Elucidating\nTertiary Structures of Affibody in Vacuo\nUsing Genetic Code Expansion and FRIPS Mass Spectrometry

Radical-directed protein fragmentation techniques, particularly\nfree radical-initiated peptide sequencing (FRIPS) mass spectrometry\n(MS), offer significant potential for elucidating protein structures\nin the gas phase. This study presents a novel approach to protein\nstructural analysis in vacuo, combining FRIPS MS with genetic code\nexpansion (GCE) technology. By incorporating unnatural amino acids\n(UAAs) at specific sites within an Affibody protein, we effectively\nintroduced a radical precursor at six distinct positions. The study\nexplores structural information derived from radical-directed fragmentations\nby analyzing the proximity and pathways of radical transfer within\nthe protein’s tertiary structure. Our findings reveal that\nin the lowest charge state (+5), the Affibody retains a folded conformation\nresembling its native structure, with significant radical-directed\nfragmentations occurring through both “through-sequence”\nand “through-space” mechanisms. These results demonstrate\nthe potential of FRIPS MS to provide residue-specific insights into\nprotein folding and structural information in the gas phase, paving\nthe way for a more detailed protein structure analysis.