Multiplex Trifluoromethyl and Hydroxyl Radical Chemistry Enables High-Resolution Protein Footprinting

Abstract

Hydroxyl radical-based protein footprinting (HRPF) coupled with mass spectrometry is a valuable medium-resolution technique in structural biology, facilitating the assessment of protein structure and molecular–level interactions in solution conditions. In HRPF with X-rays (XFP), hydroxyl radicals generated by water radiolysis covalently label multiple amino acid (AA) side chains. However, HRPF technologies face challenges in achieving their full potential due to the broad (>10³) dynamic range of AA reactivity with ^•OH and difficulty in detecting slightly modified residues, most notably in peptides with highly reactive residues like methionine, or where all residues have low ^•OH reactivities. To overcome this limitation, we developed a multiplex labeling chemistry that utilizes both CF₃ radicals (^•CF₃) produced from a trifluoromethylation (TFM) reagent and OH radicals (^•OH), under controlled and optimized radiolysis doses generated by X-rays. We optimized the dual ^•CF₃/^•OH chemistry using model peptides and proteins, thereby extending the existing ^•OH labeling platform to incorporate simultaneous ^•CF₃ labeling. We labeled >50% of the protein sequence and >80% of protein solvent-accessible AAs via multiplex TFM labeling resulting in high-resolution footprinting, primarily by enhancing the labeling of AAs with low ^•OH reactivity via the ^•CF₃ channel, while labeling moderate and highly ^•OH-reactive AAs in both ^•CF₃ and ^•OH channels. Moreover, the low reactivity of methionine with ^•CF₃ enabled the detection and quantification of additional AAs labeled by ^•CF₃ within methionine-containing peptides. Finally, we found that the solvent accessibility of protein AAs directly correlated with ^•CF₃ labeling, demonstrating that multiplex TFM labeling enables a high-resolution assessment of molecular interactions for enhanced HRPF.