Combining data-independent acquisition (DIA) with trapped ion mobility separation (TIMS) introduces an additional separation dimension, effectively reducing sample complexity and enhancing system peak capacity. The dia-PASEF scanning technique utilizes fixed quadrupole isolation windows in both the m/z and mobility domains. To further improve analytical efficiency, diagonal-PASEF has been introduced, which dynamically tracks the precursor ion cloud along its diagonal trajectory for optimal acquisition.
While existing data processing platforms accommodate diagonal-PASEF workflows, targeted data extraction remains underrepresented. Here, we describe the integration of diagonal-PASEF data processing within Skyline, enabling robust targeted quantitation.
For method evaluation, we employed the ApoEdge panel (ProteomEdge, Sweden), comprising heavy isotope-labeled Lys and Arg (13C, 15N) quantitative recombinant protein standards (qRePS) for all 18 human apolipoproteins. Samples were either directly digested or spiked into non-depleted plasma. Tryptic digests underwent separation on an 8 cm × 75 µm C18 column (IonOpticks) using nanoElute HPLC (Bruker), coupled to timsTOF HT (Bruker) via a CaptiveSpray source with a 10-minute acetonitrile gradient. The diagonal-PASEF protocol implemented six 50 Da slices, with a 50 ms accumulation/ramp time per slice, yielding a total cycle time of 0.4 seconds (including one MS1 scan). For targeted extraction in Skyline, libraries were generated by analyzing standards with dia-PASEF and Spectronaut (Biognosys).
Our results demonstrate effective diagonal-PASEF data processing in Skyline for targeted proteomics. The six-slice, 50 Da diagonal-PASEF approach enabled extraction of all 18 heavy-labeled apolipoproteins in both pure standards and plasma backgrounds. Analysis across five replicates showed high reproducibility. Endogenous protein concentrations were estimated using single-point calibration. Future work will extend targeted extraction to triple proteome samples.