Validation of Thin-diaPASEF for maximizing proteome coverage

We optimized the parameters of CCS range, polygon scan region, ramp time, and isolation window width to achieve high-depth proteome analysis using the timsTOF HT. As a result, we developed Thin-diaPASEF, which was optimized with a CCS range of 0.7–1.3, a ramp time of 150 ms, an isolation window width of 26 Th (with an overlap width of 1 Th), and a polygon area focused on regions where precursors accumulate. Thin-diaPASEF was then compared with existing methods, including py-diAID PASEF, slice-PASEF, and synchro-PASEF. The results demonstrated that Thin-diaPASEF achieved superior protein identification.

Performance evaluation of Thin-diaPASEF revealed the identification of approximately 9,400 proteins with a sample injection of 500 ng and an analysis time of 100 min. Finally, we applied Thin-diaPASEF to plasma proteome analysis. We compared three sample preparation methods: an improved LEL method based on our previously reported plasma and serum preparation approach, Seer’s nanoparticle-based method, and the conventional Top14 column-based method. As a result, the improved LEL method successfully identified 5,378 proteins at an analysis throughput of 24 samples per day.