Transmembrane proteome analysis of frozen mouse lung tissues by LC-MS using metal organic framework-based protein...

Membrane proteins (MPs) play crucial roles in cellular functions and are key targets for drug discovery and cancer research. However, due to their poor solubility, high hydrophobicity, and low abundance, MPs present significant analytical challenges, particularly with regard to their dissolution, isolation, and characterization. Efficiently extracting MPs from frozen tissue samples remains challenging, which limits our ability to study their functions in pathological processes. In this study, we developed a metal-organic frameworks (MOFs)-assisted bio-separation strategy using Zeolitic Imidazolate Framework-67 (ZIF-67) for efficient membrane proteome isolation from frozen mouse lung tissue. We optimized key parameters for MPs extraction, including protein extraction approaches (tissue grinding versus enzymolysis), material-to-protein ratio, and SDS concentration. Our results demonstrated that ZIF-67 strategy with enzymolysis dramatically improved the enrichment efficiency of MPs with multi-transmembrane domains (TMs≥2) than the commercial kit method. In particular, among the 500 most abundant MPs isolated by the optimized ZIF-67 strategy, 133 were MPs with TMs≥2, which was 2.18 times that of the kit method. We further applied this strategy to analyze two distinct types of clinical lung cancer tissue samples (ground glass opacity nodules and solid nodules). Quantitative proteomics analysis revealed distinct membrane protein profiles and dysregulated pathways in the two lung cancer nodules. The enhanced capability of the ZIF-67 strategy for analyzing multi-transmembrane proteome provided valuable biological information for investigating the underlying molecular mechanisms underlying distinct nodule types. The strategy's compatibility with frozen tissues underscores its broad applicability in translational and biomedical studies, paving the way for in-depth exploration of MPs in disease mechanisms and therapeutic development.