Data-independent acquisition (DIA) mass spectrometry has emerged as a powerful analytical approach, systematically fragmenting all precursor ions within predefined isolation windows across a selected mass-to-charge (m/z) range. Unlike data-dependent acquisition (DDA), which preferentially selects precursor ions based on intensity, DIA provides enhanced identification and quantification opportunities for lower-intensity peptides, significantly improving proteome coverage. Nevertheless, standard DIA methodologies have not been widely applied to isobaric-labeled peptides, primarily due to challenges in accurately quantifying reporter ions arising from co-isolation interference from co-eluting peptides, negatively impacting quantitative precision and accuracy. In this study, we introduce an optimized strategy integrating narrow-window DIA with isobaric labeling, employing the Orbitrap Astral mass analyzer. Leveraging its exceptional high-speed acquisition capabilities of up to 200 Hz and high-resolution MS/MS scans at 80K, the analyzer enabled ultra-narrow isolation windows (0.6 Th), thereby significantly enhancing isolation specificity comparable to data-dependent acquisition (DDA). We evaluated the performance of our proposed method using ovarian cancer tissue peptides labeled with 18plex-TMTpro reagents and compared it directly to conventional DDA-based analysis. Our findings demonstrate that the narrow-window DIA method consistently identified a greater number of peptides, achieving deeper proteomic coverage while maintaining quantitative precision and accuracy equivalent to DDA. This comprehensive approach offers substantial improvements for multiplexed proteomics, holding significant potential for large-scale population proteomics studies.
