Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation
Abstract:Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusio… Show more
“…Since CD63, CD9, FLOT1 and HSC70 are enriched in Vn96 samples compared to ME and Scr samples, these findings suggest that Vn96 does not merely sediment circulating proteins at 17,000 × g under these conditions, but rather enriches for these EV-specific proteins. Recent publications have also demonstrated co-isolation of lipoproteins (ApoB and ApoA1) with both UCF and Vn96 15 , 27 , suggesting that the proteomic profile of material isolated by Vn96 may be similar to UCF. Figure 2 Characterization of plasma EVs isolated by UCF, scUCF and Vn96 methods, all from the same donor plasma (n = 3).…”
Extracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and ‘omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.
“…Since CD63, CD9, FLOT1 and HSC70 are enriched in Vn96 samples compared to ME and Scr samples, these findings suggest that Vn96 does not merely sediment circulating proteins at 17,000 × g under these conditions, but rather enriches for these EV-specific proteins. Recent publications have also demonstrated co-isolation of lipoproteins (ApoB and ApoA1) with both UCF and Vn96 15 , 27 , suggesting that the proteomic profile of material isolated by Vn96 may be similar to UCF. Figure 2 Characterization of plasma EVs isolated by UCF, scUCF and Vn96 methods, all from the same donor plasma (n = 3).…”
Extracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and ‘omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.
“…Common problems when isolating EVs from blood plasma and other biological fluids are co-isolation of albumin, lipoproteins and other non-EV proteins [ 40 ]. In a recent study, EV isolation from plasma using SEC and ultracentrifugation was compared.…”
Section: Discussionmentioning
confidence: 99%
“…In a recent study, EV isolation from plasma using SEC and ultracentrifugation was compared. It was shown that SEC isolation resulted in higher yield and less contamination of albumin, but more lipoproteins compared to ultracentrifugation due to the overlap in size [ 40 ]. Similar to the blood plasma, lymphatic drainage fluid is a complex fluid which makes the isolation of pure EVs challenging.…”
Background
Breast cancer is the most common cancer, and the leading cause of cancer-related deaths, among females world-wide. Recent research suggests that extracellular vesicles (EVs) play a major role in the development of breast cancer metastasis. Axillary lymph node dissection (ALND) is a procedure in patients with known lymph node metastases, and after surgery large amounts of serous fluid are produced from the axilla. The overall aim was to isolate and characterize EVs from axillary serous fluid, and more specifically to determine if potential breast cancer biomarkers could be identified.
Methods
Lymphatic drain fluid was collected from 7 patients with breast cancer the day after ALND. EVs were isolated using size exclusion chromatography, quantified and detected by nanoparticle tracking analysis, electron microscopy, nano flow cytometry and western blot. The expression of 37 EV surface proteins was evaluated by flow cytometry using the MACSPlex Exosome kit.
Results
Lymphatic drainage exudate retrieved after surgery from all 7 patients contained EVs. The isolated EVs were positive for the typical EV markers CD9, CD63, CD81 and Flotillin-1 while albumin was absent, indicating low contamination from blood proteins. In total, 24 different EV surface proteins were detected. Eleven of those proteins were detected in all patients, including the common EV markers CD9, CD63 and CD81, cancer-related markers CD24, CD29, CD44 and CD146, platelet markers CD41b, CD42a and CD62p as well as HLA-DR/DP/DQ. Furthermore, CD29 and CD146 were enriched in Her2+ patients compared to patients with Her2- tumors.
Conclusions
Lymphatic drainage exudate retrieved from breast cancer patients after surgery contains EVs that can be isolated using SEC isolation. The EVs have several cancer-related markers including CD24, CD29, CD44 and CD146, proteins of potential interest as biomarkers as well as to increase the understanding of the mechanisms of cancer biology.
“…In addition, Vn96 isolates EVs with reduced contamination from protein aggregates or lipoproteins, compared to other methods of EV isolation (i.e. ultracentrifugation) ( 57 ) . Briefly, 1 ml of EV-containing media was incubated with 40 μl (2.5 mg/ml) of Vn96 for 1 h, rotating, at room temperature.…”
Cell-derived extracellular vesicles (EVs) participate in cell-cell communication
via
transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily “monocyte-like” at Day 1 to primarily “macrophage-like” at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.
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