Elucidating Tertiary Structures of Affibody in Vacuo Using Genetic Code Expansion and FRIPS Mass Spectrometry.

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