AbstractProteins are the fundamental building blocks of all living cells and they carry out nearly all cell functions. Proteins predominantly act as part of multiprotein complexes to carry out these functions. The association of protein isoforms and post-translationally modified forms in protein complexes can influence their subcellular location, activity and substrate specificity. It is therefore crucial to characterise protein complexes at the level of the protein isoforms and post-translationally modified forms they contain to fully decipher the network of signalling and regulatory pathways within cells.
The aim of my work has been to develop a technique to study protein complexes through the use of size exclusion chromatography (SEC) combined with tandem mass spectrometry. Combining these approaches has enabled an in-depth analysis of protein complexes in U2OS cells, including those involving post-translationally modified proteins and protein isoforms. The data presented in this thesis provide a proof of concept, together with forming a useful resource, which can be used alongside pull-down analyses to differentiate the interaction partners involved in different protein complexes. This combined approach minimises the need for multiple IP analyses and facilitates a more targeted approach in dissecting the components of individual protein complexes.
I developed this technique further by utilising in vivo crosslinking prior to denaturing SEC. This approach enabled more efficient recovery and detection of proteins previously underrepresented using native SEC analysis, including many membrane complexes, thereby providing a more complete picture of endogenous protein complexes.
I have applied the native SEC/MS approach in a study of the interactions between the MRFAP1-MORF4L1 proteins. I demonstrated that this complex is distinct from the larger complex involving interactions between the MRGBP-MORF4L1 proteins. In addition, I also demonstrate that the native SEC/MS technique can be extended to assess the effect of drugs on protein-protein interactions.
Overall, the methods I present in this thesis enable the rapid, proteome-wide analysis of endogenous protein complexes, which will advance the future study of protein complexes in biology.
|Date of Award||2014|
|Supervisor||Angus Lamond (Supervisor)|