Optimizing Digestion for Integral Membrane Protein Footprinting and Bottom-Up Mass Spectrometry Analysis

Integral membrane proteins (IMPs), which constitute 50–60% of drug targets, play es-sential roles in numerous biological processes but remain underrepresented in conven-tional bottom-up and in structural proteomics owing to their hydrophobicity and re-sistance to proteolysis. Although advances in IMPs proteomics have improved global IMPs detection, most efforts focus on proteome-scale protein identification rather than targeted structural analysis. Protein footprinting and cross-linking, two approaches in structural proteomics, require high sequence coverage and protein digestion to pep-tides of suitable length for structural elucidation, necessitating optimized digestion condition for individual IMPs. Here, we describe a digestion protocol tailored for structural mass spectrometry, applying it to an amphipathic IMP with distinct ex-tramembrane and transmembrane domains, as a model system. We evaluated the use of various protease–additive combinations and applied filter-aided sample prepara-tion (FASP) to remove detergents and surfactants efficiently prior to MS analysis. The optimized conditions consistently yielded >90% sequence coverage. Guided by MS re-tention time calibration and hydrophobic factor simulations, we identified a “sweet spot” for transmembrane peptide detection. Notably, although cleavable surfactants can enhance proteome-wide coverage, our results show that they are not essential for single protein studies as in structural proteomics. Instead, detergent removal, protease selection, and generation of suitably sized peptides are critical for enabling reliable bottom-up structural analysis of IMPs. The protocol developed here was successfully applied across several footprinting methods for structural studies of IMPs.