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Automated Proteomics Sample Preparation of Phosphatidylserine-Positive Extracellular Vesicles from Human Body Fluids.

ABSTRACT: Extracellular vesicles (EVs) are ubiquitously secreted by almost every cell type and are present in all body fluids. Blood-derived EVs can be used as a promising source for biomarker monitoring in disease. EV proteomics is currently being analyzed in clinical specimens. However, their EV proteomics preparation methods are limited in throughput for human subjects. Here, we introduced a novel automated EV isolation and sample preparation method using a magnetic particle processing robot for automated 96-well processing of magnetic particles for EV proteomics analysis that can be started with a low volume of multiple clinical samples. The automation of EV purification reduced the coefficient of variation of protein quantification from 3.5 to 2.2% compared with manual purification, enabling the quantification of 1120 proteins in 1 h of MS analysis. This automated proteomics EV sample preparation is attractive for processing large cohort samples for biomarker development, validation, and routine testing.

SUBMITTER: Muraoka S

PROVIDER: S-EPMC9670285 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Automated Proteomics Sample Preparation of Phosphatidylserine-Positive Extracellular Vesicles from Human Body Fluids.

Muraoka Satoshi S Hirano Masayo M Isoyama Junko J Ishida Mimiko M Tomonaga Takeshi T Adachi Jun J

ACS omega 20221103 45

Extracellular vesicles (EVs) are ubiquitously secreted by almost every cell type and are present in all body fluids. Blood-derived EVs can be used as a promising source for biomarker monitoring in disease. EV proteomics is currently being analyzed in clinical specimens. However, their EV proteomics preparation methods are limited in throughput for human subjects. Here, we introduced a novel automated EV isolation and sample preparation method using a magnetic particle processing robot for automa ...[more]

PMID: 36406491

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Project description:Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

Project description:Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze-thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated. Plasma is the most commonly studied body fluid in EV research. We mainly focused on EVs originating from platelets and erythrocytes and investigated the behaviour of these 2 types of EVs independently as well as in plasma samples of healthy subjects. EVs in urine and saliva were also studied for comparison. All samples were analysed simultaneously before and after freeze-thawing by resistive pulse sensing, nanoparticle tracking analysis, conventional flow cytometry (FCM) and transmission (scanning) electron microscopy. Our main finding is that the effect of centrifugation markedly depends on the cellular origin of EVs. Whereas erythrocyte EVs remain present as single EVs after centrifugation, platelet EVs form aggregates, which affect their measured concentration in plasma. Single erythrocyte and platelet EVs are present mainly in the range of 100-200 nm, far below the lower limit of what can be measured by conventional FCM. Furthermore, the effects of single freeze-thaw cycle, time delay between blood collection and plasma preparation up to 1 hour and storage up to 1 year are insignificant (p>0.05) on the measured concentration and diameter of EVs from erythrocyte and platelet concentrates and EVs in plasma, urine and saliva. In conclusion, in standard protocols for EV studies, centrifugation to isolate EVs from collected body fluids should be avoided. Freezing and storage of collected body fluids, albeit their insignificant effects, should be performed identically for comparative EV studies and to create reliable biorepositories.

Dataset Information

Automated Proteomics Sample Preparation of Phosphatidylserine-Positive Extracellular Vesicles from Human Body Fluids.

Publications

Automated Proteomics Sample Preparation of Phosphatidylserine-Positive Extracellular Vesicles from Human Body Fluids.

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