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Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuick^TM Isolation.

ABSTRACT: Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuick^TM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuick^TM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuick^TM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

SUBMITTER: Gemoll T

PROVIDER: S-EPMC7290673 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tissue factor (TF) is the main initiator of coagulation and procoagulant phospholipids (PPL) are key components in promoting coagulation activity in blood. Both TF and PPL may be presented on the surface of extracellular vesicles (EVs), thus contributing to their procoagulant activity. These EVs may constitute a substantial part of pathological hypercoagulability that is responsible for triggering a higher risk of thrombosis in certain patients. The aim of this study was to describe a model system for the isolation of EVs required for investigating their effect on coagulation. Differential ultracentrifugation (DUC) with and without a single washing step was used to isolate and evaluate the procoagulant capacity of EVs from healthy volunteers through analysis of thrombin generation and PPL activity. Ultracentrifugation at 20,000 × g and 100,000 × g resulted in pellets containing larger vesicles and smaller vesicles, respectively. Isolation yield of particle concentration was assessed by nanoparticle tracking analysis. Immunoelectron microscopy and western blotting revealed vesicles positive for the commonly used EV-marker CD9. Plasma proteins and lipoproteins were co-isolated with the EVs; however, application of a washing step clearly diminished the amount of contaminants. The isolated EVs were capable of enhancing thrombin generation, mainly due to PPL predominantly present in pellets from 20,000 × g centrifugation, and correlated with the activity measured by a PPL activity assay. Thus, DUC was proficient for the isolation of EVs with minimal contamination from plasma proteins and lipoproteins, and the setup can be used to study EV-associated procoagulant activity. This may be useful in determining the procoagulant activity of EVs in patients at potentially increased risk of developing thrombosis, e.g. cancer patients. Abbreviations: TF: Tissue factor; PL: Phospholipids; EVs: Extracellular vesicles; FXa: Activated coagulation factor X; TGA: Thrombin generation assay; PPL: Procoagulant phospholipids; DUC: Differential ultracentrifugation; NTA: Nanoparticle tracking analysis; TEM: Transmission electron microscopy; SPP: Standard pool plasma; CTI: Corn trypsin inhibitor; 20K: 20,000 × g; 100K: 100,000 × g; FVIII: Coagulation factor VIII.

Project description:BackgroundCirculating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins and plasma proteins. Previous studies suggested combinations of different sEVs isolation methods could improve the yield and purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC), and their combination in a proteomic perspective.ResultsPlasma samples were collected from healthy individuals, and sEVs were separated by one-step SEC, one-step UC, and combining SEC with UC, respectively. Here we exhibited that the purity of sEVs was improved by SEC in contrast to traditional UC. Furthermore, by conducting a SEC procedure followed by UC, we separated sEVs with the highest purity. In the proteomic analysis, 992 protein species were identified in the plasma sEVs isolated by our novel separation method, of which several proteins are sEVs-associated proteins but hitherto never been identified in the previous studies and database, much more than plasma sEVs isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, plasma sEVs isolated by the new procedure kept 584 previously identified sEVs-associated proteins and 360 other proteins that have not been detected before. Detailed analysis suggested that more kinds of sEVs biomarkers, such as CD9, ALIX, and FLOT1, could be identified in plasma sEVs isolated by the novel isolation method as compared to one-step UC/SEC. Furthermore, the lower abundance ranks of common contaminants, such as lipoproteins and IgG chains, in the sEVs fractions obtained by our new method as compared to one-step UC/SEC also demonstrated the purity of sEVs had been improved.ConclusionsCombining SEC with UC could significantly improve the performance of mass spectrometry-based proteomic profiling in analyzing plasma-derived sEVs.

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Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuick^TM Isolation.

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Dataset Information

Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation.

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OmicsDI is part of the ELIXIR infrastructure

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Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuick^TM Isolation.