Advances in protein topology mapping methods are urgently needed to complement the wealth of interactome data that is presently being generated at a rapid pace. Chemical crosslinking followed by mass spectrometry (MS) has evolved over the last decade as an attractive method for protein topology and interface mapping, and holds great promise as a counterpart to modern interactome studies in the field of proteomics. Furthermore, stabilization of proteins and protein complexes with crosslinking offers many advantages over high-resolution structural mapping methods, including the ability to study protein topologies in vivo. The reliance on direct detection of crosslinked peptides, however, continues to pose challenges to protein topology and interface mapping with chemical crosslinking plus MS. The present body of work aimed to develop a novel generic methodology that utilizes chemical crosslinking, cyanogen bromide (CNBr) cleavage and MS for the low-resolution mapping of protein topologies and interfaces. Through such low-resolution mapping of crosslinked regions, this novel strategy overcomes limitations associated with the direct detection of crosslinked peptides. Following optimization of various steps, the present method was validated with the bacterial DNA-directed RNA polymerase core complex and was subsequently applied to probe the tetrameric assembly of yeast Skp1p-Cdc4p heterodimers. Further improvements were made through the enrichment of crosslinked CNBr-cleaved protein fragments prior to their identification via MS. Two enrichment strategies were developed which depended upon the conjugation of tags to CNBr-cleaved peptide C-termini followed by either tandem affinity purification or tandem reversed-phase HPLC purification. These strategies were successfully applied for the efficient purification of disulfide-linked peptides from peptide mixtures. It is expected that the potential to achieve sensitive mapping of topologies and interfaces of multi-subunit protein complexes in vivo, in combination with further enhancements to permit studies on complex protein samples, will extend the utility of this method to complement large-scale interactome studies.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31969 |
| Date | 11 January 2012 |
| Creators | Weerasekera, Rasanjala Kumari |
| Contributors | Schmitt-Ulms, Gerold |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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