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Jens Mattow

Publications and source records attributed to Jens Mattow.

11 recordsLinked to original sources
PubMed2006

Selenium-binding protein 2, the major hepatic target for acetaminophen, shows sex differences in protein abundance.

Liver samples from female and male mice of two subspecies, Mus musculus musculus and Mus musculus domesticus, were investigated by a combination of 2-DE and MALDI-MS. The image analysis of the generated 2-DE patterns revealed several protein spots with significant differences in intensity/abundance between the sexes. Seven protein spots, which were prominent in 2-DE patterns of male mice, but which showed very low intensities in females, were identified as selenium-binding protein 2 (SBP2) also known as 56-kDa acetaminophen-binding protein. Edman degradation indicated that at least three of these protein spots represent N-terminally truncated SBP2 variants. Furthermore, it was shown that the observed differences in SBP2 abundance correlate with sex differences in transcription of the gene encoding SBP2, selenbp2, as revealed by RT-PCR and restriction digest as well as sequence analysis of the products. Since SBP2 has been described as the major target for acetaminophen in mouse liver cytosol, these findings are discussed with respect to their possible relevance for sex differences in acetaminophen-mediated toxicity, which have been described in a variety of mammals including mice and rats.

Acetaminophen↗
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

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers.

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.

Bacterial Proteins↗
PubMed2004

Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis.

Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost "on top" of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.

Animals↗
PubMed2003

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology.

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG Copenhagen.

A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793).

Bacterial Proteins↗
PubMed2003

Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis.

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving many unassigned after a first database search. A number of these are caused by experiment-specific contaminants or by neighbor spots, as well as by additional proteins or post-translational modifications. To understand the complete protein composition of a spot we suggest an iterative procedure based on large numbers of PMFs, exemplified by PMFs of 480 Helicobacter pylori protein spots. Three key iterations were applied: (1) Elimination of contaminant mass peaks determined by MS-Screener (a software developed for this purpose) followed by reanalysis; (2) neighbor spot mass peak determination by cluster analysis, elimination from the peak list and repeated search; (3) re-evaluation of contaminant peaks. The quality of the identification was improved and spots previously unidentified were assigned to proteins. Eight additional spots were identified with this procedure, increasing the total number of identified spots to 455.

Cluster Analysis↗
PubMed2003

The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization.

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.

Antigens, Bacterial↗
PubMed2002

Isoaspartyl dipeptidase activity of plant-type asparaginases.

Recombinant plant-type asparaginases from the cyanobacteria Synechocystis sp. PCC (Pasteur culture collection) 6803 and Anabaena sp. PCC 7120, from Escherichia coli and from the plant Arabidopsis thaliana were expressed in E. coli with either an N-terminal or a C-terminal His tag, and purified. Although each of the four enzymes is encoded by a single gene, their mature forms consist of two protein subunits that are generated by autoproteolytic cleavage of the primary translation products at the Gly-Thr bond within the sequence GTI/VG. The enzymes not only deamidated asparagine but also hydrolysed a range of isoaspartyl dipeptides. As various isoaspartyl peptides are known to arise from proteolytic degradation of post-translationally altered proteins containing isoaspartyl residues, and from depolymerization of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin), plant-type asparaginases may not only function in asparagine catabolism but also in the final steps of protein and cyanophycin degradation. The properties of these enzymes are compared with those of the sequence-related glycosylasparaginases.

Amidohydrolases↗