Comparative Proteomic Analysis of Capillary and Micropillar Array Columns Using DIA: Insights from the TKO Yeast Standard

The effective separation of complex peptide mixtures is a cornerstone of mass spectrometry-based proteomics analysis as it enhances the accuracy and depth of proteomic analyses. Here, we compare data sets collected of whole-cell tryptic peptides, which were fractionated by either conventional flame-pulled, C18 packed-bed microcapillary columns or a microfabricated pillar array column (μPAC). Sixteen samples that included four different yeast strains (Δ<i>met6</i>, Δ<i>pfk2</i>, Δ<i>ura2</i>, and wildtype) were analyzed in quadruplicate using data-independent acquisition. Each column enabled the quantification of >4700 proteins, with >95.4% overlap between column formats. The μPAC showed higher MS1 and MS2 signal intensities while maintaining similar peptide characteristics as the capillary column. The capillary column favored slightly longer and more hydrophobic peptides. Both columns achieved high data completeness at the protein level (>95%) and reproducible quantification, with μPAC offering slight improvements. Principal component and correlation analyses confirmed the capture of yeast strain-specific differences, with hierarchical clustering prioritizing strain over column effects. Protein quantification validated gene knockouts in both column formats, demonstrating similar accuracy of quantification. These findings highlight the μPAC as a standardized and robust alternative to capillary columns in proteomic analysis.