Improving Proteomic Identification Using Narrow Isolation Windows with Zeno SWATH Data-Independent Acquisition.

Data-independent acquisition (DIA) techniques such as sequential window acquisition of all theoretical mass spectra (SWATH) acquisition have emerged as the preferred strategies for proteomic analyses. Our study optimized 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 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 5916 and 7719 protein groups on the digested peptides, corresponding to 400 ng of protein from mouse liver and HEK293T cells, respectively, improving identification by 7.52 and 4.99%, respectively, compared to conventional SWATH. The median coefficient of variation of the quantified values was less than 6%. We further analyzed 200 ng of benchmark samples comprising peptides from known ratios of Escherichia coli , yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to those of conventional SWATH, identifying an average of 95,456 precursors and 9342 protein groups across three benchmark samples, representing 12.6 and 9.63% improved identification compared to conventional SWATH. The nSWATH method improved identification at various loading amounts of benchmark samples, identifying 40.7% more protein groups at 25 ng. These results demonstrate the improved performance of nSWATH, contributing to the acquisition of deeper proteomic data from complex biological samples.