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Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics.

Junho Park1, Fengchao Yu2, James M Fulcher3

  • 1Department of Pharmacology, School of Medicine, CHA University, Seongnam-si, Gyeonggi-do, Seongnam 13488, Republic of Korea.

Analytical Chemistry
|January 13, 2023
PubMed
Summary
This summary is machine-generated.

This study introduces an improved mass spectrometry method for single-cell proteomics, enhancing protein quantification accuracy by using MS3 and a linear ion trap. The new approach increases proteome coverage and enables deeper insights into cellular processes like immune activation.

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Cell Biology

Background:

  • Single-cell proteomics demands high-throughput and sensitivity.
  • Isobaric labeling methods suffer from ratio compression, distorting quantification.
  • MS3-based quantification can mitigate ratio compression but often reduces proteome coverage.

Purpose of the Study:

  • To improve MS3-based single-cell proteomics for higher accuracy and coverage.
  • To address limitations of previous MS3 methods, including ion losses and long duty cycles.
  • To apply an optimized MS3 method for studying immune activation in single macrophages.

Main Methods:

  • Developed an improved MS acquisition method using a linear ion trap for reporter ion measurement in MS3-based single-cell proteomics.
  • Optimized the real-time search MS3 method.
  • Applied the method to analyze immune activation in 126 single macrophages.

Main Results:

  • The linear ion trap integration increased proteome coverage for single-cell peptides using the MS3 method.
  • Quantified over 1200 proteins in single cells with 50% nonmissing data and over 1000 proteins with 75% nonmissing data.
  • Identified limitations of low-resolution ion trap detectors and proposed solutions for multiplexed single-cell proteomics.

Conclusions:

  • The improved MS3 method enhances proteome coverage and quantification accuracy in single-cell proteomics.
  • This method provides a valuable tool for studying cellular heterogeneity and function, such as immune activation.
  • Further optimization is needed to overcome detector limitations for broader single-cell proteomic applications.