A Protocol for Robust Discovery Proteomics Using Nano Liquid Chromatography Using Pillar‐Array Column Technology and High‐Resolution Mass Spectrometry With Data‐Independent Acquisition

ABSTRACT Nano liquid chromatography–mass spectrometry has come to be a key enabling technology in shotgun proteomics due to the combination of exceptional separation power, sensitivity, and comprehensiveness. However, the know‐how of setting up proteomics methods to deliver robust, reliable, and meaningful results to large‐scale life science experiments has remained somewhat ambiguous. This protocol outlines guidance for establishing nano‐LC–MS/MS workflows focusing on comprehensive and untargeted deep proteome profiling, using state‐of‐the‐art column technology and mass spectrometry. Employing a second‐generation micropillar‐array column, a trade‐off is demonstrated between analysis time and chromatographic resolving power, which in turn impacts peptide and protein identification scores from a commercial HeLa reference standard. Furthermore, a straightforward workflow to develop a data‐independent acquisition (DIA)‐parallel accumulation—serial fragmentation (PASEF) analytical method is proposed, with a special focus on the optimization of the ESI source settings. Besides the method development, the study discusses the use of segmented gradients, and an MS‐compatible surfactant in the sample diluent is also explored. Finally, the robustness of the developed method is demonstrated through consistently identifying 7558 protein groups (CV = 0.3%) as maintaining high repeatability peptide retention times (mean CV = 0.2%) and system pressure (CV = 0.4%) over 21 consecutive analyses.