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. 2022 Dec 16;3(4):101725.
doi: 10.1016/j.xpro.2022.101725. Epub 2022 Sep 26.

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

Christa P Baker  1 , Iain R Phair  2 , Alejandro J Brenes  1 , Abdelmadjid Atrih  1 , Dylan G Ryan  3 , Roland Bruderer  4 , Albena T Dinkova-Kostova  2 , Douglas J Lamont  1 , J Simon C Arthur  5 , Andrew J M Howden  6
Affiliations

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

Christa P Baker et al. STAR Protoc. .

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).

Keywords: Bioinformatics; Immunology; Mass spectrometry; Protein biochemistry; Proteomics.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Workflow summary of the main steps in proteomic data acquisition
Figure 2
Figure 2
Expected outcomes for DIA proteomic analysis (A–C) Comparisons of the data obtained using DIA and TMT-DDA proteomic methods for unstimulated bone marrow derived macrophages. (A) Venn Diagram comparing DIA and TMT total protein hits. (B) Comparison of the detection of Toll-like receptors (TLRs) between DIA and TMT-DDA datasets. (C) Schematic diagram showing potentially relevant examples of the proteins identified in the DIA dataset, displaying the total number of hits in each cellular component in parentheses, sorted by function using the DAVID Bioinformatics Database. (D) Shows an example volcano plot comparing the proteome of macrophages from nuclear factor erythroid 2-related factor 2 knockout mice (Nrf2 KO) and macrophages from mice with a knockdown of its negative regulator Kelch-like ECH-associated protein 1 Keap1 knockdown (Keap1 KD) obtained using DIA methods. X axis shows the of Log2 fold change in estimated copy numbers, and y axis the -Log10 p-value (Student's t-test, n=4). Both datasets have been published previously in (Ryan et al., 2022) and are available in PRIDE (accession number PXD030455).
See this image and copyright information in PMC

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