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Optimization and Modeling of Quadrupole Orbitrap Parameters for Sensitive Analysis toward Single-Cell Proteomics.

Bingyun Sun1, Jessica Rae Kovatch1, Albert Badiong1

  • 1Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Faculty of Health Science, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.

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Summary

Optimizing mass spectrometry (MS) parameters, specifically precursor ion isolation window and injection time, significantly enhances single-cell proteomics. This research reveals optimal settings for increased protein detection in trace samples.

Keywords:
LC-MS/MSOrbitrapion injection timemass isolation windowshotgun proteomicssingle-cell proteomicstandem mass spectrometrytrace proteomics

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Cellular Biology

Background:

  • Single-cell proteomics requires highly sensitive analysis due to limited sample amounts.
  • Mass spectrometry (MS) for single cells needs optimized instrumentation and parameters for sensitivity and throughput.
  • Unlike nucleic acids, proteins lack amplification potential, making MS parameter optimization critical.

Purpose of the Study:

  • To investigate the impact of precursor ion isolation window (IW) and maximum precursor ion injection time (ITmax) on single-cell shotgun proteomics.
  • To determine optimal MS parameters for enhanced peptide sequencing and protein detection in ultra-trace samples.
  • To improve proteomic depth for a better understanding of cellular functions.

Main Methods:

  • Experiments were conducted on an ultrahigh-field quadrupole Orbitrap (Q-Exactive HF).
  • Two key parameters, precursor ion isolation window (IW) and maximum precursor ion injection time (ITmax), were systematically varied.
  • Data were analyzed using modeling to identify optimal settings for different sample quantities.

Main Results:

  • An isolation window (IW) of 4.0 Th was found to be optimal for sample quantities from 1 ng to 100 ng.
  • A sample-quantity dependent maximum precursor ion injection time (ITmax) exceeding 250 ms was optimal for 1-ng samples.
  • Optimized parameters (IW=4.0 Th, ITmax >250 ms) increased detected protein groups by up to 300% compared to benchmark conditions (IW=2.0 Th, ITmax=50 ms) for 1-ng samples.

Conclusions:

  • Optimized MS parameters, particularly IW and ITmax, are crucial for advancing single-cell and trace proteomics.
  • The identified optimal parameters enable deeper proteome penetration, aiding the study of cellular functions like signaling and cell adhesion.
  • This work provides guidance for rational selection of MS parameters in sensitive proteomic analyses.