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Optimized Digestion Conditions for Membrane Protein Foot-printing and Mass Spectrometry Analysis

ABSTRACT: Integral membrane proteins (IMPs), which constitute 50–60% of drug targets, play essen-tial roles in numerous biological processes but remain underrepresented in conventional bottom-up and in structural proteomics owing to their hydrophobicity and resistance 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 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 evalu-ate its performance using a single amphipathic IMP model featuring distinct extramem-brane 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 hydropho-bic 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. In-stead, 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 IMPs charac-terization.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ming Cheng

LAB HEAD: Michael L Gross

PROVIDER: PXD072461 | Pride | 2026-07-02

REPOSITORIES: Pride

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			Action	DRS
	Additives_ProteaseMAX01.raw	Raw
	Additives_ProteaseMAX01_CM830_2R.raw_20260425_Byonic.xlsx	Xlsx
	Additives_ProteaseMAX02.raw	Raw
	Additives_ProteaseMAX02_CM830_2.raw_20260425_Byonic.xlsx	Xlsx
	Additives_Rapigest01.raw	Raw

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Optimized Digestion Conditions for Membrane Protein Foot-printing and Mass Spectrometry Analysis

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