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Helmut E Meyer

Publications and source records attributed to Helmut E Meyer.

At least 37 records · Page 2Linked to original sources
PubMed2006

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

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

Algorithms↗
PubMed2006

HUPO Brain Proteome Project: summary of the pilot phase and introduction of a comprehensive data reprocessing strategy.

The Human Proteome Organisation (HUPO) initiated several projects focusing on the proteome analysis of distinct human organs. The Brain Proteome Project (BPP) is the initiative dedicated to the brain, its development and correlated diseases. Two pilot studies have been performed aiming at the comparison of techniques, laboratories and approaches. With the help of the results gained, objective data submission, storage and reprocessing workflow have been established. The biological relevance of the data will be drawn from the inter-laboratory comparisons as well as from the re-calculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort will be summarised and compared, showing the added value of this concerted work.

Animals↗
PubMed2006

The power of cooperative investigation: summary and comparison of the HUPO Brain Proteome Project pilot study results.

Within the pilot phase of the HUPO Brain Proteome Project, nine participating laboratories analysed human (epilepsy and/or post mortem material) and mouse brain samples (embryonic, juvenile and adult), respectively, using a variety of different state of the art techniques. Thirty-seven different analytical approaches were accomplished. Of these analyses, 17 were done differentially, i.e. the protein expression patterns of the different samples (human or mouse) were compared. A catalogue of all proteins present in the respective sample was built in 20 analyses (mapping). All data were collected in the Data Collection Center in Bochum, Germany, and were reprocessed according to thoroughly defined parameters. In this report, a summary of all results and inter-laboratory comparisons with respect to the number of identified proteins, the analysed organism, and the used techniques is presented.

Animals↗
PubMed2006

Changes of the hepatic proteome in murine models for toxically induced fibrogenesis and sclerosing cholangitis.

We investigated the changes in the hepatic proteome in murine models for toxic-induced fibrogenesis and sclerosing cholangitis. A comprehensive comparison of protein changes observed is made and the mechanistical basis of the expression changes is discussed. Hepatic fibrosis was induced by repetitive intraperitoneal CCl4 treatment of BALB/c mice or developed spontaneously in BALB/c-ATP-binding cassette, subfamily B, member 4 (Abcb4) knock out mice. Fibrosis was verified by a morphometric score and assessment of hydroxyproline content of liver tissue, respectively. The innovative difference in-gel electrophoresis (DIGE) technique was used to analyse protein expression levels of the mouse proteome. Results were confirmed by Western blotting and real-time RT-PCR. In CCl4-induced fibrosis 20 out of 40 and in BALB/c-Abcb4(-/-) mice 8 out of 28 differentially expressed proteins were identified utilizing DIGE. Only two proteins, selenium-binding protein (Sbp2) and carbonic anhydrase 3, have been unidirectionally expressed (i.e. down-regulated) in both models. Relevant differences in the pathogenesis of toxically induced liver fibrosis and sclerosing cholangitis exist. The only novel protein with regard to liver fibrosis depicting a unidirectional expression pattern in both animal models was Sbp2. An explicit protein function could not be clarified yet.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2005

Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins.

Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron-sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.

Amino Acid Sequence↗
PubMed2005

Influence of ACE-inhibition and mechanical unloading on the regulation of extracellular matrix proteins in the myocardium of heart transplantation candidates bridged by ventricular assist devices.

BACKGROUND: Whether adverse structural changes in the myocardium due to remodelling can be reversed by ventricular assist device (VAD) support in patients with end-stage heart failure is controversial. AIMS: To investigate the effect of VAD support on the extra-cellular matrix. METHODS: We analysed the collagen content in terminal failing ventricles of VAD-patients and donor hearts using 4-hydroxyproline for total collagen and real time RT-PCR for fibronectin (FN), collagen I alpha 1 (Col1A1), III alpha 1 (Col3A1) and TGF beta 1 analysis. RESULTS: Compared to donor hearts we found similar increases in Col1A1 and TGF beta1 but not Col3A1 and FN mRNAs, which were similar in the myocardium from patients receiving a VAD or heart transplant. However, patients receiving ACE-I during VAD-support had lower Col1A1 mRNA content at transplantation. The total collagen content was not influenced by mechanical unloading or by ACE-I medication. CONCLUSION: Mechanical unloading by VAD does not reduce the collagen content of the terminal failing ventricle possibly due to increased TGF beta1 levels. However, Col1A1 production may be reduced by ACE-I medication during VAD support.

Adult↗
PubMed2005

The mono-ADP-ribosyltransferases Alt and ModB of bacteriophage T4: target proteins identified.

Infection of Escherichia coli by bacteriophage T4 leads to the expression of three phage mono-ADP-ribosyltransferases (namely, Alt, ModA, and ModB), each of which modifies a distinct group of host proteins. To improve understanding of these interactions and their consequences for the T4 replication cycle, we used high-resolution two-dimensional gel electrophoresis and mass-spectrometry to identify some of the putative target proteins ADP-ribosylated in vitro by Alt (total approximately 27) and ModB (total approximately 8). E. coli trigger factor and the elongation factor EF-Tu were 2 targets of ModB action, and these proteins were among the 10 identified as targets of Alt, hinting that these factors are involved in phage replication.

ADP Ribose Transferases↗
PubMed2005

Inactivation of the mitochondrial heat shock protein zim17 leads to aggregation of matrix hsp70s followed by pleiotropic effects on morphology and protein biogenesis.

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.

Dimerization↗
PubMed2005

Profiling of alopecia areata autoantigens based on protein microarray technology.

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Adult↗
PubMed2005

"Subcellular proteomics" of neuromelanin granules isolated from the human brain.

"Subcellular proteomics" is currently the most effective approach to characterize subcellular compartments. Based on the powerful combination of subcellular fractionation and protein identification by LC-MS/MS we were able for the first time to 1) isolate intact neuromelanin granules from the human brain and 2) establish the first protein profile of these granules. This compartment containing neuromelanin (NM) is primarily located in the primate's substantia nigra, one of the main brain regions that severely degenerates in Parkinson disease. We used mechanic tissue disaggregation, discontinuous sucrose gradient centrifugation, cell disruption, and organelle separation to isolate NM granules from human substantia nigra. Using transmission electron microscopy we demonstrated that the morphological characteristics of the isolated NM granules are similar to those described in human brain tissue. Fundamentally we found numerous proteins definitely demonstrating a close relationship of NM-containing granules with lysosomes or lysosome-related organelles originating from the endosome-lysosome lineage. Intriguingly we further revealed the presence of endoplasmic reticulum-derived chaperones, especially the transmembrane protein calnexin, which recently has been located in lysosome-related melanosomes and has been suggested to be a melanogenic chaperone.

Calnexin↗
PubMed2005

Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17.

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.

Amino Acid Sequence↗
PubMed2005

Identification of dynamic proteome changes upon ligand activation of Trk-receptors using two-dimensional fluorescence difference gel electrophoresis and mass spectrometry.

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

Brain-Derived Neurotrophic Factor↗
PubMed2005

Great mood in proteomics: Beijing and the HUPO Human Brain Proteome Project.

More than 1200 attendees came together at the 3(rd) HUPO World Congess in Beijing, October 25-27, 2004. In numerous different sessions the wide range of proteomic areas became visible. The HUPO Brain Proteome Project (HUPO BPP) organized an evening session on October 23, presenting the first results of two pilot studies as well as the newest, very positive international development in this field. The rising importance became even more apparent in the plenary presentation of all HUPO initiatives and the following congress activities.

Animals↗
PubMed2005

Differential proteome analysis of conditioned media to detect Smad4 regulated secreted biomarkers in colon cancer.

Smad4 is a tumor suppressor gene primarily involved in carcinogenesis of the pancreas and colon. The functional inactivation of Smad4 is a late step genetically. In pancreatic carcinogenesis, loss of Smad4 marks the transition to invasive growth. In colorectal cancers, the frequency of Smad4 inactivation is markedly increased in metastatic cancers. We have established cell biological models, re-expressing Smad4 in deficient human cancer cells, in which we could show that Smad4 is adequate to suppress tumor growth through suppression of angiogenic and invasive properties. Thus, pairs of Smad4-re-expressing and Smad4-deficient cells are prone to model the progression from premalignant stages to carcinomas in the carcinogenic process and may provide access to Smad4 targets of high clinical relevance. We present here a "differential secretome analysis", comparing all the proteins released in vitro from the Smad4-deficient and Smad4-re-expressing SW480 human colon carcinoma cells. The differential secretome catalog comprises more than 25 proteins including proteases and protease inhibitors, as well as established tumor biomarkers. In conclusion, this approach proved to be a sensitive tool to specifically detect Smad4 targets relevant for tumor-stroma interactions. It is also able to reflect complex alterations of cellular physiology. Moreover, the results support our hypothesis that human tumor markers detectable in serum may be identified through differential secretome analyses.

Biomarkers, Tumor↗
PubMed2005

Tryptic transpeptidation products observed in proteome analysis by liquid chromatography-tandem mass spectrometry.

Commonly, prior to mass spectrometry based analysis of proteins or protein mixtures, the proteins are subjected to specific enzymatic proteolysis. For this purpose trypsin is most frequently used. However, the process of proteolysis is not unflawed. For example, some side activities of trypsin are known and have already been described in the literature (e.g., chymotryptic activity). Here, we describe the occurrence of transpeptidated peptides during standard proteome analysis using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric protein identification. Different types of transpeptidated peptides have been detected. The most frequently observed transpeptidation reaction is N-terminal addition of arginine or lysine to peptides. Furthermore, addition of two amino acids to the N-terminus of a peptide has also been detected. Another transpeptidation that we observed, is combination of two peptides, which were originally located in different regions of the analyzed protein. Currently, the full amount of peptides generated by transpeptidation is not clear. However, it should be recognized that protein information is presently lost as these effects are not detectable with available database search software.

Animals↗
PubMed2005

Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.

In order to identify new molecular markers for pancreatic intra-epithelial neoplasias (PanINs), the precursor lesions of pancreatic ductal adenocarcinoma, we established a proteomics approach analysing microdissected PanIN cells. Due to the limited amount of proteins available from microdissection, we developed a procedure including fluorescence dye saturation labelling in combination with high resolution two-dimensional gel electrophoresis. With this procedure we were able to analyse proteins extracted from 1000 microdissected cells with a high resolution of up to 2500 protein spots. Using protein lysates from the pancreatic carcinoma tissue as a reference proteome, we were able to successfully identify the proteins. Thus, we could match approximately 2200 protein spots (92%) of the microdissected sample proteome to the reference proteome for protein identification using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and nanoliquid chromatography-electrospray ionisation tandem mass spectrometry after in-gel digestion. The first proteome analysis of microdissected PanIN-2 grades revealed eight differentially expressed proteins. The differential expression of the three actin filament-associated proteins--transgelin, vimentin and MRCL3 as well as actin itself--indicates a relevant role of the actin cytoskeleton during pancreatic tumour progression. Additionally, two members of the annexin family (annexin A2 and A4) implicate a functional contribution of exocytotic and endocytotic pathways at that stage.

Adenocarcinoma↗
PubMed2005

HUPO Brain Proteome Project Pilot Studies: bioinformatics at work.

The data acquisition phase of initial pilot studies (human and mouse brain samples) of the Human Proteome Organisation (HUPO) Brain Proteome Project (BPP) is now complete and the data generated by the participating laboratories has been submitted to the central Data Collection Center. The BPP Bioinformatics Group met on 8th April 2005 at the European Bioinformatics Institute (Hinxton, UK) to discuss strategies for the reanalysis of the pooled data from all the participating laboratories. A summary of the results of the data reprocessing will be presented at the 4th HUPO World Congress that will be held in August/September 2005.

Animals↗