Elucidating the structural consequences of phosphorylation by mass spectrometry, ion mobility and molecular modelling

Phosphorylation is known to affect the structure and function of proteins and peptides, affecting their intramolecular non-covalent interactions and ability to interact with other molecules. The work presented in this thesis aims to develop mass spectrometry (MS), ion mobility spectrometry (IMS) and molecular modelling (MM) methods in order to study phosphorylation in model protein and peptide systems, with a focus on the effect of phosphorylation on the non-covalent interaction pattern and structure of these model systems. An electron capture dissociation (ECD)-guided ion mobility spectrometry/molecular modelling (IMS/MM) workflow is described for the simulation of large ensembles of phosphopeptides and their fragments under mass spectrometry conditions. This workflow was facilitated by the production of forcefield parameters for several amino acid residue sets (neutral C-terminal residues, neutral N-terminal residues, neutral arginine residues, neutral phosphorylated residues and residues adjacent to the cleavage site in c+ and z• fragments). The ECD-guided IMS/MM workflow was applied to the study of the 14-3-3-ζ-derived peptide VVGARRS(pS)WRVVSSI (pS denotes phosphorylated Ser), which contains the phosphorylation motif RRSsWR. Variants were generated by successive arginine-to-leucine substitutions within the phosphorylation motif. Structures were proposed for each of the peptides investigated. In the GSK3β-derived peptide Ac-QLVRGEPNVS(pY)I(Calk)SRYYR-Am (pY denotes phosphorylated Tyr, Calk denotes cysteine-S-acetamide), substitution of specific amino acids (pTyr → pSer, Cys-alk → Ala, and Glu → Ala) allowed the ECD behaviour of the wild-type peptide to be understood, and the effect of the substituted residues to be probed. The ECD-guided IMS/MM workflow allowed insights from the ECD behaviour to be related to the structures of these peptides, and further structural features of the peptides to be probed. By studying the ECD and IMS behaviour of fragments of the GSK3β-derived peptides and the phosphoprotein β-casein, it was proposed that electron transfer dissociation (ETD) fragments might be better model systems than derived peptides to infer information about the non-covalent interactions contained in the whole peptide or protein. Applying the ECD-guided IMS/MM workflow to the z172+ fragment of the GSK3β-pY11 peptide showed that the fragment had a similar structure to that of the intact ion. The inclusion of travelling wave ion mobility spectrometry (TWIMS) in ETD experiments produced similar levels of fragment coverage to ECD fragmentation of β-casein, and allowed the identification of a subset of fragments in the GSK3β-pY11 spectrum that were produced by a slow dissociation process from a charge reduced species. Isolation and ECD fragmentation experiments of GSK3β-derived peptides demonstrated that the ETD- and ECD-generated z172+ fragment had different ECD behaviours. This observation was related to the different energetics and structures of ETD-generated and ECD-generated fragments, and set into the context of differences between the mechanisms of the two fragmentation methods. Supplementary data for this thesis can be found on the University of Birmingham eData repository at: https://doi.org/10.25500/edata.bham.0000066