Modern proteomics increasingly demands workflows that combine sensitive targeted quantitation with comprehensive, unbiased discovery. While the Orbitrap Astral analyser offers exceptional speed and sensitivity, Astral scans historically could not drive data-dependent decisions, limiting support for targeted methods like SureQuant. Here we introduce a new method execution framework and filter library that enable intelligent data-dependent decisions on Astral scans, unlocking SureQuant and Hybrid-DIA on the Orbitrap Astral Zoom mass spectrometer. This approach integrates targeted quantitation and DIA within a single acquisition, delivering high coverage with minimal trade-offs and establishing a platform for scalable, data-driven acquisition strategies.
Classical HeLa proteomics samples were prepared by spiking 18 heavy stable isotope–labelled peptides (10 fmol each) into 100 ng tryptic HeLa digest. LC-MS analysis was performed using a Thermo Scientific Vanquish Neo UHPLC, an IonOpticks Aurora Rapid XT column, and a Thermo Scientific FAIMS Pro Duo interface set to −40 V. Methods were configured with MS1 resolution of 120,000 over 380–980 m/z. The SureQuant layer monitored internal standards to trigger quantitation on endogenous peptides, followed by a DIA cycle using 5 m/z isolation windows and a 3 ms maximum injection time (hybrid cycle time ~1.2 s). Reference DIA runs without spike-in used 2 m/z windows and the same 3 ms maximum injection time. A predicate-based filter engine and optimised scheduling strategies supported real-time candidate selection in ~1 ms to maintain high scan rates (up to 270 Hz) without interrupting parallel acquisition.
Preliminary results show robust quantification and broad proteome coverage. Across heavy/light peptide pairs, extracted ion chromatograms show over 10 data points with preserved fragment ion ratios, while hybrid-DIA maintained a mean experimental cycle time of 0.7 s, with only 0.1 s dedicated to SureQuant—indicating capacity for larger target panels. From a single hybrid-DIA run, 6,900 protein groups were identified using a library-based DIA analysis, compared to 7,400 protein groups in optimised reference DIA, confirming minimal loss in discovery performance despite integrating targeted scans.
These advances show that the Orbitrap Astral Zoom MS can seamlessly combine targeted and untargeted acquisition in complex samples, enabling sensitive quantitation alongside comprehensive discovery. The new framework and filter library provide the foundation for future intelligent acquisition innovations, delivering high performance without compromising throughput or data quality.