Optimized Digestion Conditions for Membrane Protein Footprinting and Mass Spectrometry Analysis

Integral membrane proteins (IMPs), which constitute 50–60% of drug targets, play essential roles in numerous biological processes but remain underrepresented in conventional bottom-up and structural proteomics owing to their hydrophobicity and resistance to proteolysis. Although advances in IMP proteomics have improved global IMP 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 peptides of suitable length for structural elucidation, necessitating optimized digestion condition for individual IMPs. Here, we report a digestion protocol tailored for structural mass spectrometry and evaluate its performance by using a single amphipathic IMP model featuring distinct extramembrane and transmembrane domains. We evaluated the use of various protease–additive combinations and applied filter-aided sample preparation (FASP) to remove detergents and surfactants efficiently prior to MS analysis. The optimized conditions consistently yielded >90% sequence coverage. Guided by MS retention 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 they are 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 provides a practical framework for optimizing digestion conditions in IMP characterization.