MassIVE MSV000098694

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Microscaled Cell Surface Proteomics for Cryo-preserved Cells and Tissue Samples

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Description

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. 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. 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. 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. [doi:10.25345/C5BG2HP68] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Cell Surface Proteomics ; DatasetType:Proteomics

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Principal Investigators:
(in alphabetical order) 		Steven A. Carr, Broad Institute of MIT and Harvard, United States
Submitting User: malpap1
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Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

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Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

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Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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