Leveraging Adaptive RT on the Orbitrap Ascend MultiOmics Tribrid mass spectrometer for improved sensitivity and throughput in targeted workflows

Significance of the topic

Adaptive retention-time (RT) alignment directly addresses a major bottleneck in high-throughput targeted proteomics: losses of targets and reproducibility caused by chromatographic drift. By dynamically mapping background elution in real time and adjusting scheduled acquisition windows, Adaptive RT enables much narrower windows, higher multiplexing, increased injection time per target and reduced need for external RT standards. This improves sensitivity, throughput and data completeness for workflows that move from discovery proteomics to quantitative targeted assays on a single mass spectrometer platform.

Goals and study overview

The technical note evaluates the Adaptive RT feature implemented on the Thermo Scientific Orbitrap Ascend MultiOmics Tribrid mass spectrometer (Tune v4.3). The objective was to demonstrate a complete discovery-to-target workflow that combines data-independent acquisition (DIA) reference mapping with scheduled parallel-reaction monitoring (PRM) to: increase assay multiplexing, preserve sensitivity and reproducibility under chromatographic shifts, and avoid the use of spiked RT calibration standards. Performance was characterized using spectral libraries from gas-phase fractionation DIA, PRM conductor-driven method building, PQ500 plasma reference peptides and HeLa digest standards.

Used instrumentation

• UHPLC: Thermo Scientific Vanquish Neo with trap-and-elute configuration; EASY-Spray PepMap RSLC C18 column; 60 samples-per-day (24 min) gradient; column temp 45°C; sampler 7°C.
• Mass spectrometer: Thermo Scientific Orbitrap Ascend MultiOmics Tribrid MS (Tune v4.3) with EASY-Spray source.
• Consumables and standards: Pierce HeLa Digest/PRTC, Biognosys PQ500 peptide kit, digested plasma matrix, LC-MS grade solvents and formic acid.
• Software: Proteome Discoverer 3.1 SP1 (CHIMERYS search) for library generation; Skyline-daily (with PRM Conductor) for assay building and refinement.

Methodology

Workflow summary:

• Discovery library generation: Gas-phase fractionation (GPF)-DIA experiments collected over successive 100 m/z precursor bands (Ion Trap 1 Th windows; Orbitrap 4 Th windows) to produce high-quality chromatogram libraries processed with CHIMERYS and Proteome Discoverer.
• Reference mapping: An Adaptive RT DIA experiment was acquired with the Acquire Reference option enabled. The instrument compresses background retention information into a .rtbin reference file for later Align-to-Reference scheduling.
• Target selection and method creation: Library results were imported into Skyline-daily; PRM Conductor was used to create unscheduled PRM lists to map empirical elution, refine targets, and export scheduled PRM methods. A balance-load strategy limited peptides per protein to optimize acquisition resources.
• Targeted acquisition: Final scheduled PRM methods used Adaptive RT to dynamically shift acquisition windows (examples used 0.8 min windows) and exploited either Ion Trap or Orbitrap MSn detection modes depending on the required throughput and resolution.
• Data processing: PRM data and GPF-DIA libraries were handled in Proteome Discoverer and Skyline; metrics such as CV, limit of detection (LOD), and limit of quantitation (LOQ) were estimated from serial-dilution experiments of PQ500 peptides in plasma.

Main results and discussion

Key findings:

• Adaptive RT workflow reliably generated a .rtbin reference and used it to align scheduled PRM windows in real time, compensating for chromatographic perturbations including intentional LC flow changes and column aging.
• Even substantial RT shifts (reported up to ~5 minutes within a 24-min gradient) were corrected so that targets remained inside narrow acquisition windows; without Adaptive RT these targets would be missed.
• Assay reproducibility was high: median CV values for final assays were approximately 6% across replicates, indicating robust quantitation with narrow RT windows.
• Multiplexing capability: on a 24-min gradient the platform could accommodate ~3,000 qualified precursors in an Ion Trap-based PRM assay or ~2,200 precursors in Orbitrap-based PRM, reflecting trade-offs between speed and resolving power.
• Sensitivity: for an example peptide (SALVLQYLR) in the PQ500/plasma matrix, the on-column limit of detection was estimated at ~2.2 attomoles and the limit of quantitation at ~6.6 attomoles, with acceptable accuracy and CV across dilution points.

Benefits and practical applications

The presented approach combines discovery and targeted phases on one instrument with several practical advantages:

• Eliminates the need for external spiked RT calibration standards, reducing cost, preparation steps and potential interferences.
• Enables narrower scheduled windows, increasing the number of targets that can be measured without compromising signal quality.
• Maintains quantitative completeness by avoiding missing values that arise from shifted chromatographic peaks.
• Provides flexible assay development paths: using Ion Trap detection maximizes throughput and multiplexing, while Orbitrap detection supports higher resolution where needed.
• Applicable to clinical or large-cohort targeted proteomics where robustness to chromatographic variability and high multiplexing are critical.

Future trends and potential uses

Projected developments and applications include:

• Broader adoption of real-time RT alignment across vendor platforms and experiment types (DIA, PRM, hybrid acquisitions) to increase throughput in clinical proteomics and biomarker verification.
• Integration of Adaptive RT with automated method-creation tools and cloud-based libraries to streamline end-to-end pipelines from discovery to validated assays.
• Further optimization of acquisition strategies that combine Ion Trap speed and Orbitrap resolution dynamically within runs to maximize both depth and quantitation precision.
• Application to other omics modalities (e.g., phosphoproteomics, metabolomics) where retention variability and high multiplexing present similar challenges.

Conclusion

The Adaptive RT feature on the Orbitrap Ascend MultiOmics Tribrid MS enables robust, high-multiplex targeted workflows by using a DIA-based reference mapping (.rtbin) to dynamically align scheduled PRM windows. This approach improves assay sensitivity and reproducibility, supports very large numbers of precursors per assay (thousands depending on detector choice), and reduces dependence on external RT standards. The workflow demonstrated attomole-level sensitivity for representative peptides and consistent quantitative CVs around 6%, illustrating suitability for demanding targeted proteomics applications.

References

1. Remes PM, Yip P, MacCoss MJ. Highly Multiplex Targeted Proteomics Enabled by Real-Time Chromatographic Alignment. Analytical Chemistry. 2020;92(17):11809-11817. doi:10.1021/acs.analchem.0c02075
2. Remes PM, Jacob CC, Heil LR, Shulman N, MacLean BX, MacCoss MJ. Hybrid Quadrupole Mass Filter - Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics. Journal of Proteome Research. 2024;23(12):5476-5486. doi:10.1021/acs.jproteome.4c00599