Inline HIC-HRPF Enables Conformer-Specific Structural Analysis of Apo and Holomyoglobin.

Hydroxyl radical protein footprinting (HRPF) is a method for protein higher-order structure analysis that oxidizes solvent-accessible side chains of amino acids. One major limitation of HRPF is that results represent an ensemble average of all conformers in solution, preventing resolution of conformationally heterogeneous systems. Here, we established an inline hydrophobic interaction chromatography (HIC)-HRPF workflow that enables conformer-specific labeling while correcting for gradient-dependent differences in radical scavenging. Significant radical scavenging disparity exists between ammonium sulfate and phosphate buffers, and we compensated for this with 7 mM HEPES supplementation, restoring effective radical dose parity across the gradient. Using apo and holomyoglobin as a benchmark, we achieved baseline chromatographic separation of conformers. Multi-dose HRPF revealed conformer-specific oxidation patterns: apomyoglobin displayed enhanced labeling in peptide 81-97, specifically residue H83, consistent with destabilization of the F-helix and opening of the heme pocket, while holomyoglobin exhibited increased oxidation in peptide 120-154 corresponding to M132, E137, and L138, suggesting hinge-like rearrangements in the H-helix upon heme incorporation. These results validate inline HIC-HRPF as a robust, conformer-specific probe of higher-order structure and highlight the necessity of scavenging parity across the gradient for reliable structural interpretation. The method establishes a framework for extending HRPF to dynamic conformationally heterogeneous systems resolvable by HIC.