Proteome study to optimize Zeno SWATH DIA

Data-independent acquisition (DIA) techniques, such as SWATH acquisition, have emerged as the preferred strategy for proteomic analysis. Our study optimizes the SWATH-DIA method using a narrow isolation window placement approach improving its proteomic performance. We optimized the acquisition parameter combinations of narrow isolation windows with different widths (1.9 Da and 2.9 Da) on a ZenoTOF 7600 (Sciex); the acquired data were analyzed using DIA-NN (version 1.8.1). Narrow SWATH (nSWATH) identified 5,916 and 7,719 protein groups on the digested peptides corresponding to 400 ng of protein from mouse liver and HEK293T cells, improving identification by 7.52% and 4.99%, respectively, compared to that in conventional SWATH. The median coefficient of variation (CV) of the quantified values was less than 6%. We further analyzed benchmark samples comprising peptides from known ratios of E. coli, yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to conventional SWATH, identifying an average of 95,456 precursors and 9,342 protein groups across three benchmark samples, representing 12.6% and 9.63% improved identification compared to conventional SWATH. Furthermore, nSWATH detected lower-abundance proteins than conventional SWATH.