different bacteria to range from 1.8 to 3%. BOMP is available at http://www.bioinfo.no/tools/bomp.
Journal of the American Society for Mass Spectrometry, 2008
This book is timely in that there is currently little availability of textbooks in the area of co... more This book is timely in that there is currently little availability of textbooks in the area of computational mass spectrometry-based proteomics. It contains material important for anyone who would like to understand or develop methods to evaluate proteomics data. This book has four authors, all of whom are authors of various algorithms and tools in computational proteomics (e.g., MASorter and PepMerger) and have a well-established knowledge base in mass spectrometry.
Current Pharmaceutical Biotechnology, 2006
Discovery of disease specific biomarkers in human body fluids has become an important challenge i... more Discovery of disease specific biomarkers in human body fluids has become an important challenge in clinical proteomics. Facing the increasing threat of degenerative and disabling diseases like cancer, cardiovascular, neurological and inflammatory diseases in large parts of the world's population, there is an urgent need to improve early diagnostics. In this review we discuss possibilities and limitations connected to using mass spectrometry based proteomics in the search for novel biomarkers, with focus on multiple sclerosis as a typical representative for the large group of non-curable degenerative and disabling disease with the lack of specific tests for early diagnosis. Careful control of the pre-analytical phase including sampling, storage and fractionation of samples, in addition to a thoroughly considered patient selection, is important in order to avoid false biomarkers to appear in the resulting mass spectra. Furthermore, advanced computational tools are needed in order to discover potential biomarkers from the enormous data amounts generated by the mass spectrometers. The development of such computer tools is a research field currently in the start phase and could prove to be a bottle neck in the biomarker discovery the next years. Therefore, a rather detailed review of the most used computational and pre-analytical methods is given in this review. Mass spectrometry based biomarker discovery is undoubtedly still in its early infancy. However, in light of the potential of this technology to provide deep coverage of the body fluid proteomes, it will certainly consolidate its role in developing molecular medicine into clinical practice.
Archives of Microbiology, 2006
High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identi... more High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting beta-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP ( http://www.bioinfo.no/tools/bomp ) predicted 43 beta-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8-3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins ( http://www.bioinfo.no/tools/lipo ). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.
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Papers by Kristian Flikka