- Development of enrichment methods for cross-linked peptides to study the dynamic topology of large protein complexes by mass spectrometry
- Award date
- 2 December 2013
- Number of pages
- Document type
- PhD thesis
- Faculty of Science (FNWI)
- Swammerdam Institute for Life Sciences (SILS)
In the cell, most proteins carry out their functions as subunits of larger assemblies. Knowledge about the folding and mutual interactions of composing subunits is crucial to comprehend the dynamics and structures of such protein complexes and the molecular mechanisms underlying biological processes. Unfortunately, large complexes are often not amenable to techniques used to obtain detailed protein structural information like x-ray crystallography and nuclear magnetic resonance spectroscopy. In this thesis chemical protein cross-linking coupled with mass spectrometric analysis (CXMS) has been utilized as a core approach to obtain structural information of proteins. With CXMS the identity of cross-linked amino acids and their position in the protein amino acid sequence is determined, yielding spatial distance restraints, since the linked residues cannot be father away in a cross-linked protein complex than the length of the spacer of the used crosslinker. With this information a topological map of subunit arrangements can be obtained. Although CXMS has been applied for more than a decade, the rareness and low abundance of cross-linked peptides in digests forms a main analytical challenge, since this circumstance hampers mass spectrometric analysis. In this thesis methodologies aimed at enrichment of cross-linked peptides have been developed and applied. These studies were performed both in isolated complexes and in complicated protein mixtures. In Chapter 2, 3 and 4, the development of enrichment approaches intended to isolate azide-containing cross-linked peptides of proteins/complexes for mass spectrometric analysis are described. In Chapter 5, the characterization of novel interaction using the methodology developed in this thesis is shown.
- Research conducted at: Universiteit van Amsterdam
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library, or send a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.