Mass spectrometry-assisted protease substrate screening
ABSTRACT Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown. Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed-phase chromatography they are analyzed by tandem mass spectrometry and the substrates identified by database searching. The proof of principle in this study is demonstrated by incubating immobilized human plasma proteins with thrombin and by identifying by tandem mass spectrometry the fibrinopeptides, released by the action of thrombin from their natural substrate fibrinogen, in the reaction mixture.
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ABSTRACT: The second Dagstuhl Seminar on Computational Proteomics took place from March 3rd to 7th, 2008 in Schloss Dagstuhl-Leibniz Cen- ter for Informatics. This highly international meeting brought together researchers from computer science and from proteomics to discuss the state of the art and future developments at the interface between exper- iment and theory. This interdisciplinary exchange covered a wide range of topics, from new experimental methods resulting in more complex data we will have to expect in the future to purely theoretical studies of what level of experimental accuracy is required in order to solve certain problems. A particular focus was also on the application side, where the participants discussed more complex experimental methodologies that are enabled by more sophisticated computational techniques. Quantita- tive aspects of protein expression analysis as well as posttranslational modifications in the context of disease development and diagnosis were discussed. The seminar sparked a number of new ideas and collaborations and has resulted in several joint grant applications and paper submis- sions. This paper describes the seminar topics, its goals and results. The ex- ecutive summary is followed by the abstracts of the presentations given. Links to extended abstracts or full papers are provided, if available.
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ABSTRACT: This review covers recent developments in mass spectrometry-based applications dealing with functional proteomics with special emphasis on enzymology. The introduction of mass spectrometry into this research field has led to an enormous increase in knowledge in recent years. A major challenge is the identification of "biologically active substances" in complex mixtures. These biologically active substances are, on the one hand, potential regulators of enzymes. Elucidation of function and identity of those regulators may be accomplished by different strategies, which are discussed in this review. The most promising approach thereby seems to be the one-step procedure, because it enables identification of the functionality and identity of biologically active substances in parallel and thus avoids misinterpretation. On the other hand, besides the detection of regulators, the identification of endogenous substrates for known enzymes is an emerging research field, but in this case studies are quite rare. Moreover, the term biologically active substances may also encompass proteins with diverse biological functions. Elucidation of the functionality of those-so far unknown-proteins in complex mixtures is another branch of functional proteomics and those investigations will also be discussed in this review.Analytical and Bioanalytical Chemistry 07/2011; 402(2):625-45. DOI:10.1007/s00216-011-5236-4 · 3.58 Impact Factor
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ABSTRACT: The use of nanocrystalline titanium dioxide films as affinity targets for the selective isolation and enrichment of phosphopeptides with subsequent analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry is described. A strong affinity of phosphopeptides to anatase titanium dioxide surfaces is observed, and a standard protocol for the selective isolation and enrichment of phosphopeptides on titanium dioxide films using a proteolytic digest of alpha- and beta-casein was developed. All washing and elution procedures using these films can be processed directly on the MALDI target, thereby avoiding sample contamination and losses. In addition, the enrichment of the phosphopeptides was improved due to a considerable enlargement of the surface. Several film substrates compatible with routine inlet systems of mass spectrometers, as conductive glass, aluminum, and silicon, have been manufactured and tested. A biological application was examined by the human fibrinogen-thrombin system. For a quantification and comparison of different expression levels of phosphoproteins in biological systems, the peptides were labeled with S-methyl thioimidate reagents. The capability of this method for high-throughput applications make the use of mesoporous titanium dioxide films as an affinity MALDI target a promising tool in phosphoproteomics. A combination of an amidation protocol showed that a quantification of phosphorylated peptides can easily be performed using TiO(2) films.Analytical Chemistry 02/2010; 82(3):1047-53. DOI:10.1021/ac902403m · 5.83 Impact Factor