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

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. Sin-gle-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).

Automated summary

Here, an optimized protocol for analyzing murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics is described. The protocol includes sample preparation utilizing the S-Trap method, mass spectrometry data acquisition, and data analysis approaches. The single-shot DIA protocol achieves comparable proteomic depth with data-dependent MS approaches, making it suitable for large sample numbers with high experimental reproducibility.