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Quantitative shotgun proteome analysis by direct infusion.

Jesse G Meyer1,2,3,4, Natalie M Niemi5,6,7, David J Pagliarini5,6,7,8,9

  • 1Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA. jesmeyer@mcw.edu.

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|November 24, 2020
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Summary
This summary is machine-generated.

Gas-phase separation enables rapid proteome analysis, bypassing liquid chromatography-mass spectrometry (LC-MS) bottlenecks. This direct infusion-shotgun proteome analysis (DI-SPA) method significantly increases throughput for large-scale proteomic studies.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biotechnology

Background:

  • Liquid chromatography-mass spectrometry (LC-MS) is a standard for peptide analysis in proteomics.
  • However, LC-MS analysis is time-consuming, limiting high-throughput studies.
  • There is a need for faster proteomic analysis methods to accelerate discovery.

Purpose of the Study:

  • To develop and validate a novel gas-phase separation technique for rapid proteome analysis.
  • To demonstrate the feasibility of direct infusion-shotgun proteome analysis (DI-SPA) for high-throughput quantification.
  • To assess the utility of DI-SPA in a complex multifactorial biological study.

Main Methods:

  • Implemented direct infusion-shotgun proteome analysis (DI-SPA) coupled with data-independent acquisition mass spectrometry (DIA-MS).
  • Utilized gas-phase peptide separation as an alternative to liquid chromatography.
  • Applied the method to analyze protein expression in cultured human cells under various conditions.

Main Results:

  • Achieved targeted quantification of over 500 proteins per minute (~3.5 proteins/sec).
  • Successfully performed a complex multifactorial proteomic study involving nutrients, genotype, and toxins.
  • Generated over 45,000 quantitative protein measurements from 132 samples in approximately 4.4 hours of MS data acquisition.

Conclusions:

  • DI-SPA offers a fast, label-free, and unbiased approach for proteome quantification without LC.
  • This method significantly enhances throughput, making it suitable for large-scale studies.
  • DI-SPA is a valuable tool for accelerating drug discovery and biomarker identification requiring proteomic analysis of numerous samples.