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Peter Roepstorff

Publications and source records attributed to Peter Roepstorff.

At least 55 records · Page 3Linked to original sources
PubMed2004

IntAct: an open source molecular interaction database.

IntAct provides an open source database and toolkit for the storage, presentation and analysis of protein interactions. The web interface provides both textual and graphical representations of protein interactions, and allows exploring interaction networks in the context of the GO annotations of the interacting proteins. A web service allows direct computational access to retrieve interaction networks in XML format. IntAct currently contains approximately 2200 binary and complex interactions imported from the literature and curated in collaboration with the Swiss-Prot team, making intensive use of controlled vocabularies to ensure data consistency. All IntAct software, data and controlled vocabularies are available at http://www.ebi.ac.uk/intact.

Animals↗
PubMed2004

Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry.

A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in-gel digestion by trypsin. In addition, the N-terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2-D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting.

Carbohydrates↗
PubMed2004

Proteome analysis of barley seeds: Identification of major proteins from two-dimensional gels (pI 4-7).

Germination of monocotyledonous plants involves activation and de novo synthesis of enzymes that degrade cell walls and starch and mobilize stored endosperm reserves for embryo growth. Two-dimensional (2-D) gel electrophoresis and mass spectrometry were applied to identify major water-soluble proteins in extracts of mature barley (Hordeum vulgare) seeds and to follow their fate during germination. About 1200 and 600 spots of pI 4-7 were detected on 2-D gels by silver staining and colloidal Coomassie Brilliant Blue staining, respectively. About 300 spots were selected for in-gel digestion followed by matrix-assisted laser desorption/ionization-mass spectrometry-peptide map fingerprint analysis. Database searches using measured peptide masses resulted in 198 identifications of 103 proteins in 177 spots. These include housekeeping enzymes, chaperones, defence proteins (including enzyme inhibitors), and proteins related to desiccation and oxidative stress. Sixty-four of the identifications were made using expressed sequence tags (ESTs). Numerous spots in the 2-D gel pattern changed during germination (micromalting) and an intensely stained area which contained large amounts of the serpin protein Z appeared centrally on the 2-D gel. Spots containing alpha-amylase also appeared. Identification of 22 spots after three days of germination represented 13 different database entries and 11 functions including hydrolytic enzymes, chaperones, housekeeping enzymes, and inhibitors.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2004

UDP-glucose pyrophosphorylase is upregulated in carriers of the porcine RN- mutation in the AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy metabolism in eukaryotic cells acting as a metabolic sensor. In its activated form AMPK inhibits ATP consuming pathways and stimulates ATP generating pathways. A dominant mutation, denoted RN(-), in the porcine PRKAG3 gene, encoding the regulatory gamma3 subunit of AMPK, results in hyperaccumulation of glycogen in glycolytic skeletal muscle cells. To study the effects of this mutation on protein expression patterns in skeletal muscle, comparative proteome analysis of muscle samples from 12 animals (6 rn (+)/rn (+) and 6 RN(-)/rn (+)) was performed. The major finding of the proteome analysis was that the key enzyme in the synthesis of glycogen, UDP-glucose pyrophosphorylase, was significantly up-regulated in RN(-) carriers. This observation was subsequently supported by studies of enzyme activity and Northern blot analysis. Furthermore, the expression patterns of enzymes related to glycolysis and the citric acid cycle were also affected. Our data suggests that hyperaccumulation of glycogen mediated by the RN(-) mutation is due to an increased synthesis of glycogen.

Animals↗
PubMed2003

Postmortem proteome changes of porcine muscle related to tenderness.

Proteome analysis was used to investigate the relation between changes in postmortem proteome of porcine muscle and tenderness development. Muscle samples were taken at slaughter and 72 h postmortem, and the registered changes in the proteome were related to Warner-Bratzler shear force. One hundred and three protein spots were found to change significantly (P < 0.01) over time, and of these the 27 most pronounced changes were identified. Eleven out of the 27 changes were fragments of actin. Other identified myofibril proteins or fragments included myosin heavy chain, titin, myosin light chain I, myosin light II, CapZ, and cofilin. Correlation analysis revealed significant correlations between three of the identified actin fragments and the myosin heavy chain fragment to shear force. Moreover, myosin light chain II and triose phosphate isomerase I were also found to correlate significantly to shear force. The results clearly demonstrate that postmortem degradation of actin and myosin heavy chain is related to meat tenderness.

Animals↗
PubMed2003

Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange.

Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37 degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock promoters. The correlated exchange is shown to constitute a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy. We propose that this gradual decrease in stabilization energy of domain sigma 2 with increasing temperatures facilitates the unfolding of sigma 32 by the AAA+ protease FtsH thereby decreasing its half-life. Taken together our data show that the sigma 2 domain of sigma 32 can act as a thermosensor, which might be important for the heat shock regulation.

Amides↗
PubMed2003

The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end.

14-3-3 proteins constitute a family of well conserved proteins interacting with a large number of phosphorylated binding partners in eukaryotic cells. The plant plasma membrane H+-ATPase is an unusual target in that a unique phosphothreonine motif (946YpTV, where pT represents phosphothreonine) in the extreme C-terminal end of the H+-ATPase interacts with the binding cleft of 14-3-3 protein (Wurtele, M., Jelich-Ottmann, C., Wittinghofer, A., and Oecking, C. (2003) EMBO J. 22, 987-994). We report binding of 14-3-3 protein to a nonphosphorylated peptide representing the 34 C-terminal residues of the Arabidopsis plasma membrane H+-ATPase isoform 2 (AHA2). Following site-directed mutagenesis within the 45 C-terminal residues of AHA2, we conclude that, in addition to the 946YpTV motif, a number of residues located further upstream are required for phosphorylation-independent binding of 14-3-3. Among these, Thr-924 is important for interaction with 14-3-3 protein even when Thr-947 is phosphorylated. We suggest that the role of phosphorylation, which is accentuated by fusicoccin, is to stabilize protein-protein interaction between 14-3-3 protein and several residues of the H+-ATPase C-terminal domain.

14-3-3 Proteins↗
PubMed2003

Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research.

The application of state-of-the-art proteomics and functional genomics technologies to the study of cancer is rapidly shifting toward the analysis of clinically relevant samples derived from patients, as the ultimate aim of translational research is to bring basic discoveries closer to the bedside. Here we describe the essence of a long-term initiative undertaken by The Danish Centre for Translational Breast Cancer Research and currently underway for cancer biomarker discovery using fresh tissue biopsies and bio-fluids. The Centre is a virtual hub that brings together scientists working in various areas of basic cancer research such as cell cycle control, invasion and micro-environmental alterations, apoptosis, cell signaling, and immunology, with clinicians (oncologists, surgeons), pathologists, and epidemiologists, with the aim of understanding the molecular mechanisms underlying breast cancer progression and ultimately of improving patient survival and quality of life. The unifying concept behind our approach is the use of various experimental paradigms for the prospective analysis of clinically relevant samples obtained from the same patient, along with the systematic integration of the biological and clinical data.

Academies and Institutes↗
PubMed2003

New insights into the rat spermatogonial proteome: identification of 156 additional proteins.

Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4-9 was created, and protein entities fractionated in a pH 5-6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.

Animals↗
PubMed2003

Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman degradation and mass spectrometric techniques.

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.

Amino Acid Sequence↗
PubMed2003

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes.

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.

Adenosine Triphosphate↗
PubMed2003

Characterization of plant oligosaccharides by matrix-assisted laser desorption/ionization and electrospray mass spectrometry.

Structural characterization of arabinoxylans from wheat by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry using a Q-TOF mass analyser (ESI-Q-TOF) or an ion trap (IT) mass analyser is presented. An arabinoxylan sample digested with endoxylanase A was analysed using MALDI-TOF mass spectrometry (MS), resulting in the identification of molecular ions for structures with up to 22 monosaccharide residues. As the two-component monosaccharides xylose and arabinose are isobaric, structures differing in the number of arabinose branching residues were indistinguishable based on molecular mass and also fragmentation pattern upon collision-induced dissociation (CID). Permethylation followed by ESI-CID analyses using ITMS was performed to obtain structural information regarding the number of arabinose branching residues and their spatial arrangement along the xylose backbone. Analysis of the signal corresponding to an oligomer with six monosaccharide residues showed the presence of at least four isomeric structures differing in degree of branching and position of the branched residue relative to the cleavage site of the enzyme. This is the first demonstration of the use of ESI-ITMS for the structural characterization of arabinoxylan mixtures.

Carbohydrate Sequence↗
PubMed2003

Proteome analysis of the purine stimulon from Lactococcus lactis.

A comparative expression proteome analysis was carried out by analyzing differential expression patterns of pulse-labelled proteins on two-dimensional gels under standard conditions and during purine nucleotide starvation, followed by mass spectrometric identification of regulated proteins. Based upon the expression patterns, three stimulons could be identified in Lactococcus lactis subsp. cremoris. The Psu proteins (purine starvation up-regulated) had increased synthesis during purine depletion in a purine auxotroph. Among these proteins were enzymes of the purine biosynthesis pathways (PurE, PurS, PurM, PurL), and enzymes involved in the generation of C1 units (GlyA, Fhs). C1 units are primarily required for purine biosynthesis. Upon analysis of the nucleotide sequence preceding the structural genes for these proteins in the L. lactis IL1403 genome sequence showed that all contained PurBox-Pribnov box structures resembling the PurR activated promoters for the purDEK and purCSQLF operons. Most, and possibly all members of the Psu stimulon are thus members of the PurR regulon. Five Psu proteins could not be identified. The second stimulon, the Psd stimulon (purine starvation decreased), whose members are down-regulated during purine depletion, contained proteins related to protein synthesis (PpsB, EF-TS, trigger factor), or to GTPases (FtsZ, EF-TS); or are involved in energy metabolism (GapB, CcpA). No common regulatory elements could be found for members of this stimulon. Two Psd proteins escaped identification. The last, Dcu (decoynine up-regulated), stimulon contained proteins whose synthesis escaped the severe general depression during inhibition of the GMP synthetase by decoynine. This regulon was comprised of mostly glycolytic enzymes (fructose bisphosphate aldolase, enolase, pyruvate kinase) and translation elongation factors (GTPases: EF-TU, EF-G). Two Dcu proteins could not be identified. Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L. lactis subsp. lactis was available. The two subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium. A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus.

Adenosine↗
PubMed2003

Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis.

A new sample preparation method for MALDI based on the use of a mixture of the two commonly used matrices, DHB and CHCA, is described. The matrix mixture preparation results in increased sequence coverage and spot-to-spot reproducibility for peptide mass mapping compared to the use of the single matrix components. This results in more reliable protein identification in proteomics studies and facilitates automated data acquisition. This method shows better tolerance towards salts and impurities, eliminating the need for pre-purification of the samples. It has also been found to be advantageous for the analysis of intact proteins, and especially for glycoproteins. The mixture allows the presence of rather high concentrations of urea in the sample solutions.

Glycoproteins↗
PubMed2003

Using optimized collision energies and high resolution, high accuracy fragment ion selection to improve glycopeptide detection by precursor ion scanning.

Glycosylation is the most widespread protein modification and is known to modulate signal transduction and several biologically important interactions. In order to understand and evaluate the biological role of glycosylation it is important to identify the glycosylated protein and localize the site glycosylation under particular biological conditions. To identify glycosylated peptides from simple mixtures, i.e., in-gel digests from single SDS PAGE bands we performed high resolution, high accuracy precursor ion scanning using a quadrupole TOF instrument equipped with the Q(2) pulsing function. The high resolving power of the quadrupole TOF instrument results in the selective detection of glycan specific fragment ions minimizing the interference of peptide derived fragment ions with the same nominal mass. Precursor ion scanning has been previously described for these glycan derived ions. However the use of this method has been limited by the low specificity of the method. The analysis using precursor ion scanning can be applied to any peptide mixture from a protein digest without having previous knowledge of the glycosylation of the protein. In addition to the low femtomole (nanomolar) detection limits, this method has the advantage that no prior derivatization or enzymatic treatment of the peptide mixtures is required.

Amino Acid Sequence↗
PubMed2003

Tissue distribution, intracellular localization and proteolytic processing of rat 4-hydroxyphenylpyruvate dioxygenase.

4-hydroxyphenylpyruvate dioxygenase (HPD) is an important enzyme involved in tyrosine catabolism. HPD was shown to be identical to a protein named the F-antigen, exploited by immunologists because of its unique immunological properties. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. Decreased expression of HPD is often observed in association with the severe type I tyrosinemia, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. In this study we present a comprehensive analysis of tissue specific expression and intracellular localization of HPD in the rat. By combined use of in situ hybridization and immunohistochemistry we confirm previously known sites of expression in liver and kidney. In addition, we show that HPD is abundantly expressed in neurons in the cortex, cerebellum and hippocampus. By using immunoelectron microscopy and confocal laser scanning microscopy, we provide evidence that HPD contrary to earlier assumptions specifically localizes to membranes of the endoplasmic reticulum and the Golgi apparatus. Detailed mass spectrometric analyses of HPD purified from rat liver revealed N-terminal and C-terminal processing of HPD, and expression of recombinant HPD suggested that C-terminal processing enhances the enzymatic activity.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed2003

Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

Actins↗