Profiling Protein Post-Translational Modifications in Plasma-derived Extracellular Vesicles as Fingerprints for Breast Cancer Subtypes

Addressing tumor heterogeneity in breast cancer (BC) research is crucial, given the distinct subtypes like triple-negative (TN), luminal A/B (LAB), and HER2, requiring precise differentiation for effective treatment. This study introduces a non-invasive method by analyzing post-translationally modified proteins in plasma extracellular vesicles (EVs), which play a role in immune regulation and intercellular communication. Examining modifications like phosphorylation, acetylation and glycosylation in EVs provides insights into BC dynamics. One hundred and one plasma samples from LAB BC, TN BC and healthy individuals underwent discovery and validation experiments. The study identified over 28,000 unique non-modified peptides, 5,000 phosphopeptides, 680 acetyl peptides and 1,300 glycopeptides that were successfully characterized. Bioinformatics analyses revealed significant overexpression of 815 non-modified proteins, 3,958 phosphopeptides, 352 acetyl peptides and 895 glycopeptides in LAB BC or TN BC subtypes. Phosphorylated and glycosylated PD-L1 peptides emerged as potential markers for BC, regardless of subtype. Aligning the findings with literature and PAM50 gene signatures highlighted markers correlated with lower survival rates. The study also conducted 123 scheduled parallel reaction monitoring (PRM) analyses, leveraging machine learning to pinpoint a panel of specific modification sites with high accuracy for subtype differentiation. This research reveals diagnostic markers and enhances understanding of the molecular landscape, contributing to more effective and personalized BC diagnostics and treatments.