Biomedical subjects

Rune Matthiesen

Publications and source records attributed to Rune Matthiesen.

At least 19 recordsLinked to original sources

PubMed2007

Differential fragmentation patterns of pectin oligogalacturonides observed by nanoelectrospray quadrupole ion-trap mass spectrometry using automated spectra interpretation.

Oligogalacturonides of different degrees of polymerization (DP) and methyl esterification (DE) were structurally analyzed by nanoESI quadrupole ion-trap mass spectrometry. The fragmentation patterns of the oligogalacturonides were compared using the program 'Virtual Expert Mass Spectrometrist' (VEMS) for structural annotation. In the analyzed oligogalacturonides of lower DP, the generation of C/Y ions, i.e. ions retaining the glycosidic oxygen, was higher than that of B/Z ions. In general, with oligogalacturonides of higher DP, the B/Z ions were generated more abundantly. Oligogalacturonides with free carboxylic acid groups underwent higher water loss compared to fully methyl-esterified oligogalacturonides under the same fragmentation conditions. Cross-ring cleavage, in which fragmentation occurs across the ring system of the galacturonate residue and signified by unique mass losses, was observed to be higher in fully methyl-esterified oligogalacturonides than in non-methyl-esterified ones. This study demonstrates the different fragmentation patterns of oligogalacturonides as influenced by the presence or absence of methyl ester groups. For a detailed analysis of unknown oligogalacturonides, cross-ring fragmentation gives more structural information than glycosidic bond cleavage. One implication of this is that more structural information is obtained when analyzing methyl-esterified oligogalacturonides than non-methyl-esterified ones in an ion-trap instrument. This is of particular importance in pectin chemistry, where mass spectrometry has become the technique of choice for structural determination. Although this study was not designed to explain the mechanisms of oligogalacturonide fragmentation, possible explanations for why non-methyl-esterified oligogalacturonides undergo more water loss than methyl-esterified ones will be postulated. In addition, the VEMS program was extended to automatically interpret and assign the fragment ions peaks generated in this study.

Nanotechnology↗

PubMed2007

Introduction to proteomics.

Mass spectrometry (MS) has recently become one of the most informative methods for studying proteins. Albiet, MS cannot compete with the detailed structural information obtained by methods such as nuclear magnetic resonance and X-ray crystallography. However, MS is much easier to automate and use as a large-scale technique. Large-scale proteomic methods are valuable for studying the dynamics of the proteins and their posttranslational modification in living cells. Despite the great potential of mass spectrometers, many laboratories are struggling with data analysis and data storage. The complexity of the data analysis stems from the large number of experiments that can be performed by various mass spectrometers. In addition, many mass spectrometers have their own data formats. Performing data analysis on MS data, therefore, requires a rather extensive setup of algorithms and data parsers. In recent years it has become evident that the proteomics society needs standard formats for storing and exchanging data. This has triggered a new problem, which is the invention of several different standard formats. In this chapter, an overview of the most common proteomics experiments with MS, together with an overview of data formats, is presented.

Amino Acid Sequence↗

PubMed2007

Virtual Expert Mass Spectrometrist v3.0: an integrated tool for proteome analysis.

The number of tools described in the literature for analysis of proteome data is growing fast. However, most tools are not able to communicate or exchange data with other tools. In Virtual Expert Mass Spectrometrist (VEMS) v3.0 an effort has been made to interface and export to already existing tools. In this chapter, an outline of how to use the VEMS program to search tandem mass spectrometry data against databases is described. Additionally, examples on how to extend the analysis with other external tools are given.

Calibration↗

PubMed2007

Quantitation with virtual expert mass spectrometrist.

Quantitative analysis of proteins and peptides by mass spectrometry has been greatly advanced by the development of proteomic technologies within recent years. Particularly, labeling of peptides and proteins with stable isotopes such as 2H, 15N, and 13C facilitated the unbiased comparison of protein amounts in distinct samples in a single mass spectrometric experiment. These methods can be applied to detect quantitative changes in protein amounts and posttranslational modifications such as phosphorylation. Quantitation of mass spectra requires accurate and efficient bioinformatics tools, which can match corresponding peptides, determine peak intensities, and calculate relative amounts. In this chapter, we describe the use of virtual expert mass spectrometrist for the quantitation of mass spectra from samples with peptides and proteins encoded with stable isotopes.

Computational Biology↗

PubMed2007

Sequence handling by sequence analysis toolbox v1.0.

The fact that mass spectrometry have become a high-throughput method calls for bioinformatic tools for automated sequence handling and prediction. For efficient use of bioinformatic tools, it is important that these tools are integrated or interfaced with each other. The purpose of sequence analysis toolbox v1.0 was to have a general purpose sequence analyzing tool that can import sequences obtained by high-throughput sequencing methods. The program includes algorithms for calculation or prediction of isoelectric point, hydropathicity index, transmembrane segments, and glycosylphosphatidyl inositol-anchored proteins.

Computational Biology↗

PubMed2007

Interpretation of collision-induced fragmentation tandem mass spectra of posttranslationally modified peptides.

Tandem collision-induced dissociation (CID) mass spectrometry (MS) provides a sensitive means of analyzing the amino acid sequence of peptides. Modern MS instrumentation is capable of rapidly generating many thousands of tandem mass spectra, and protein database search engines have been developed to cope with this avalanche of data. In most studies, there is a schism between discarding perfectly valid data and including nonsensical peptide identifications--this is currently a major bottleneck in data analysis and it calls for manual evaluation of the data. Especially for posttranslationally modified peptides, there is a need for manual validation of the data because search algorithms seldom have been optimized for the identification of modified peptides and because there are many pitfalls for the unwary. This chapter describes some of the issues that should be considered when interpreting and validating low-energy CID tandem mass spectra and gives some useful tables to aid this process.

Amino Acid Sequence↗

PubMed2007

Organization of proteomics data with YassDB.

In recent years the organization of mass spectrometry (MS) data obtained in large-scale proteomics projects became an important issue. This has catalyzed the development of a few different database schemes for storing MS data, as well as some dedicated user interfaces. However, many of these projects are still rather immature and often do not cover all needs. Because our needs were quite specific, it was necessary to build a database that accommodates all the major types of experiments generated in house and that could be easily extended by new modules made by collaborators or students. A database application named "YassDB" will be described in this chapter. The application is implemented in a "three-tier" application architecture, with a database layer, a middle layer consisting of web services and a client layer, containing the user interface. This offers high flexibility: it allows other applications, written in any language, to be written as clients to the database. The setup and use of the YassDB database application with two client programs "pProRep" and "VEMS" will be outlined.

Databases, Protein↗

PubMed2007

Analysis of carbohydrates by mass spectrometry.

The analysis method described in this chapter demonstrates the structural characterization of carbohydrates based on their molecular mass, as well as the mass of their respective fragment ions using mass spectrometry (MS). The carbohydrate molecules are first converted into gaseous ions, under vacuum, after which their mass-to-charge ratio is measured. The mass-to-charge ratio provides information on their preliminary identification, which is further elucidated by fragmenting the ions under a process of collision-induced dissociation. The masses obtained in the first stage of MS together with those obtained in the subsequent stages (MSn) are combined into a mass list that is loaded into the program, Virtual Expert Mass Spectrometrist (VEMS) v3.0. The mass lists obtained are then used by VEMS to search a database of glycans to give the identity of the carbohydrate and the correct assignment of the fragment ions.

Carbohydrates↗

PubMed2007

Useful mass spectrometry programs freely available on the internet.

The intention with this chapter is to give an overview of a broad range of freely available programs on the internet which are useful for analyses of mass spectrometry data. The list is by no means a full list of free proteomics tools on the net and I apologize if there are other good tools on the internet that have been missed. The presented programs should cover the needs for the most general tasks in data analysis of proteomics data and have to a limited extend been tested. The links provided in this chapter will over time become invalid. In such cases it is worth while to try a World Wide Web search using the program packages names. This will often reveal the updated links. Some of the links presented in this chapter will also be maintained at http://yass.sdu.dk.

Computational Biology↗

PubMed2007

Extracting monoisotopic single-charge peaks from liquid chromatography-electrospray ionization-mass spectrometry.

Peak extraction from raw data is the first step in analysis of mass spectrometry (MS) data. The quality of this procedure is very important because it affects the quality of all subsequent analysis, such as database searches and peak quantitation. Many methods have been proposed in the literature, yet the number of practical solutions in terms of available software is rather limited. Virtual Expert Mass Spectrometrist (VEMS) v3.0 includes an algorithm for extracting monoisotopic single-charged peaks and their corresponding retention time from liquid chromatography (LC)-MS data. The extracted peaks can subsequently be exported to other programs or used internally by VEMS to perform peptide mass fingerprinting searches or peptide quantitation. Additionally, VEMS interfaces the commercial program ProteinLynx Global server v2.0.5 for automatic peak extraction from MS/MS spectra obtained by LC-MS/MS.

Chromatography, Liquid↗

PubMed2006

Integration of gel-based proteome data with pProRep.

UNLABELLED: pProRep is a web application integrating electrophoretic and mass spectral data from proteome analyses into a relational database. The graphical web-interface allows users to upload, analyse and share experimental proteome data. It offers researchers the possibility to query all previously analysed datasets and can visualize selected features, such as the presence of a certain set of ions in a peptide mass spectrum, on the level of the two-dimensional gel. AVAILABILITY: The pProRep package and instructions for its use can be downloaded from http://www.ptools.ua.ac.be/pProRep. The application requires a web server that runs PHP 5 (http://www.php.net) and MySQL. Some (non-essential) extensions need additional freely available libraries: details are described in the installation instructions.

Computational Biology↗

PubMed2006

Quantitative proteomic analysis of post-translational modifications of human histones.

Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.

Acetylation↗

PubMed2006

Celecoxib inhibits interleukin-12 alphabeta and beta2 folding and secretion by a novel COX2-independent mechanism involving chaperones of the endoplasmic reticulum.

Celecoxib (CE) is a nonsteroidal anti-inflammatory drug (NSAID) that is a specific inhibitor of cyclooxygenase 2 (COX2). It is indicated for a variety of chronic inflammatory conditions, including rheumatoid arthritis. Over the last few years, adverse cardiovascular effects and increased risk for heart attacks have been associated with this drug. In addition, evidence is emerging for COX2-independent molecular targets. CE has been shown to induce apoptosis in various cancer cells lines through a COX2-independent mechanism that seems to involve inactivation of protein kinase Akt and inhibition of endoplasmic reticulum (ER) Ca2+ ATPase. In this study, we show that both CE and an analog devoid of COX2 inhibitory activity [1-(4-sulfamoyl phenyl)-3-trifluoromethyl-5-(4-trifluoromethylphenyl)pyrazole, CEA] inhibit the secretion of the dimeric interleukin-12 (IL-12) alphabeta and beta2 forms with identical IC50 values of 20 and 30 microM, respectively, whereas no such effect was seen with rofecoxib. Reverse transcription-polymerase chain reaction analysis showed that this inhibition was not due to a blockage of transcription of the alpha- and beta-chain expression cassettes. Secretion of the beta monomer form was less strongly inhibited, suggestive for a mechanism primarily targeting dimer assembly in the ER. Analysis of intracellular fractions revealed that both CE and CEA increased the association of IL-12 with calreticulin, an endoplasmic reticulum-resident chaperone involved in the retention of misfolded cargo proteins while blocking interaction with ERp44. Our findings reveal a previously undescribed effect of celecoxib on oligomer protein folding and assembly in the endoplasmic reticulum and ensuing secretion and suggest that celecoxib-driven alteration of the secretome may be involved in some of its clinical side effects.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant.

Proteomic screening of strawberry (Fragaria ananassa) yielded a 58% success rate in protein identification in spite of the fact that no genomic sequence is available for this species. This was achieved by a combination of MALDI-MS/MS de novo sequencing of double-derivatized peptides and indel-tolerant searching against local protein databases built on both EST and full-length nucleotide sequences. The amino acid sequence of a strawberry allergen, homologous to the well-known major birch pollen allergen Bet v 1, was partially determined. This strawberry allergen, named Fra a 1 according to the nomenclature for allergen proteins, showed sequence identity of 54 and 77%, respectively, with corresponding allergens from birch and apple. Differential expression, as evaluated by 2-D DIGE, occurred in 10% of protein spots when red strawberries were compared to a colorless (white) strawberry mutant. White strawberries, known to be tolerated by individuals affected by allergy, were found to be virtually free from the strawberry allergen. Also several enzymes in the pathway for biosynthesis of flavonoids, to which the red color pelargonidin belongs, were down-regulated. This approach to assess differential protein expression without access to genomic sequence information can also be applied to other crop plants and phenotypic traits.

Allergens↗

PubMed2006

Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E.

Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins associated with the translation initiation eIF4F complex that may affect complex formation were identified and quantitated under distinct growth conditions. Site-specific phosphorylation of eIF4E and eIF4G and elevated levels of eIF4G:eIF4E complexes in phorbol ester treated HEK293 cells, and in serum-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP complexes, for functions related to RNP assembly, remodeling and/or transition from active translation to mRNA degradation. Our results demonstrate that our quantitative proteomic strategy can be applied to the identification and quantitation of protein complex components in human cells grown under different conditions.

Amino Acid Sequence↗

PubMed2005

Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry.

Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([2H3]-leucine, [13C6]arginine, and [2H4]lysine), [13C6]arginine proved to be the most suitable. Incorporation of [13C6]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [13C6]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling.

Amino Acid Sequence↗

PubMed2004

Use of performic acid oxidation to expand the mass distribution of tryptic peptides.

Significant identification of proteins by mass fingerprinting and partial sequencing of tryptic peptides is central to proteomics. However, peptide masses cluster with distances of approximately 1 Da. Expanding these clusters will give more peptides of unique masses, thereby identifying proteins with a higher significance. The mass clusters can be expanded downward by including more oxygen atoms in the peptides. Classic performic acid oxidation modifies three residues, Cys to CysO(3), Met to MetO(2), and Trp to TrpO(2). In this study, we compare the mass distributions of tryptic peptides computed from the predicted proteomes of Bacillus subtilis, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens modified by oxidation, reduction, and reduction followed by carboxymethylation, carboxamidomethylation, or pyridylethylation. Forty to 46% of the eukaryotic tryptic peptides contain Cys, Met, or Trp. Additionally, the importance of mass accuracy of differentially modified tryptic peptides for significant protein identification by database searches was analyzed. The results show that performic acid oxidation gives markedly extended mass distributions at mass accuracies from +/-0.002 to +/-0.25 Da for the eukaryotes. The effect of the expanded mass distribution on significant protein identification was illustrated by searching simulated mass peak lists against the databases containing oxidized and reduced tryptic peptides. The specificity of formic acid oxidation was tested experimentally, and no general adverse effects were detected. Tryptic peptides provided a 100% sequence coverage of oxidized barley grain peroxidase by LC-MS, and the sequence coverages of oxidized and carboxymethylated bovine serum albumin were similar by MALDI-TOF MS analyses.

Amino Acids↗

PubMed2004

Database-independent, database-dependent, and extended interpretation of peptide mass spectra in VEMS V2.0.

The Virtual Expert Mass Spectrometrist (VEMS) program package was developed for flexible, automated, and manual de novo tandem mass spectrometry (MS/MS) protein sequencing, and includes accessory programs for matrix-assisted laser desorption/ionization-mass spectrometry (MS) interpretation, and generation of protein and peptide databases. VEMS V2.0 has been developed into a fast tool for combining database-independent and -dependent protein assignments in an extended analysis of MS/MS-peptide data. MS or MS/MS data can be directly recalibrated after the first search by fitting the data to the best search result using polynomial equations. The score function is an improvement of known scoring algorithms and can be adapted for any MS instrument type. In addition, VEMS offers a novel statistical model for evaluating the significance of the protein assignment. The novel features are illustrated by the analysis of the fragmentation spectra obtained by liquid chromatrography-MS/MS analysis of peptides from an anionic peroxidase enriched protein fraction from potato root tissue. The extended analysis mode resulted in the additional assignment of spectra for nine modified tryptic peptides and nine miscleaved peptides, in addition to the 45 spectra from regular tryptic peptides. Of the nine modified peptides, three were glycosylated.

Algorithms↗