Biomedical subjects

Walter Weiss

Publications and source records attributed to Walter Weiss.

7 recordsLinked to original sources

PubMed2007

Two-dimensional electrophoresis for plant proteomics.

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteome analysis. 2-DE allows separation of highly complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance and delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or posttranslational modifications. 2-DE can resolve more than 5000 proteins simultaneously (approx 2000 proteins routinely) and can detect and quantify <1 ng of protein per spot. Today's 2-DE technology with IPGs has overcome the former limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5 and 12 has allowed the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow pH range IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, permit the detection of low abundance proteins. In this article we provide a comprehensive protocol of the current 2-DE technology for plant proteome analysis and describe in detail the individual steps of this technique.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Guidelines for the next 10 years of proteomics.

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

Biomarkers↗

PubMed2005

Identification, cloning, expression, and characterization of the gene for Plasmodium knowlesi surface protein containing an altered thrombospondin repeat domain.

Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias.

Amino Acid Sequence↗

PubMed2004

Current two-dimensional electrophoresis technology for proteomics.

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where Mr and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037-1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5-12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2-DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two-dimensional protein databases.

Animals↗

PubMed2004

Measuring the effects of an ever-changing environment on malaria control.

The effectiveness of malaria control measures depends not only on the potency of the control measures themselves but also upon the influence of variables associated with the environment. Environmental variables have the capacity either to enhance or to impair the desired outcome. An optimal outcome in the field, which is ultimately the real goal of vaccine research, will result from prior knowledge of both the potency of the control measures and the role of environmental variables. Here we describe both the potential effectiveness of control measures and the problems associated with testing in an area of endemicity. We placed canaries with different immunologic backgrounds (e.g., naïve to malaria infection, vaccinated naïve, and immune) directly into an area where avian malaria, Plasmodium relictum, is endemic. In our study setting, canaries that are naïve to malaria infection routinely suffer approximately 50% mortality during their first period of exposure to the disease. In comparison, birds vaccinated and boosted with a DNA vaccine plasmid encoding the circumsporozoite protein of P. relictum exhibited a moderate degree of protection against natural infection (P < 0.01). In the second year we followed the fate of all surviving birds with no further manipulation. The vaccinated birds from the first year were no longer statistically distinguishable for protection against malaria from cages of naïve birds. During this period, 36% of vaccinated birds died of malaria. We postulate that the vaccine-induced protective immune responses prevented the acquisition of natural immunity similar to that concurrently acquired by birds in a neighboring cage. These results indicate that dominant environmental parameters associated with malaria deaths can be addressed before their application to a less malleable human system.

Animals↗

PubMed2002

High pressure effects step-wise altered protein expression in Lactobacillus sanfranciscensis.

In this study we investigated the cellular response to the application of high hydrostatic pressure. High pressure is increasingly used for food preservation. With high resolution 2-D electrophoresis we compared the protein patterns of atmospherically grown Lactobacillus sanfranciscensis with those pressure treated up to 200 MPa. We performed the comparative study by using overlapping immobilized pH gradients covering the pH range from 2.5 up to 12 in order to maximize the resolution for the detection of stress relevant proteins. For improved quantitative analysis, staining with SyproRuby was used in addition to silver staining. By computer aided image analysis we detected more than a dozen spots within the pH range from 3.5 to 9 that were more than two-fold increased or 50% decreased in their intensity upon high pressure treatment. Two of them (approx. values: pI 4.0 and 4.2, respectively; M(r) approximately 15 000) have almost identical matrix-assisted laser desorption/ionization-time of flight mass spectrometry spectra and were identified by liquid chromatography-tandem mass spectrometry as putative homologs/paralogs to cold shock proteins of Lactococcus lactis. Their expression is opposed (i.e. the more acidic one is repressed, while the other one is induced); this effect is maximal at 1 h, 150 MPa. It was further remarkable that by monitoring the barosensitivity of the cells within 25 MPa steps, we observed a differential pressure induction or repression of the detected proteins as well. For example one protein (approx. values: pI 4.2, M(r) approximately 15 000) shows a maximum induction after 1 h, 150 MPa while another one (pI 7.5, M(r) approximately 25 000) is maximally induced after 1 h, 50/75 MPa. This indicates a successive cell response and different signalling pathways for these responses.

Amino Acid Sequence↗

PubMed2002

Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels.

Due to their heterogeneity and huge differences in abundance, the detection and identification of all proteins expressed in eukaryotic cells and tissues is a major challenge in proteome analysis. Currently the most promising approaches are sample prefractionation procedures prior to narrow pH range two-dimensional gel electrophoresis (IPG-Dalt) to reduce the complexity of the sample and to enrich for low abundance proteins. We recently developed a simple, cheap and rapid sample prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated gels. Complex sample mixtures are prefractionated in Sephadex gels containing urea, zwitterionic detergents, dithiothreitol and carrier ampholytes. After IEF, up to ten gel fractions alongside the pH gradient are removed with a spatula and directly applied onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of two-dimensional (2-D) gel electrophoresis. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and the full compatibility with subsequent IPG-IEF, since the prefactionated samples are not eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with subsequent IPG-IEF. Prefractionation allows loading of higher protein amounts within the separation range applied to 2-D gels and facilitates the detection of less abundant proteins. Also, this system is highly flexibile, since it allows small scale and large scale runs, and separation of different samples at the same time. In the current study, this technology has been successfully applied for prefractionation of mouse liver proteins prior to narrow pH range IPG-Dalt.

Animals↗