Utilization of Fluoroalkylation for Structural Analysis of Proteins

The structural characterization of proteins is essential for understanding their function, particularly in pharmaceutical research where protein conformation plays a critical role in biological activity and drug binding. Fast fluoroalkylation of proteins (FFAP) has recently emerged as a cost-effective and experimentally simple alternative to established protein footprinting techniques, such as Fast photochemical oxidation of proteins (FPOP), for probing protein structure. In this thesis, FFAP-based strategies were applied using horse heart apomyoglobin as a model system, employing both established and newly designed Togni reagents. In the first part, an azide containing Togni reagent was used for fluoroalkylation, followed by Strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry conjugation with a novel Bicyclo[6.1.0]non-4-yne (BCN) probe. Protein modifications were analyzed using Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). While the initial fluoroalkylation step was successful, the outcome of the subsequent click chemistry reaction could not be conclusively assessed due to loss of the protein signal in the mass spectrum, potentially related to the high hydrophobicity of the BCN reagent. In the second part, newly designed Togni reagents were investigated as FFAP probes. Successful labelling of apomyoglobin was achieved and characterized by ESI FT-ICR MS. Additionally, formation of a side product was observed, indicating competing reaction pathways. A qualitative bottom-up analysis using Trapped ion mobility Time-of-flight mass spectrometry (TIMS-TOF MS) enabled identification of modified amino acid residues, providing insight into the selectivity of the probes. Overall, this study demonstrates both the potential and current limitations of FFAP as a tool for protein structural analysis. The results highlight the importance of reagent design, particularly hydrophobicity, and support further development of FFAP-based approaches for investigating the conformation of pharmacologically relevant proteins.