Development and Application of Novel Electron Transfer Dissociation-based Technologies for Proteomics

Proteins are key actors in all cellular processes and pathways and almost all diseases are linked to perturbations of proteins, their modification state or interaction networks. Mass spectrometry-based proteomics has matured to a high-throughput quantitative technology, aiming to provide sensitive, accurate and complete information on protein abundance, interactions and networks. In the current workflow in MS-based proteomics, proteins are cleaved into peptides by proteolytic digestion followed by MS analysis. Peptide sequencing is key to any MS-based proteomic experiment and provides the foundation for structural analysis. Collision induced dissociation (CID) is the most established dissociation technique and is nowadays routinely used for peptide sequencing. Over the last years, electron-driven approaches such as electron transfer dissociation (ETD) have evolved as valuable alternative fragmentation techniques. In this thesis the development of novel tandem mass spectrometry technologies based on electron-transfer dissociation is described. First, a new method that combines CID and ETD with either ion trap or Orbitrap detection in a data-dependent decision tree approach is introduced. This method enables selection of the most appropriate combination of fragmentation technique and mass analyzer ‘on-the-fly’, in turn leading to more confident peptide identification. Next, a novel fragmentation method combining higher-energy collision dissociation (HCD) and ETD in a single MS/MS event is described. This method, coined EThcD, provides extensive peptide backbone fragmentation and substantially increases the overall peptide sequence coverage. Furthermore, the higher quality in EThcD spectra facilitates the analysis of post-translational modifications, e.g. phosphorylation or glycosylation. Localization of post-translational modifications is challenging because it requires observation of site-determining fragment ions. This is exemplified for the analysis of phosphorylated peptides, which revealed superior phosphorylation site assignment using EThcD compared to current fragmentation methods. Another part of this thesis describes a novel instrumental setup to perform ETD reactions in the HCD collision cell on an Orbitrap Velos instrument by applying a static DC gradient axially to the rods. This gradient enables simultaneous three dimensional, charge sign independent, trapping of cations and anions, initiating electron transfer reactions in the center of the HCD cell where oppositely charged ions clouds overlap. The data shows that performing ETD in the HCD cell provides similar fragmentation as ion trap-ETD but will require further optimization to match performance of ion trap-ETD. The benefits of ETD are further highlighted in a study of endogenous neuropeptides derived from rat brain. Neuropeptides are key players in food intake regulation. Here, we analyze brain extract from two different brain areas. Based on the results from chapter 2, we utilized HCD and ETD fragmentation to increase the overall peptide identification rate. Overall we identified more than 1700 unique endogenous peptides, including virtually all neuropeptides that are known to be involved in food intake regulation. The quantitative data highlights differential processing of endogenous peptides that are derived from the same precursor protein. Moreover, we found comparable upregulation of orexigenic and anorexigenic potentially indicates conflicting signaling in rats that were fed on a high fat/high sucrose diet