Wide Window Acquisition and AI-based data analysis to reach deep proteome coverage for a wide sample range, including single cell proteomic inputs – Co-immunoprecipitation samples

A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yielded impressive results, current studies suffer from a limited proteomic depth and dynamic range therefore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns (µPAC™) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYS™ search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. Using this optimized workflow on immunoprecipitation samples of Smarca5/SNF2H, we found 32 additional interaction partners compared to the original workflow utilizing a packed bed column. These additional interaction partners include previously described interaction partners of Smarca5 like Baz2b as well as undescribed interactors including Arid1a, which is also involved in chromatin remodeling and has been described as key player in neurodevelopmental and malignant disorders.