A robust “bottom up” proteomics pipeline is integral for assessing protein structure using hydroxyl radical protein footprinting mass spectrometry.

Hydroxyl Radical Protein Footprinting (HRPF) monitors macromolecular structure and dynamics by utilizing hydroxyl radicals to probe the solvent-accessible side chains of proteins. Hydroxyl radicals form irreversible covalent bonds with protein side chains based on their solvent accessibility and intrinsic reactivity. Following labeling, bottom-up proteomics which involves protease digestion and liquid chromatography (LC-MS/MS) coupled with mass spectrometry, is routinely employed to detect and quantify the modified protein side chains. The HRPF technique has been a breakthrough in the field of structural biology, enabling the assessment of structures and interrelationships between proteins, protein-drug complexes or such macromolecular mixtures. It is now being extended to complex applications such as in-cell and in-vivo studies. This perspective focuses on detailing aspects of peptide separations technology in HRPF, with a particular emphasis on chromatography. The discussion further encompasses the HRPF methodology, its current limitations, recent developments, and proposed ideas for future developments for selected research fields.