PXD072461-1

PXD072461 is an original dataset announced via ProteomeXchange.

Title	Optimized Digestion Conditions for Membrane Protein Foot-printing and Mass Spectrometry Analysis
Description	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.
HostingRepository	PRIDE
AnnounceDate	2026-07-02
AnnouncementXML	Submission_2026-07-01_16:38:33.156.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ming Cheng
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	carbamoylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2025-12-28 09:52:57	ID requested
⏵ 1	2026-07-01 16:38:33	announced

10.3390/MEMBRANES16060215;

submitter keyword: membrane protein

vitamin K epoxide reductase (VKOR)

mass spectrometry

protein footprinting

filter-aided sample preparation (FASP), membrane protein digestion

Michael L Gross
contact affiliation	Washington University in St. Louis
contact email	mgross@wustl.edu
lab head
Ming Cheng
contact affiliation	Shanghai Institute of Materia Medica Chinese Academy of Sciences
contact email	chengming@simm.ac.cn
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD072461
     1. Label: PRIDE project
     2. Name: Optimized Digestion Conditions for Membrane Protein Foot-printing and Mass Spectrometry Analysis