Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nanoliquid chromatography–electrospray ionization-tandem mass spectrometry (nano-LC–ESI-MS/MS) is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples. However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nanoflow high-pressure liquid chromatography (HPLC), current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology. With the recent introduction of the μPAC system, which provides perfectly ordered micropillar array based chromatographic support materials, completely new chromatographic concepts for optimization toward the needs of ultrasensitive proteomics become available. Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micropillar array column to a widely used nanoflow HPLC column for the proteomics analysis of 10 ng of tryptic HeLa cell digest. Comparative analysis of LC–MS/MS-data corroborated that micropillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, and increased sensitivity in the analysis of low-input proteomics samples and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nanoflow HPLC columns.

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 (μPACTM) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYSTM search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. Our data shows that μPACTM columns clearly outperform classical packed bed columns boosting peptide IDs by up to 140%. Already at classical narrow isolation widths CHIMERYSTM boosted ID rates by a factor of 2.6 compared to the conventional search engine MS Amanda 2.0. By combining CHIMERYSTM with WWA, even a 4.6-fold increase in ID rates could be achieved. Using our optimized workflow, we were further able to identify more than 10,000 proteins from a single 2 h gradient shotgun analysis. We further investigated the applicability of WWA for single cell inputs and found that the choice of the optimal isolation window width depends on sample input and complexity. Using a short 5.5 cm column and very high flow rates during loading and column equilibration we improved sample throughput to ~100 samples per day while maintaining high protein ID numbers. We believe that this is especially important for the single cell field where throughput is one of the most limiting factors. Finally, we applied our optimized workflow on immunoprecipitations of Smarca5/SNF2H and found 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.