Diving into cellular signalling: Functional proteome analysis by mass spectrometry based approaches

The main aim of my work as described in this thesis has been to improve on proteomics technologies to study protein phosphorylation, protein oxidation and interactions between proteins and drug molecules. In chapter two, we were the first to perform thermal proteome profiling on zebrafish lysate. Thermal proteome profiling is a valuable technique in which mass spectrometry is used to find the on- and off-targets of small ligands, such as drugs. By finding the toxic off-targets of drugs, their development may be improved. Traditionally, thermal proteome profiling experiments were performed on single cell types, causing information on tissue specific proteins to be lost. Here, we improved on this methodology by performing thermal proteome profiling on zebrafish embryo lysate, which harbors all tissue specific proteins. We first showed, as a proof of principle, that we could detect ligand induced stability changes in pervanadate treated lysate, after which we extended this to the selective STAT3 inhibitor napabucasin. Using our approach, we validated the mode of action of napabucasin, while simultaneously finding aldehyde dehydrogenases as off-targets. In chapter three, we investigated the labile post-translational modification phosphohistidine. Phosphohistidine has been very difficult to study in the past, due to not being compatible with standard enrichment strategies. However, recently a novel approach has been developed which allows the identification of this PTM on a proteome wide scale. It was shown that phosphohistidine plays an important role in bacteria such as E.coli, however the importance and scope of this PTM in mammalian systems is not known. Here, we investigated the extent of phosphohistidine in mammalian cells using this optimized workflow. Many novel sites were found, but the validity of these was questioned. Therefore, acidification of the samples was used as a negative control. In E.coli, this drastically decreased the presence of phosphohistidine, while in mammalian samples this behavior was not replicated. Therefore, we concluded that the sites found in our experiments are false positives, and that the contribution of phosphohistidine in mammalian systems is extremely limited. In chapter four, we investigated the oxidative behavior of the catalytic cysteine of the tyrosine phosphatase SHP2 and its mutants. The oxidation of the catalytic cysteine of SHP2 is a known mechanism to (ir)reversibly inactivate it, but we were curious how the rates of oxidation differ between the wildtype phosphatase and the catalytically more active Noonan mutant. This mutant is in a more open conformation compared to the wildtype, which might cause it to be more readily oxidized. Indeed, through a differential alkylation approach we showed that the Noonan mutant is more readily oxidized compared to the wildtype. Additionally, we showed that the addition of catalase to SHP2 in a fusion protein can efficiently protect the catalytic cysteine against hydrogen peroxide. In the future, these fusion proteins may be used to determine the oxidation status of SHP2 in vivo. Lastly, in chapter five I share my view on the future of proteomics. In addition, a lay summary of this thesis and my acknowledgements can be found here.