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Improved Surfactant-Assisted One-Pot Sample Preparation for Robust Convenient Single-Cell Proteomics.

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Updated: Jan 13, 2026

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier
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Improved surfactant-assisted one-pot sample preparation for robust convenient single cell proteomics.

Reta Birhanu Kitata, Zhangyang Xu, Nadia Bayou

    Biorxiv : the Preprint Server for Biology
    |January 7, 2026
    PubMed
    Summary

    A new single-cell proteomics (SCP) method, iSOP, uses a low microliter processing volume for robust and convenient analysis. This improved surfactant-assisted one-pot processing enhances cellular heterogeneity characterization.

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    Area of Science:

    • Proteomics
    • Cell Biology
    • Analytical Chemistry

    Background:

    • Single-cell proteomics (SCP) technologies have advanced cellular heterogeneity characterization.
    • Current SCP methods often use small volumes (sub-µL to 1 µL), limiting accessibility and robustness due to evaporation.
    • Specialized devices or 384-well plates with water addition are common but have drawbacks.

    Purpose of the Study:

    • To develop a robust and convenient SCP method using a low microliter processing volume.
    • To overcome limitations of existing SCP techniques regarding accessibility and analytical robustness.
    • To enable cost-effective, routine quantitative SCP analysis.

    Main Methods:

    • Developed iSOP (improved Surfactant-assisted One-Pot processing) using a 384-well plate with tight sealing.
    • Optimized single-cell processing conditions, selecting a 3 µL processing volume with trypsin and Lys-C enzymes.
    • Utilized a standard liquid chromatography-mass spectrometry (LC-MS) platform for analysis.

    Main Results:

    • iSOP-MS detected and quantified approximately 1,200-1,800 protein groups from single HeLa or MCF7 cells.
    • Analysis of neuroblastoma cell lines (BE2-C and SK-N-SH) identified an average of ~1,700 and ~2,050 protein groups, respectively.
    • Demonstrated precise characterization of cellular heterogeneity within and between distinct cell types.

    Conclusions:

    • iSOP provides a robust, convenient, and cost-effective solution for routine quantitative single-cell proteomics.
    • The method overcomes evaporation issues associated with low-volume SCP techniques.
    • iSOP facilitates detailed cellular heterogeneity analysis, advancing cell biology research.