{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:32:27Z","timestamp":1785407547065,"version":"3.56.0"},"posted":{"date-parts":[[2025,7,18]]},"group-title":"Cell Biology","reference-count":29,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,18]],"date-time":"2025-07-18T00:00:00Z","timestamp":1752796800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["U24CA270823"],"award-info":[{"award-number":["U24CA270823"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["U01CA271402"],"award-info":[{"award-number":["U01CA271402"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Dr Miriam and Sheldon G. Adelson Medical Research Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,20]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Cell surface proteins (CSPs) regulate key cellular functions and represent valuable targets for diagnostics and therapeutics. Despite advances in proteomic workflows, CSP analysis from cryopreserved or low-input clinical samples remains limited by technical constraints, including reduced membrane integrity, inefficient labeling, and high background. To address these challenges, we optimized and benchmarked two complementary surface enrichment strategies compatible with low-input applications (fewer than 1 million cells) and real-world sample types, including fresh, viably cryopreserved, and dissociated solid tissues.<\/jats:p>\n                <jats:p>We systematically compared oxidation-based N-glycoprotein capture and WGA-HRP-mediated proximity labeling across a range of input amounts using both solid tumor (A549) and hematologic cancer (KMS-12-BM) cell lines. The N-glycopeptide method yielded superior specificity in low-input contexts, while WGA-HRP captured complementary CSP subsets. Together, the methods identified more than 700 CSPs, with approximately 175 unique identifications per protocol. Both workflows detected dynamic EGFR internalization following EGF stimulation and maintained high reproducibility (Pearson correlation greater than 0.9) between fresh and cryopreserved preparations.<\/jats:p>\n                <jats:p>To extend these findings to tissue-derived samples, we optimized dissociation protocols for healthy endometrium and applied the N-glycopeptide method to cryopreserved dissociated endometrium from three healthy donors. Enzymatic dissociation enabled accurate CSP profiling from fewer than 1 to 2 million cells.<\/jats:p>\n                <jats:p>This study provides a systematic comparison of two leading surface proteomics approaches, validates their performance on cryopreserved and low-input specimens, and demonstrates applicability to clinically relevant tissues. Our optimized workflows enable robust surfaceome characterization in translational settings where sample quantity and preservation methods are often limiting, opening new avenues for biomarker discovery and patient stratification.<\/jats:p>","DOI":"10.1101\/2025.07.18.664488","type":"posted-content","created":{"date-parts":[[2025,7,19]],"date-time":"2025-07-19T00:50:11Z","timestamp":1752886211000},"source":"Crossref","is-referenced-by-count":0,"title":["Microscaled Cell Surface Proteomics for Cryo-preserved Cells and Tissue Samples"],"prefix":"10.64898","author":[{"given":"John R.","family":"Thorup","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah A.","family":"Johnston","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moe","family":"Haines","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edwin","family":"Sedodo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathleen","family":"O\u2019Neill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6912-6977","authenticated-orcid":false,"given":"Ronald","family":"Drapkin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Namrata D.","family":"Udeshi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael A.","family":"Gillette","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven A.","family":"Carr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6040-4854","authenticated-orcid":false,"given":"Shankha","family":"Satpathy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025072301250588000_2025.07.18.664488v2.1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0121314"},{"key":"2025072301250588000_2025.07.18.664488v2.2","doi-asserted-by":"crossref","first-page":"864","DOI":"10.1038\/nbt0909-864a","article-title":"Erratum: Corrigendum: Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins","volume":"27","year":"2009","journal-title":"Nat. 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