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Related Experiment Video

Updated: Aug 1, 2025

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier
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Sample Preparation Methods for Targeted Single-Cell Proteomics.

Azad Eshghi1, Xiaofeng Xie2, Darryl Hardie1

  • 1University of Victoria - Genome BC Proteomics Centre, Victoria, British Columbia V8Z 5N3, Canada.

Journal of Proteome Research
|April 24, 2023
PubMed
Summary
This summary is machine-generated.

Comparing cell isolation and proteomic sample preparation methods for single-cell analysis, this study found the CellenONE device best for quantifying hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes.

Keywords:
HbA1cSingle-cell proteomicscarboxymethyl hemoglobinglycated hemoglobinhemoglobinone-potquantitativered blood celltargeted

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Area of Science:

  • Proteomics
  • Cell Biology
  • Analytical Chemistry

Background:

  • Accurate quantification of proteins like hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes is crucial for diagnosing and monitoring various health conditions.
  • Single-cell analysis offers high resolution for studying biological heterogeneity, but requires optimized cell isolation and sample preparation techniques.

Purpose of the Study:

  • To compare the efficacy of three distinct cell isolation methods combined with two proteomic sample preparation strategies for single-cell and near-single-cell analysis of Hb and gly-Hb in erythrocytes.
  • To identify the most reliable method for quantifying protein content at the single-cell level and assessing protein heterogeneity within red blood cell populations.

Main Methods:

  • Three cell isolation techniques were evaluated: Fluorescence-Activated Cell Sorting (FACS) with automated preparation in one-pot (autoPOTS), limited dilution microscopy with a novel rapid one-pot method, and CellenONE-based isolation with the same rapid one-pot method.
  • Targeted mass spectrometry and stable isotope-labeled standard peptides were employed to quantify Hb and gly-Hb in isolated erythrocytes.
  • The study utilized whole blood from N=10 donors, analyzing both single red blood cells (RBCs) and small populations (5-25 RBCs).

Main Results:

  • The CellenONE device consistently isolated single-cells, enabling accurate Hb quantification (540-660 amol/RBC), comparable to the calculated reference range.
  • FACS and limited dilution isolated single-digit cell numbers, suitable for quantifying cytomegalovirus (CMV)-Hb heterogeneity but less precise for absolute quantification.
  • Analysis of 5-25 RBCs provided a more efficient alternative to single-cell analysis for measuring protein heterogeneity, revealing unique multimodal distributions for each individual.

Conclusions:

  • The CellenONE system is the most effective method for routine single-cell isolation and precise Hb quantification in erythrocytes.
  • While FACS and limited dilution can assess heterogeneity, analyzing small cell populations (5-25 RBCs) offers a practical and efficient approach to study protein heterogeneity and individual variability.