High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time

High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time | 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 Article High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time Ryan Kelly, Chao Wang, Hsien-Jung Lin, Siqi Huang, Garrett Haynie, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8042176/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Mass spectrometry (MS)-based single-cell proteomics (SCP) enables proteome-wide analysis at single-cell resolution, offering insights into cellular heterogeneity, biological processes, and disease mechanisms. However, conventional nanoLC-MS workflows are constrained by long gradients and low throughput, limiting their application to large cohorts. Here, we present a multicolumn nanoLC–MS platform that achieves 5-minute separation windows with 100% duty cycles at ~ 100 nL/min, enabling the analysis of up to 288 single cells per day with minimal additional hardware. The system provides stable peptide separation, negligible carryover, and robust retention-time reproducibility across 2,000 consecutive injections. Over the course of this study, we successfully analyzed more than 4,000 samples at nearly 288 samples per day (SPD) throughput. The platform identified ~ 4,400 proteins per 250 pg digest injection and an average of ~ 3,200 proteins per single HeLa cell with maxima exceeding 4,300, which is comparable to state-of-the-art longer-gradient workflows. Quantitative benchmarking with mixed-species standards confirmed accurate measurements across ~ 6,000 proteins. The workflow distinguished proteome profiles across hundreds of single cells, and profiling of RAW264.7 macrophages revealed LPS-induced markers and macrophage activation pathways. Together, these results establish a robust and scalable platform for high-throughput SCP, demonstrating the feasibility of thousands of single-cell analyses within a single study while maintaining deep proteome coverage and biological interpretability. Biological sciences/Biochemistry/Proteomics Biological sciences/Biological techniques/Analytical biochemistry/Liquid chromatography Full Text Additional Declarations Yes there is potential Competing Interest. TT, XX and RTK have a financial interest in MicrOmics Technologies, which provided electrospray emitters used in this study. Supplementary Files SupplementaryInformationsubmit.docx Supplementary Information - Supplementary Figures 1 and 2 and Supplementary Table AccesstoMSrawdata.docx Access to MS raw data Supplementaryvideo1.mp4 Supplementary video 1 - Sample traveling through Channel 1 Supplementaryvideo2.mp4 Supplementary video 2 - Sample traveling through Channel 2 Supplementaryvideo3.mp4 Supplementary video 3 - Sample traveling through both channels Cite Share Download PDF Status: Under Review Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8042176","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":546071730,"identity":"147557ac-051e-431f-807e-3e84ea45b586","order_by":0,"name":"Ryan Kelly","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYNCCAwwyfAzMx2Ac4rTwsDGwpZGshceMOC3mM5KfPWA4Y8PDJpHz7TFvG4Mc340E/FpkbqSZGzDcSANqyd1uDNRiLElIi4REgpkEw4fDIC3bpIFaEjcQ1pL+DajlP8hhz0Ba6onQkgO05cYBkBY2kJYEA4JaeN6USSScSeZh43lmbjjnnIThzDMPCGhhT98m8eGYnRw/OzDo3pTZyPMdJ2ALgwBQQQKUwcTDIEFAOQjwH0AwGH8QoWEUjIJRMApGHgAA1uk+9qmCZ1wAAAAASUVORK5CYII=","orcid":"","institution":"Brigham Young University","correspondingAuthor":true,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Kelly","suffix":""},{"id":546071731,"identity":"4a8148bb-e63e-4552-a31e-86fd4fc7ca69","order_by":1,"name":"Chao Wang","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Wang","suffix":""},{"id":546071732,"identity":"6b392eb6-6c58-4f0d-aafa-a8760c8a3d39","order_by":2,"name":"Hsien-Jung Lin","email":"","orcid":"https://orcid.org/0000-0003-4704-3692","institution":"Brigham Young University","correspondingAuthor":false,"prefix":"","firstName":"Hsien-Jung","middleName":"","lastName":"Lin","suffix":""},{"id":546071733,"identity":"33785102-120e-430f-aef5-fc748c5ced30","order_by":3,"name":"Siqi Huang","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Huang","suffix":""},{"id":546071734,"identity":"f262c118-1d2e-45de-9485-a52da45cb08b","order_by":4,"name":"Garrett Haynie","email":"","orcid":"https://orcid.org/0009-0004-4547-5330","institution":"Brigham Young University","correspondingAuthor":false,"prefix":"","firstName":"Garrett","middleName":"","lastName":"Haynie","suffix":""},{"id":546071735,"identity":"c4de9728-72bd-4b98-a4f5-4098e26c2065","order_by":5,"name":"Kenneth Triggs","email":"","orcid":"https://orcid.org/0009-0001-5091-7058","institution":"Brigham Young University","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Triggs","suffix":""},{"id":546071736,"identity":"4cc0611e-ab84-4065-85e2-1154bc1d1fbf","order_by":6,"name":"YenJou Chang","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"prefix":"","firstName":"YenJou","middleName":"","lastName":"Chang","suffix":""},{"id":546071737,"identity":"ae713297-cf96-4324-8722-b3f6efb08e33","order_by":7,"name":"David Hansen","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Hansen","suffix":""},{"id":546071738,"identity":"f16400be-fd29-43a6-9838-f79ca3345c06","order_by":8,"name":"Thy Truong","email":"","orcid":"","institution":"MicrOmics Technologies","correspondingAuthor":false,"prefix":"","firstName":"Thy","middleName":"","lastName":"Truong","suffix":""},{"id":546071739,"identity":"857b1825-2a5a-4700-992d-54a2702510d9","order_by":9,"name":"Xiaofeng Xie","email":"","orcid":"","institution":"MicrOmics Technologies","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-11-05 23:50:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8042176/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8042176/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96204442,"identity":"a4d6f10c-043f-43a4-a30a-7fac66e0bd22","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4828834,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptMulticolumnsubmit.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/6a8a087e671118b36a6399f7.docx"},{"id":96252985,"identity":"6b307b86-1c50-410e-baf2-c87a20a41cbc","added_by":"auto","created_at":"2025-11-19 07:41:46","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11122,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2588897.json","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/8a76c5774f5b999848f0bf6a.json"},{"id":96204439,"identity":"01b82a40-e306-4763-8093-5b82b3585d76","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2366320,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationsubmit.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/b6b1a8cf763b9b20d9f171ea.docx"},{"id":96204440,"identity":"05df3dff-939a-4493-b53e-f856577a636a","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150361,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25888970enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/5f027ab826f6aa680720d8e5.xml"},{"id":96250382,"identity":"d5c91db3-a032-4f9d-a0f0-248f38cbc88d","added_by":"auto","created_at":"2025-11-19 07:38:14","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":799434,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/0a32305c673e50425c80fa1f.png"},{"id":96252411,"identity":"a5943478-1332-4399-aa8b-6174027f5615","added_by":"auto","created_at":"2025-11-19 07:40:54","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1010779,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/90d8b56850bd870588c1a2dc.png"},{"id":96204450,"identity":"07211aee-1c0e-46e6-8282-57610266ee95","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82996,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/5d94601729e386810470047e.png"},{"id":96204451,"identity":"cc3ae3b5-e118-4f44-ab11-8b5a78779e44","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96866,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/9060a4ed69cfef2f3c200859.png"},{"id":96252311,"identity":"41a1e684-6ec9-4d32-a0ee-6281aa7d8493","added_by":"auto","created_at":"2025-11-19 07:40:46","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96281,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/5ae8a22ec5484e9ec8b24ec6.png"},{"id":96204458,"identity":"7092fe88-24c5-4cbe-8add-088e9a1b10ac","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":253987,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/4f19d715e6580ea5a8cb77b9.png"},{"id":96204464,"identity":"2e17a7dc-8717-4b9a-80c4-245db52befe6","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":215585,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/163b01286c599ec9abb1833e.png"},{"id":96250360,"identity":"a8c186cf-c47b-4495-ae21-fe1a159a4d1d","added_by":"auto","created_at":"2025-11-19 07:38:12","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86930,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/3dd924bc43b92a0635e2195b.png"},{"id":96204449,"identity":"1ca9fa3b-d223-4472-82ec-5c45aa801e6b","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105917,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/3195e43859023af41a396725.png"},{"id":96204461,"identity":"638b3842-cd49-495b-b200-f63f035045e5","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192732,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/ce1a5c1dd02f966ccbb39b62.png"},{"id":96204475,"identity":"a29795fc-2d6a-4a45-bc68-7ae127a9a102","added_by":"auto","created_at":"2025-11-18 17:00:31","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17452,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/f434251250388ad653a8290e.png"},{"id":96204462,"identity":"23533e54-5cbb-44af-be77-8a93789a9ef8","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22526,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/c5382f6bbf5023ded419b57b.png"},{"id":96204480,"identity":"27315cba-ba4f-46f6-b959-6392e40acbdd","added_by":"auto","created_at":"2025-11-18 17:00:31","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22742,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/59f24d67231da8f2b6a983b1.png"},{"id":96204446,"identity":"46c24970-b905-4691-ae95-6a977e167bc7","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60506,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/39a876fd0079c85197a59a08.png"},{"id":96204474,"identity":"dd0c92cc-62e8-4658-915d-0a7cb6404e43","added_by":"auto","created_at":"2025-11-18 17:00:31","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49012,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/30f35e56c99a941c7c8ddb4c.png"},{"id":96204469,"identity":"fabcccc8-22a3-45de-9037-53359e00324f","added_by":"auto","created_at":"2025-11-18 17:00:31","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24937,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/f3ea132a09c860e3d215bd97.png"},{"id":96204478,"identity":"25204244-cd40-46a1-9313-b874fb14e69e","added_by":"auto","created_at":"2025-11-18 17:00:31","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148668,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25888970structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/a1614c76576ef5cc8747bd9c.xml"},{"id":96252425,"identity":"6132cbae-209e-4442-844f-88ca0c11172f","added_by":"auto","created_at":"2025-11-19 07:40:56","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161167,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/ae82a211c2e1e0fb140ece8f.html"},{"id":96257071,"identity":"88bf5275-5287-40a4-8a3c-8e4d6b9cfe05","added_by":"auto","created_at":"2025-11-19 07:51:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1328575,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptMulticolumnsubmit.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1_covered_396636cc-c6e0-4cc7-b44e-3737125a1965.pdf"},{"id":96204445,"identity":"342ddcf7-ecd3-4c60-ae30-855deffd3121","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2366320,"visible":true,"origin":"","legend":"Supplementary Information - Supplementary Figures 1 and 2 and Supplementary Table","description":"","filename":"SupplementaryInformationsubmit.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/9d227aaaa730c46e7e4e2490.docx"},{"id":96204438,"identity":"e2c0d723-ca36-4a21-b9db-698c5ae5c8bf","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13884,"visible":true,"origin":"","legend":"Access to MS raw data","description":"","filename":"AccesstoMSrawdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/956cf3dae9df60129bedbe58.docx"},{"id":96204452,"identity":"4910b86c-f96c-4958-9360-7b68c452ef0d","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8832516,"visible":true,"origin":"","legend":"Supplementary video 1 - Sample traveling through Channel 1","description":"","filename":"Supplementaryvideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/323ceda6ee1972a851edf051.mp4"},{"id":96204455,"identity":"b1578aee-5247-417b-837e-8710d20ac264","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8788868,"visible":true,"origin":"","legend":"Supplementary video 2 - Sample traveling through Channel 2","description":"","filename":"Supplementaryvideo2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/5f055094fe81b155c54bb5f2.mp4"},{"id":96204465,"identity":"575c7eff-a33d-4269-bba3-8bcde5bd4aa0","added_by":"auto","created_at":"2025-11-18 17:00:30","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16662870,"visible":true,"origin":"","legend":"Supplementary video 3 - Sample traveling through both channels","description":"","filename":"Supplementaryvideo3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8042176/v1/4baab1a4690eff581ca8dda1.mp4"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nTT, XX and RTK have a financial interest in MicrOmics Technologies, which provided electrospray emitters used in this study.","formattedTitle":"High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8042176/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8042176/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMass spectrometry (MS)-based single-cell proteomics (SCP) enables proteome-wide analysis at single-cell resolution, offering insights into cellular heterogeneity, biological processes, and disease mechanisms. However, conventional nanoLC-MS workflows are constrained by long gradients and low throughput, limiting their application to large cohorts. Here, we present a multicolumn nanoLC\u0026ndash;MS platform that achieves 5-minute separation windows with 100% duty cycles at ~\u0026thinsp;100 nL/min, enabling the analysis of up to 288 single cells per day with minimal additional hardware. The system provides stable peptide separation, negligible carryover, and robust retention-time reproducibility across 2,000 consecutive injections. Over the course of this study, we successfully analyzed more than 4,000 samples at nearly 288 samples per day (SPD) throughput. The platform identified\u0026thinsp;~\u0026thinsp;4,400 proteins per 250 pg digest injection and an average of ~\u0026thinsp;3,200 proteins per single HeLa cell with maxima exceeding 4,300, which is comparable to state-of-the-art longer-gradient workflows. Quantitative benchmarking with mixed-species standards confirmed accurate measurements across ~\u0026thinsp;6,000 proteins. The workflow distinguished proteome profiles across hundreds of single cells, and profiling of RAW264.7 macrophages revealed LPS-induced markers and macrophage activation pathways. Together, these results establish a robust and scalable platform for high-throughput SCP, demonstrating the feasibility of thousands of single-cell analyses within a single study while maintaining deep proteome coverage and biological interpretability.\u003c/p\u003e","manuscriptTitle":"High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 17:00:25","doi":"10.21203/rs.3.rs-8042176/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f347918c-29cb-4e2f-907d-20cfa483d20a","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58088220,"name":"Biological sciences/Biochemistry/Proteomics"},{"id":58088221,"name":"Biological sciences/Biological techniques/Analytical biochemistry/Liquid chromatography"}],"tags":[],"updatedAt":"2025-12-01T18:16:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 17:00:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8042176","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8042176","identity":"rs-8042176","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}