DIA label-free proteomic analysis of murine bone-marrow-derived macrophages

Christa P. Baker, Iain R. Phair, Alejandro J. Brenes, Abdelmadjid Atrih, Dylan G. Ryan, Roland Bruderer, Albena T. Dinkova-Kostova, Douglas J. Lamont, J. Simon C. Arthur (Lead / Corresponding author), Andrew J. M. Howden (Lead / Corresponding author)

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Abstract

Here, we describe an optimized protocol to analyze murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics. We provide a complete step-by-step protocol describing sample preparation utilizing the S-Trap approach for on-column digestion and peptide purification. We then detail mass spectrometry data acquisition and approaches for data analysis. Single-shot DIA protocols achieve comparable proteomic depth with data-dependent MS approaches without the need for fractionation. This allows for better scaling for large sample numbers with high inter-experimental reproducibility. For complete details on the use and execution of this protocol, please refer to Ryan et al. (2022).

Original languageEnglish
Article number101725
Number of pages17
JournalSTAR Protocols
Volume3
Issue number4
Early online date26 Sep 2022
DOIs
Publication statusE-pub ahead of print - 26 Sep 2022

Keywords

  • Bioinformatics
  • Immunology
  • Mass spectrometry
  • Protein biochemistry
  • Proteomics

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