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Multiplex protein profiling method for extracellular vesicle protein detection.

ABSTRACT: Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.

SUBMITTER: Sun L

PROVIDER: S-EPMC8203679 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

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Multiplex protein profiling method for extracellular vesicle protein detection.

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