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Related Experiment Video

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Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis
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Multiplexed peptide analysis using data-independent acquisition and Skyline.

Jarrett D Egertson1, Brendan MacLean1, Richard Johnson1

  • 1Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

Nature Protocols
|May 22, 2015
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Summary
This summary is machine-generated.

This study introduces data-independent acquisition (DIA) mass spectrometry for peptide quantification in complex mixtures using Skyline software. DIA enables comprehensive peptide detection and accurate quantification, improving upon data-dependent acquisition methods.

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Analytical Chemistry

Background:

  • Data-dependent acquisition (DDA) in mass spectrometry (MS) only identifies a subset of peptides.
  • Selected reaction monitoring (SRM) is limited by predetermined target peptides.
  • There is a need for comprehensive and targeted peptide quantification in complex biological samples.

Purpose of the Study:

  • To describe a protocol for peptide detection and quantification in complex mixtures using data-independent acquisition (DIA) on a Q-Exactive mass spectrometer.
  • To demonstrate the utility of the Skyline Targeted Proteomics Environment for analyzing DIA data.
  • To provide a quality control (QC) method for DIA data.

Main Methods:

  • Utilized data-independent acquisition (DIA) on a Q-Exactive mass spectrometer.
  • Generated a spectral library using data-dependent acquisition (DDA).
  • Extracted chromatograms from DIA data for targeted quantification using Skyline software.

Main Results:

  • Peptides were quantified from DIA data using targeted chromatogram extraction, similar to SRM.
  • Quantification was based on the area under the curve of extracted MS/MS chromatograms.
  • A QC method for DIA was detailed.

Conclusions:

  • Data-independent acquisition (DIA) coupled with Skyline software enables robust peptide quantification in complex mixtures.
  • The described protocol offers a comprehensive approach to targeted proteomics.
  • The method is efficient, with sample preparation and analysis completed within hours.