Dirk Wolters

Bio: Dirk Wolters is an academic researcher from Ruhr University Bochum. The author has contributed to research in topics: Proteome & Membrane protein. The author has an hindex of 33, co-authored 69 publications receiving 11283 citations. Previous affiliations of Dirk Wolters include Syngenta & Hoffmann-La Roche.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

Abstract: We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics

Abstract: We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676−682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10 000 to 1 between the most abundant and least abundant proteins/pep...

A proteomic view of the Plasmodium falciparum life cycle

Abstract: The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

Shotgun identification of protein modifications from protein complexes and lens tissue

Abstract: Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

Proteomic survey of metabolic pathways in rice

Abstract: A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

Mass spectrometry-based proteomics

Abstract: Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.

Universal sample preparation method for proteome analysis

Abstract: A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts. We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.

Functional organization of the yeast proteome by systematic analysis of protein complexes

Abstract: Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.

Abstract: We present a statistical model to estimate the accuracy of peptide assignments to tandem mass (MS/MS) spectra made by database search applications such as SEQUEST. Employing the expectation maximization algorithm, the analysis learns to distinguish correct from incorrect database search results, computing probabilities that peptide assignments to spectra are correct based upon database search scores and the number of tryptic termini of peptides. Using SEQUEST search results for spectra generated from a sample of known protein components, we demonstrate that the computed probabilities are accurate and have high power to discriminate between correctly and incorrectly assigned peptides. This analysis makes it possible to filter large volumes of MS/MS database search results with predictable false identification error rates and can serve as a common standard by which the results of different research groups are compared.