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Data Processing and Analysis for DIA-Based Phosphoproteomics Using Spectronaut.

Ana Martinez-Val1, Dorte Breinholdt Bekker-Jensen1,2, Alexander Hogrebe3

  • 1Novo Nordisk Foundation Center for Protein Research, Proteomics Program, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Methods in Molecular Biology (Clifton, N.J.)
|July 8, 2021
PubMed
Summary
This summary is machine-generated.

Data-independent acquisition (DIA) offers improved proteomics depth and reproducibility over data-dependent acquisition (DDA). This study presents a guide for analyzing phospho-DIA data and performing differential phosphorylation site analysis using Spectronaut.

Keywords:
Data-independent acquisitionPhosphoproteomicsSpectronautdirectDIA

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Phosphoproteomics

Background:

  • Data-independent acquisition (DIA) enhances proteomics data depth and reproducibility compared to data-dependent acquisition (DDA).
  • DIA overcomes limitations of DDA, such as precursor selection bias and limited dynamic range.
  • Recent advancements in mass spectrometry technology further support DIA as a superior alternative to DDA.

Purpose of the Study:

  • To extend the benefits of DIA to phosphoproteomics workflows.
  • To address computational challenges in analyzing phospho-DIA data.
  • To provide a practical guide for analyzing phospho-DIA raw data and performing differential phosphorylation site analysis.

Main Methods:

  • A step-by-step guide for analyzing phospho-DIA raw data using Spectronaut.
  • Utilizing spectral libraries or directDIA approaches within Spectronaut.
  • Implementing a protocol for differential phosphorylation site analysis on Spectronaut output.

Main Results:

  • Demonstrated increased depth, sensitivity, and reproducibility in phosphopeptide analysis using DIA.
  • Presented a user-friendly workflow for analyzing complex phospho-DIA datasets.
  • Enabled straightforward differential phosphorylation site analysis.

Conclusions:

  • DIA is a powerful strategy for phosphoproteomics, offering significant improvements in data quality.
  • The provided Spectronaut-based workflow simplifies the analysis of phospho-DIA data.
  • This guide facilitates robust differential phosphorylation site analysis, advancing phosphoproteomics research.