Mass spectrometry approaches towards the characterisation of protein post-translational modification crosstalk

Adoni, Kish R. ORCID: 0000-0001-7390-501X (2023). Mass spectrometry approaches towards the characterisation of protein post-translational modification crosstalk. University of Birmingham. Ph.D.

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Post-translational modifications (PTM) enable a given protein to access different proteoform variants in response to their environment. The modification of a protein with a PTM can facilitate the occurrence of other PTMs within the same protein, allowing it to access a specific proteoform via a mechanism known as positive PTM crosstalk. PTMs are dynamic, thus they provide the infrastructure for real-time protein regulation as the cell adapts to its surroundings. As such, to truly understand the behaviour of a given protein, all possible proteoforms that it can access must be characterised. To this end, mass spectrometry represents a powerful analytical technique for the site-localised identification of PTM decorated proteins. In this thesis, High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) was incorporated into a bottom-up proteomics workflow for the purpose of improving the identification of positive PTM crosstalk sites. A 6-fold improvement in multiple PTM containing peptides, as candidates for positive PTM crosstalk, was demonstrated in HeLa S3 cervical cancer cells, with 40% of these sites not previously reported. The technique was then leveraged against SUM52 triple negative breast carcinoma cancer cells for the investigation of positive PTM crosstalk that facilitates the cancer phenotype. 498 positive PTM crosstalk sites were suggested to play a role in generating the cancer phenotype. Further, novel positive PTM crosstalk mechanisms were proposed via the identification of specific PTM containing peptide isoforms, such as the N-terminal acetylation of cofilin, leading to its sequestration via serine-3 phosphorylation to facilitate actin polymerisation for cancer cell migration. Following this, a multimodal mass spectrometry investigation of a novel S-glutathionylation PTM site within the evolutionarily relevant light harvesting phycobilisome protein complex of cyanobacteria was performed. Native MS, native top-down MS, LC-MS/MS and LC-FAIMS-MS/MS were applied to demonstrate the relevance of this novel S-glutathionylation PTM in protecting the phycobilisome from the reactive oxygen species that are generated as by-products of photosynthesis. Further, 84 % of the S-glutathionylated peptides that were identified with LC-FAIMS-MS/MS occurred with a concurrent phosphorylation PTM, suggesting positive PTM crosstalk between S-glutathionylation and phosphorylation within cyanobacteria. Together, these findings demonstrate the relevance of mass spectrometry approaches for the aim of characterising proteoform variability within proteins, to truly understand the role of proteins in biochemistry.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department: School of Biosciences
Funders: Biotechnology and Biological Sciences Research Council
Subjects: Q Science > Q Science (General)
Q Science > QD Chemistry


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