Mass Spectrometry Insights Into Post‐Translational Modifications in Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-enclosed structures secreted by virtually all living cells, serving as essential mediators of intercellular communication in both physiological and pathological processes. There is growing interest in their potential applications as biomarkers, therapeutic targets, and drug delivery systems, which entails the need for a detailed understanding of their molecular composition. The functional cargo of EVs includes all types of biological macromolecules, among which proteins are of particular importance. As the vesicular proteome becomes increasingly mapped, research attention is gradually shifting toward post-translational modifications (PTMs), which fundamentally influence protein function and play key roles in all aspects of vesicular activity, including biogenesis, cargo sorting, recognition, and uptake. In this review, we outline recent advances in the application of mass spectrometry (MS)-based analysis of PTMs in EVs. In this context, we provide an overview of the roles of various PTMs in EV biology, discuss the impact of EV isolation methods on downstream PTM analyses, and address current challenges and approaches related to MS-based investigations. We further highlight key findings concerning specific PTMs, including glycosylation, phosphorylation, acetylation, methylation, lipidation, and small ubiquitin-like modifier (SUMOylation). Finally, we discuss studies focusing on the simultaneous analysis of multiple PTMs, as well as efforts toward multiomic data integration and single-EV characterization to resolve vesicular heterogeneity, highlighting these approaches as cutting-edge directions in the field.