Chaewon Kang (Department of Chemistry, Korea university)

Sang-Won Lee (Department of Chemistry, Korea university)

Chaewon Kang (Department of Chemistry, Korea university)

Sang-Won Lee (Department of Chemistry, Korea university)

Data-Independent Acquisition (DIA) has emerged as a powerful alternative to Data-Dependent Acquisition (DDA) in proteomics, offering improved reproducibility and comprehensiveness. Unlike DDA, which selectively fragments the most intense precursor ions, DIA fragments all ions within defined m/z windows, enabling unbiased and consistent data acquisition. However, DIA still has challenges to accurate quantification of TMT-labeled peptides due to interference from co-isolated peptides, which can compromise measurement accuracy.

In this study, we introduce a DDA-like narrow isolation window DIA method using 0.6 Th windows, employing the Astral mass analyzer, which offers acquisition speeds up to 200 Hz with high resolution and sensitivity. This DIA method was integrated with an online 2D-NCFC-RP/RPLC system for the analysis ovarian cancer peptides labeled with 18-plex TMTpro. The combination of narrow-window DIA and the DO-NCFC RP/RP LC system enabled the identification of over 400,000 unique peptides, corresponding to more than 13,000 protein groups, from the 18-plex TMT-labeled samples. Importantly, this approach maintained quantification accuracy comparable to DDA, while significantly increasing peptide identification. These results highlight the potential of narrow-window DIA on the Astral platform to substantially improve proteome coverage in isobaric labeling-based quantitative proteomics without compromising measurement precision.