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Biomedical subjects

Alexander Krah

Publications and source records attributed to Alexander Krah.

7 recordsLinked to original sources
PubMed2006

Proteins unique to intraphagosomally grown Mycobacterium tuberculosis.

Pathogenic mycobacteria persist and replicate within phagosomes of host phagocytes by inhibiting phagosome maturation at an early endosome stage. The molecular basis for this behavior is not understood. To identify proteins of Mycobacterium tuberculosis unique to the intraphagosomal phase, mycobacteria were purified from phagosomes of infected murine bone marrow-derived macrophages and analyzed by high-resolution 2-DE and MS. Protein patterns of intraphagosomally grown M. tuberculosis were compared with those of broth-cultured mycobacteria. The analysis revealed 11 mycobacterial proteins exclusively detected in intraphagosomal mycobacteria. Some of these proteins are involved in metabolism and cell envelope synthesis, such as the lipid carrier protein Rv1627c, and the conserved hypothetical protein Rv1130 that shows homology to a virulence-associated protein of Legionella pneumophila. The relevance of these proteins as factors enabling intracellular survival of M. tuberculosis is being discussed.

Bacterial Proteins↗
PubMed2005

Peptide mass fingerprinting.

Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.

Alternative Splicing↗
PubMed2004

Identification of candidate antigens for serologic detection of Helicobacter pylori-infected patients with gastric carcinoma.

Helicobacter pylori colonizes the stomach of almost half the world population and is a causative agent of gastric carcinomas and duodenal ulcers. Only a small fraction of infected people will develop these severe illnesses and a predictive test to identify people at high risk would greatly benefit disease management. Our study aimed to identify conserved bacterial antigens that may be useful for the development of such a diagnostic test. High-resolution immunoproteomics by 2-dimensional electrophoresis of H. pylori 26695 proteins was carried out with sera from infected patients with either duodenal ulcer (n=30) or gastric carcinoma (n=30), 2 clinically divergent conditions. According to their antigen recognition patterns clear groups of patients were identified. Although this classification did not correspond to the clinical status, it may be correlated to other bacterial or host factors that influence the outcome of infection. In general antigen recognition patterns were found to be highly variable, however by utilizing powerful image analysis and statistical tests the recognition of 14 antigenic protein species was found to differ significantly (p<0.01) between both diseases. Particular protein species of GroEL, HyuA, GroES and AtpA appear to be useful surrogate markers for gastric carcinoma detection and consequently should be considered for further prospective studies to assess their predictive value. For one protein species of AtpA, evidence was found that different post-translational modifications may confer different immunogenicities.

Adenocarcinoma↗
PubMed2004

Assessment of protein spot components applying correspondence analysis for peptide mass fingerprint data.

Proteins separated by two-dimensional gel electrophoresis (2-DE) may be distributed over several spots. Otherwise, one spot may contain more than one component. The same protein occurring in several spots supposedly represents differently modified protein species that might be of biological relevance. Identification of spots with peptide mass fingerprinting and database searching leads only to the detection of the major spot components. If a spot also contains additional minor protein components, quantitation of spots with protein staining techniques or antibody detection becomes misleading. In order to find spots containing minor components we applied correspondence analysis, a multivariate data exploration method, to peptide mass fingerprint data. Correspondence analysis using peak lists revealed groups of spots containing the same protein with their characteristic mass-to-charge ratio (m/z) values. In order to detect different protein spot components an interactive threshold setting and removal of m/z values with subsequent recalculation of the correspondence analysis using our software tool CorrAn are performed. The usefulness of this methodical approach was shown by a data set of peptide mass fingerprints of 284 spots of Helicobacter pylori 26695 separated by 2-DE.

Bacterial Proteins↗
PubMed2004

Immunoproteomics.

Two-dimensional electrophoresis results in an adequate resolution of the proteome of microorganisms to allow the detection and identification of specific antigens after blotting on membranes and overlaying the protein pattern with patient's sera. The complement of all identified antigens presents the immunoproteome of a microorganism. All the antigens specific for a microorganism or even for a disease are identified by mass spectrometry. For identification, peptide mass fingerprinting is used, and post-translational modifications are detected by mass spectrometry MS/MS techniques. High-resolution two-dimensional electrophoresis and unambiguous identification are prerequisites for reliable results. After statistical analysis, the resulting antigens are candidates for diagnosis or vaccination and targets for therapy.

Antibodies, Bacterial↗
PubMed2003

Analysis of automatically generated peptide mass fingerprints of cellular proteins and antigens from Helicobacter pylori 26695 separated by two-dimensional electrophoresis.

Helicobacter pylori is a causative agent of severe diseases of the gastric tract ranging from chronic gastritis to gastric cancer. Cellular proteins of H. pylori were separated by high resolution two-dimensional gel electrophoresis. A dataset of 384 spots was automatically picked, digested, spotted, and analyzed by matrix-assisted laser desorption ionization mass spectrometry peptide mass fingerprint in triple replicates. This procedure resulted in 960 evaluable mass spectra. Using a new version of our data analysis software MS-Screener we improved identification and tested reliability of automatically generated data by comparing with manually produced data. Antigenic proteins from H. pylori are candidates for vaccines and diagnostic tests. Previous immunoproteomics studies of our group revealed antigen candidates, and 24 of them were now closely analyzed using the MS-Screener software. Only in three spots minor components were found that may have influenced their antigenicities. These findings affirm the value of immunoproteomics as a hypothesis-free approach. Additionally, the protein species distribution of the known antigen GroEL was investigated, dimers of the protein alkyl hydroperoxide reductase were found, and the fragmentation of gamma-glutamyltranspeptidase was demonstrated.

Amino Acid Sequence↗