Multi-marker profiling of extracellular vesicles using streaming current and sequential electrostatic labeling

High heterogeneity in the membrane protein expression of small extracellular vesicles (sEVs) means that bulk methods relying on antibody-based capture for expression analysis have a drawback that each type of antibody may capture a different sub-population. An improved approach is to capture a representative sEV population, without any bias, and then perform a multiplexed protein expression analysis on this population. However, such a possibility has been largely limited to fluorescence-based methods. Here, we present a novel electrostatic labelling strategy and a microchip-based all-electric method for membrane protein analysis of sEVs. The method allows us to profile multiple surface proteins on the captured sEVs using alternating charge labels. It also permits the comparison of expression levels in different sEV-subtypes. The proof of concept was tested by capturing sEVs both non-specifically (unbiased) as well as via anti-CD9 capture probes (biased), and then profiling the expression levels of various surface proteins using the charge labelled antibodies. The method is the first of its kind, demonstrating an all-electrical and microchip based method that allows for unbiased analysis of sEV membrane protein expression, comparison of expression levels in different sEV subsets, and fractional estimation of different sEV sub-populations. These results were also validated in parallel using a single-sEV fluorescence technique.

Automated summary

Due to the high heterogeneity in membrane protein expression of small extracellular vesicles (sEVs), traditional antibody-based capture methods have limitations for expression analysis. In this study, an all-electric and microchip-based method was developed for unbiased analysis of sEV membrane protein expression and comparison of different sEV subsets. The method utilizes alternating charge labels to profile multiple surface proteins on captured sEVs. The results were validated using a fluorescence technique.