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

John R Thorup1, Sarah A Johnston2, Moe Haines1

  • 1Broad Institute of MIT and Harvard, Cambridge, MA, USA.

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|August 12, 2025
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Summary
This summary is machine-generated.

Optimized surface proteomics workflows accurately identify cell surface proteins (CSPs) from low-input and cryopreserved samples, enabling biomarker discovery in translational research.

Keywords:
Cell surface proteomicsN-glycoproteomeProteomics

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Area of Science:

  • Proteomics
  • Cell Biology
  • Biomarker Discovery

Background:

  • Cell surface proteins (CSPs) are crucial for cellular functions and are key targets for diagnostics and therapeutics.
  • Analyzing CSPs from limited or cryopreserved clinical samples is challenging due to technical limitations like poor membrane integrity and high background noise.

Purpose of the Study:

  • To optimize and compare two surface enrichment strategies for cell surface protein analysis.
  • To evaluate these methods on low-input samples, including fresh, cryopreserved, and dissociated tissues.
  • To enable robust surfaceome characterization in translational research settings.

Main Methods:

  • Systematic comparison of oxidation-based N-glycoprotein capture and WGA-HRP proximity labeling.
  • Application to low-input cell lines (A549, KMS-12-BM) and tissue samples (endometrium).
  • Validation of reproducibility between fresh and cryopreserved samples.

Main Results:

  • N-glycopeptide capture showed higher specificity in low-input scenarios; WGA-HRP identified complementary CSPs.
  • Over 700 CSPs identified in total, with ~175 unique to each protocol.
  • Both methods demonstrated high reproducibility (Pearson correlation > 0.9) and detected EGFR internalization.
  • Successful CSP profiling from <2 million cells in cryopreserved, enzymatically dissociated endometrium.

Conclusions:

  • Optimized workflows provide robust cell surfaceome characterization for low-input and cryopreserved samples.
  • These methods overcome common limitations in translational research, facilitating biomarker discovery and patient stratification.
  • The study validates complementary surface enrichment strategies for diverse clinical sample types.