Micro size exclusion chromatography combined with a multiplex protein profiling method for extracellular vesicle protein detection from small sample volumes

Micro size exclusion chromatography combined with a multiplex protein profiling method for extracellular vesicle protein detection from small sample volumes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Micro size exclusion chromatography combined with a multiplex protein profiling method for extracellular vesicle protein detection from small sample volumes Li Sun, David Meckes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-125398/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version 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 live 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. General Cell Biology & Physiology Cancer Biology Immuno-PCR Multiplex Exosomes Protein profiling microvesicles extracellular vesicles Figures Figure 1 Figure 1 Figure 1 Figure 1 Full Text Supplementary Files Supp.InfoLS1129.docx GraphicalAbstract.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 24 Mar, 2021 Reviews received at journal 11 Mar, 2021 Reviews received at journal 19 Jan, 2021 Reviewers agreed at journal 30 Dec, 2020 Reviewers agreed at journal 27 Dec, 2020 Reviewers invited by journal 23 Dec, 2020 Editor assigned by journal 23 Dec, 2020 Editor invited by journal 17 Dec, 2020 Submission checks completed at journal 16 Dec, 2020 First submitted to journal 09 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-125398","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":6599532,"identity":"c19cc15f-18f0-49d9-805b-fc5d80fd9334","order_by":0,"name":"Li Sun","email":"","orcid":"","institution":"Florida State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Sun","suffix":""},{"id":6599533,"identity":"b828409a-f7f6-4f27-9ed2-918bac46d74c","order_by":1,"name":"David Meckes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYDCCA0DEA2KAiA8MDIwNzAdAXGbitDDOAGlhSyCshQGmhZmHGC18x88YHnjDsE2Ov+fw4c+2OTayDWzszyQYKqwTG3BokTyTY3BwDsNtY4mzbWnSudvSjBvYeMwkGM6k49RicCAt4TAPw+3EDfw8Zsy52w4nNsj3sEkwth3GreX8M7CW+g38/J8/W277nwh2GOM/PFpuJB8AaUkw4O1hkGbcdgCohcFMgrEBtxbJG48PHJxjcNtwxpljZpK925KN29h4jC0SjqUb49LCdz6x+cObitvy/D3Jjz/83GYn28/G/vDGhxprWVxaoM5DYrOBiAS8ykfBKBgFo2AUEAIAXlVcr8A2q8wAAAAASUVORK5CYII=","orcid":"","institution":"Florida State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Meckes","suffix":""}],"badges":[],"createdAt":"2020-12-09 21:59:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-125398/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-125398/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4361386,"identity":"59219f0c-8942-4090-95ba-babde0141668","added_by":"auto","created_at":"2020-12-18 15:13:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84332,"visible":true,"origin":"","legend":"Serum EV protein profiling by micro-SEC combined with TotalSeq antibody. (A) Fraction 3 and 4 collected from micro-SEC on human serum sample were tested with TotalSeq qPCR. Equal protein was blotted on the strip. Data were normalized to serum sample. (B) Equal volume of Fraction 1-5 from the micro-SEC were run on SDS-PAGE for immunoblot on EV markers. (C) Same fraction sample as in (B) were analyzed by TotalSeq qPCR also blotted by equal volume. Data were normalized to serum sample.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1/22650f45169ab7d5e30e7d3d.jpg"},{"id":4361383,"identity":"693b4152-097c-4e38-85b6-6003d7ff4ce7","added_by":"auto","created_at":"2020-12-18 15:13:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65216,"visible":true,"origin":"","legend":"Cell surface protein profiling with TotalSeq antibody. (A) Workflow for cell samples. (B) Overlaid histogram plot flow cytometry results of 293-WT, 293-CD63 and 293-CD63-CRISPR cell stain with CD63-PE or isotype control-PE antibody. Representative result of three independent samples. (C) Result on the same cell sample as in (B) with TotalSeq qPCR method. Data were normalized to wild type sample. (D) Gene expression level was tested from RNA isolated by Trizol from cell (X axis) or cleared supernatant (Y axis). Samples were showed in triplicate.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1/39b4f0300b4b89fa2d5c621d.jpg"},{"id":4361382,"identity":"08de2a59-5fa9-4e96-8014-24f0d8fff8a1","added_by":"auto","created_at":"2020-12-18 15:13:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60972,"visible":true,"origin":"","legend":"Micro-SEC method for rapid serum EV isolation. (A) 25 uL pre-cleared and diluted serum sample was loaded on micro-SEC column and fractions were collected. (B) EV concentration were measured by NTA and protein content were assayed by OD660 (left). The purity of EV samples were calculated by particle / μg protein. CD63 Direct ELISA was used to measure the EV peak in all the fractions. All the data were collected from two individual columns.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1/10102637f84ee1d26b6f1ef7.jpg"},{"id":4361381,"identity":"072572bd-cc29-4fa7-8f32-91fd47b65473","added_by":"auto","created_at":"2020-12-18 15:13:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101720,"visible":true,"origin":"","legend":"Protein sample profiling with TotalSeq antibody. (A) Basic work flow for detection of immobilized protein sample by TotalSeq-A antibody. Structure of conjugated oligo in TotalSeq-A antibody (bottom left), reprint from BioLegend website with permission. Size and comparison of pre-cut strip and PCR tube (bottom right). (B) Melting curve of different TotalSeq antibodies on EV protein samples in triplicate. (C) Intact (left upper) or lysised (left lower) #1 EV samples were tested with TotalSeq CD63 (green) and control mIgG (blue) antibody in Triplicate. Equal amount of BSA (triangle) was used as negative control. Ct values of CD63 (white) and control mouse IgG (gray) are overlaid in column chart (right) to show the difference. (D) Serial dilution of #1 EV sample was incubated with TotalSeq antibody CD63 (square), CD81 (triangle), EGFR (diamond) and control IgG (circle). Amplification curve (bottom) of CD63 on EV dilution (green) and BSA (blue). All experiments were performed in duplicate. (E) CD63 immunoblot result of EV and whole cell lysate. (F) TotalSeq qPCR result on the same samples as in (E). Data were normalized to wild type HEK293 samples.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1/eaa734cbeeaf2107dbf0f888.jpg"},{"id":13566156,"identity":"369951b2-6c30-4e59-a1d2-de73c8372da2","added_by":"auto","created_at":"2021-09-17 03:28:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1240776,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptScientificReports12920.pdf","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1_covered.pdf"},{"id":4361387,"identity":"d8190ab5-96d5-4eb6-a448-cbf0d6786c1f","added_by":"auto","created_at":"2020-12-18 15:13:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1185453,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptScientificReports12920.pdf","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1_stamped.pdf"},{"id":4361385,"identity":"670a0fc1-9290-4975-b103-3251d1efe35f","added_by":"auto","created_at":"2020-12-18 15:13:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":473637,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.InfoLS1129.docx","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1/7c04a3404f3b851c93d1a158.docx"},{"id":4361384,"identity":"bcf5968d-3de7-4e05-b55e-3d1aa9b9d3da","added_by":"auto","created_at":"2020-12-18 15:13:06","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36406,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-125398/v1/549f0460588d27188960938d.jpg"}],"financialInterests":"","formattedTitle":"Micro size exclusion chromatography combined with a multiplex protein profiling method for extracellular vesicle protein detection from small sample volumes","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-125398/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Immuno-PCR, Multiplex, Exosomes, Protein profiling, microvesicles, extracellular vesicles","lastPublishedDoi":"10.21203/rs.3.rs-125398/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-125398/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"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 live 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.","manuscriptTitle":"Micro size exclusion chromatography combined with a multiplex protein profiling method for extracellular vesicle protein detection from small sample volumes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-18 15:13:04","doi":"10.21203/rs.3.rs-125398/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-03-24T11:28:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-11T06:20:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-01-19T08:30:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2f422d04-31f2-4fb1-b707-5a140591c63e","date":"2020-12-30T17:24:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5902267a-c196-466e-861b-8b3bbea59dcf","date":"2020-12-27T06:50:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-12-23T20:03:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-23T19:04:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-17T07:08:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-16T16:59:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2020-12-09T21:50:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cb3e5735-b10c-442b-b808-8bc362fe18a4","owner":[],"postedDate":"December 18th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1529830,"name":"General Cell Biology \u0026 Physiology"},{"id":1529831,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2021-05-21T05:29:02+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-18 15:13:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-125398","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-125398","identity":"rs-125398","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}