Proteomics, Phosphoproteomics and Mirna Analysis of Circulating Extracellular Vesicles through Automated and High-Throughput Isolation

Abstract

Extracellular vesicles (EVs) play an important role in the diagnosis and treatment of diseases because of their rich molecular contents involved in intercellular communication, regulation, and other functions. With increasing efforts to move the field of EVs to clinical applications, the lack of a practical EV isolation method from circulating biofluids with high throughput and good reproducibility has become one of the biggest barriers. Here, we introduce a magnetic bead-based EV enrichment approach (EVrich) for automated and high-throughput processing of urine samples. Parallel enrichments can be performed in 96-well plates for downstream cargo analysis, including EV characterization, miRNA, proteomics, and phosphoproteomics analysis. We applied the instrument to a cohort of clinical urine samples to achieve reproducible identification of an average of 17,000 unique EV peptides and an average of 2800 EV proteins in each 1 mL urine sample. Quantitative phosphoproteomics revealed 186 unique phosphopeptides corresponding to 48 proteins that were significantly elevated in prostate cancer patients. Among them, multiple phosphoproteins were previously reported to associate with prostate cancer. Together, EVrich represents a universal, scalable, and simple platform for EV isolation, enabling downstream EV cargo analyses for a broad range of research and clinical applications.

Extracellular vesicle proteomics and phosphoproteomics identify pathways for increased risk in patients hospitalized with COVID-19 and type 2 diabetes mellitus

“…To characterize the protein composition of plasma EV preparations purified using Tymora’s non-antibody-based affinity EVtrap™ proprietary technology (designed to quantitatively capture membrane-bound vesicles including exosomes and validated in multiple peer-reviewed publications [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] ), we assessed the enrichment in exosomal proteins curated by the ExoCarta Database. EVs were highly enriched in exosomal proteins, including 87 of the top 100 Exocarta proteins and nanoparticle tracking analysis (NTA) demonstrated size distributions consistent with preparations enriched in small EVs ( Figure 1 A-E).…”

“…EV purification was conducted as described by Nunez Lopez and colleagues [19] , using EVtrap™ (Tymora Analytical, Lafayette, IN, USA), a non-antibody- bead-based affinity technology developed to specifically and quantitatively isolate EVs. The EVtrap™ technology has been further described and validated in detail elsewhere [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] . In short, frozen plasma samples were thawed and large debris removed by centrifugation at 2500×g for 10 min.…”

High Throughput Isolation and Data Independent Acquisition Mass Spectrometry (DIA-MS) of Urinary Extracellular Vesicles to Improve Prostate Cancer Diagnosis

“…However, no difference was found in terms of age ( p = 0.275), body mass index (BMI; p = 0.534), and prostate size ( p = 0.407) ( Supplementary Table S1 ). For the isolation process, we used EVTRAP beads corresponding to automated magnetic bead separation EVrich [ 11 , 20 ] to capture and isolate the EVs for 40 samples in the discovery cohort first. A batch of 1 mL urine samples was mixed with EVTRAP beads and loaded onto the first well for 30 min of incubation, and washed with the provided wash buffer once.…”

“…The EVrich system was used as described previously [ 11 ]. Firstly, the magnetic beads and urine were added to the predetermined 96-well plate according to the ratio of 20 μL: 1 mL and mixed for half an hour, by which magnetic beads had completed EVs capture.…”

“…However, for practical clinical applications, how to extract EVs with high throughput and reproducibility is an urgent challenge to be addressed. To solve this issue, in recent years, our group has developed an automatic extraction device [ 11 ] to complement our previously developed magnetic bead EVTRAP [ 12 ]. The device, named as EVrich, uses the principle of magnetic separation to distribute and elute EVs captured by magnetic beads, which allows us to extract EVs from multiple samples in parallel.…”