Concentric-type immunoblotting coupled with LC-MS/MS for high-sensitivity analysis of exosomes secreted by sorafenib-stimulated hepatocellular carcinoma cells.
Abstract
BACKGROUND: Exosomes exhibit excellent biocompatibility and low immunogenicity, making them attractive natural drug delivery carriers. Separation and quantitative detection of exosomal proteins and cellular signaling proteins are essential to decipher exosome biological functions. Traditional Western blotting (WB) is hampered by low sensitivity and limited detection range for low-abundance proteins. Concentric-type immunoblotting (CIB) integrates circular non-uniform gradient electric field with the molecular sieving effect of polyacrylamide gels to realize efficient separation and enrichment of proteins, effectively overcoming commonly false-negative issues in WB. RESULTS: Herein, exosomes secreted by sorafenib-stimulated hepatocellular carcinoma cells (SHCC-exo) were characterized by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and CIB, verifying CIB as a high-sensitivity alternative to WB for protein separation and detection of low-abundance vesicular biomarkers in tumor-derived vesicles. LC-MS/MS confirmed that sorafenib-stimulated HCC cells actively encapsulate sorafenib, demonstrating successful fabrication of functional natural sorafenib-loaded exosomal delivery systems with intact bilayer membrane structure. Subsequent incubation of HCC cells with SHCC-exo was performed, and CIB was applied to sensitively quantify p38 and JNK proteins and their phosphorylated forms in the MAPK signaling pathway. The results indicated that SHCC-exo robustly induce HCC cell apoptosis via selective p38 activation, while the JNK signaling pathway showed no measurable activation under this specific drug treatment condition in vitro. SIGNIFICANCE: This study demonstrates that concentric-type immunoblotting is capable of detecting diverse low-abundance disease biomarker proteins within tumor-derived exosomes, and represents a prospective analytical platform for tumor diagnosis, therapy-related molecular mechanism analysis and reliable quantification of phosphorylated proteins, offering promising application prospects for versatile protein analysis in related cancer biological research.
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