Adaptive Focused Acoustics-integrated proteome profiling of macrophages uncovers low abundant proteins associated with immune homeostasis, inflammatory response, and transport

Abstract

Efficient and reproducible protein extraction is a critical step in mass spectrometry-based proteomics workflows, particularly for complex host-pathogen systems where low-abundance immune-associated proteins are difficult to detect. Probe sonication methods used for cell lysis requiring mitigation of excessive heat generation, to prevent degradation of biologically important proteins, while also limiting throughput and potentially introducing sample-to-sample variability. In this study, we evaluated adaptive focused acoustics (AFA) technology as an alternative approach for macrophage lysis and protein extraction and digestion within a standard proteomics workflow coupled with mass spectrometry. We observed that AFA technology reduced hands-on processing times and overall workflow timelines and single-sample AFA technology improves proteome coverage, dynamic range, and reproducibility. We also evaluated multiplexed AFA technology for lysis, and we observed an exclusive macrophage proteome and influence on replicate reproducibility and dynamic range detection for low abundant proteins. Moreover, multiplexed AFA technology for macrophage lysis and digestion further increased protein identifications, replicate reproducibility, and dynamic range. Considering the AFA-exclusive proteome, 86 proteins were detected across all AFA-based lysis and digestion methods, including low-abundance proteins associated with macrophage homeostasis, inflammatory response, and transport. Together, these findings demonstrate that AFA technology enhances reproducibility, throughput, and proteome depth for macrophage protein extraction while enabling the detection of biologically relevant low-abundance immune-associated proteins. These improvements provide a strong foundation for future investigation of host-pathogen infection models, where pathogen-derived proteins remain challenging to detect within complex host proteomes.