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      Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics*

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          Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.

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          Most cited references 73

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          Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.

          We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.
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            Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex.

            Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.
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              Analysis of protein complexes using mass spectrometry.

              The versatile combination of affinity purification and mass spectrometry (AP-MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP-MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP-MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein-complex assembly.

                Author and article information

                Mol Cell Proteomics
                Molecular & Cellular Proteomics : MCP
                The American Society for Biochemistry and Molecular Biology
                August 2010
                31 March 2010
                31 March 2010
                : 9
                : 8
                : 1634-1649
                From the a Institute of Molecular Systems Biology, Eidgenössiche Technische Hochschule (ETH) Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland,
                b Department of Analytical Chemistry and Food Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria,
                d Ph.D. Program in Molecular Life Sciences, University of Zurich/ETH Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland,
                g Biognosys AG, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland,
                i Faculty of Science, University of Zurich, Zurich, Switzerland, and
                j Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland
                Author notes
                k To whom correspondence should be addressed. Tel.: 41-44-633-3170; Fax: 41-44-633-1051; E-mail: aebersold@ 123456imsb.biol.ethz.ch .

                c Supported by a Schroedinger fellowship of the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung Grant J 2857-B12).

                e Both authors contributed equally to this work.

                f Supported by a European Molecular Biology Organization long term fellowship and by European Commission Grant FP7-PEOPLE-IEF.

                h Present address: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

                © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles


                Molecular biology


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