Abstract
Biological signaling is often relying on molecules of high similarity. Key examples are neuropeptides and phosphorylated proteins. Neuropeptides undergo a variety of enzymatic processing steps in the course of their biosynthesis, giving rise to a variety of molecules differing in just one amino acid or one post-translational modification. These minor
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differences, however, can lead to highly varying functionalities in the nervous system. Similarly, the phosphorylation of one individual amino acid residue on a protein can drastically alter the protein’s function, e. g. by inducing its enzymatic activity or by changing its binding partners. Additionally, these effects can be largely different depending on the exact localization of the phosphorylation on the protein backbone, even if the phosphorylation sites are in close proximity. Antibody-based research is often not able to selectively distinguish between these highly similar molecules (as evidenced by most antibodies targeting tyrosine kinases or ERK1/2 kinases). In this thesis, the power of targeted mass spectrometry was exploited to solve these analytical challenges. Selected Reaction Monitoring (SRM) was used to develop highly specific molecular assays for various neuropeptides and phosphosite localization isomers. Initially, a proof of principle study was performed to demonstrate the capability of SRM to quantify neuropeptides directly from peptide extracts of hypothalami of rats. This showed not only the capability of SRM to accurately quantify peptides with multiple basic residues, but also allowed the detection of a significantly upregulated abundance of the VGF-derived neuropeptide AQEE-30 in obese rats. Similarly, the high discrimination power of SRM for highly similar peptide sequences was shown for peptides with different phosphosite localizations. Due to their identical mass, their distinction was primarily guided by chromatographic separation achieved on an online nano-LC system with high separation power. This system allowed high confidence phosphosite localization, even upon very low signal to noise levels, as demonstrated here for phosphorylation of the protein kinase FAK at position Y560 in lung cancer cell lines. Lastly, the use of data-independent acquisition (DIA) was assessed for its use to reliably analyze phosphosite localizations on proteins in a system-wide scale. Here, the thesis demonstrates advantages of DIA over classical shotgun phosphoproteome analyses, given by the continuous measurement of all fragment ion intensities. In comparison to SRM, however, DIA still lacks sensitivity whilst offering higher multiplexing capabilities. Conclusively, this thesis demonstrates how targeted mass spectrometry can be specifically tailored to be an excellent tool to differentiate and quantify highly similar signaling molecules, which cannot be distinguished by biochemical methods alone.
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