Impact of Size Exclusion Chromatography and Ultracentrifugation on Purity and Proteomic Profiles of Extracellular Vesicles Derived from Lactobacillus reuteri

Abstract

Extracellular vesicles (EVs) produced by probiotic bacteria are increasingly recognized as crucial mediators of host-microbe communication. However, the molecular composition and biological interpretation of bacterial EV proteomes are heavily influenced by the isolation methods. In this study, we systematically compared ultracentrifugation (UC) and size exclusion chromatography (SEC) for isolating EVs from Lactobacillus reuteri, assessing their impact on EV yield, purity, and proteomic profiles. Although UC yielded significantly more EVs than SEC, it also resulted in lower purity, as evidenced by higher protein contamination and a decreased particle-to-protein ratio. In contrast, SEC improved EV purity by approximately 6.45-fold, effectively removing non-vesicular proteins. Our quantitative proteomics analysis identified 670 in UC-EVs and 858 in SEC-EVs.-- UC-EVs were primarily enriched with cytosolic metabolic enzymes, ribosomal proteins, and components associated with macromolecular complexes, indicating cosedimentation artifacts during UC. Conversely, SEC-EVs exhibited selective enrichment of membrane-associated and cell-wall-modifying proteins, reflecting their origin from envelope remodeling processes. Notably, SEC-EVs contained several proteins, including NLP/P60, peptidoglycan hydrolases, and lipoproteins linked to anti-inflammatory activities. Overall, our findings illustrate that EV proteomes are highly dependent on the isolation method and highlight SEC as a superior approach for enhancing proteomic specificity and biological interpretability in bacterial EV research.