Towards Turnkey Targeted Proteomics Solutions Using Internal Standard Triggered Acquisitions on Modified Orbitrap MS

Significance of the Topic

The introduction of high‐resolution accurate mass (HRAM) instruments has transformed targeted proteomics by enabling precise, multiplexed quantification of proteins in complex samples. The SureQuant workflow builds on parallel reaction monitoring (PRM) by using stable isotope‐labeled internal standards to trigger optimized acquisition events, delivering enhanced sensitivity, specificity, and robustness required for large‐scale protein profiling.

Objectives and Study Overview

• Implement a native internal standard triggered PRM (IS‐PRM) workflow (“SureQuant”) on Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid mass spectrometers.
• Compare performance against conventional time‐scheduled PRM in terms of acquisition efficiency, sensitivity, and tolerance to chromatographic shifts.
• Demonstrate applications in monitoring the AKT/mTOR signaling pathway and in deep plasma proteome quantification.

Methodology and Instrumentation

• Acquisition scheme: “watch mode” performs fast low‐resolution MS2 scans of spiked internal standards (IS) to track elution; detection of IS triggers “quant mode” high‐resolution, extended‐fill‐time MS2 scans of corresponding endogenous peptides.
• Chromatography: Thermo Scientific UltiMate 3000 RSLC and EASY‐nLC 1200 systems with C18 trap and analytical columns.
• Mass spectrometers: Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid under native control software with a new “Group ID” feature for method organization.
• Internal standard kits: SureQuant AKT Pathway Multiplex Panel (30 SIL peptides) and Biognosys PQ500 kit (804 SIL peptides).

Main Results and Discussion

• Productive acquisition time increased to 80–90% in SureQuant versus 10–15% in conventional PRM.
• Enhanced sensitivity enabled detection and quantification of 26 AKT/mTOR peptides versus 11 by PRM within 2.5-minute monitoring windows.
• Method demonstrated robustness against deliberate chromatographic gradient shifts, maintaining high peptide capture rates.
• In undepleted plasma, the PQ500 SureQuant assay quantified ~560 endogenous peptides (~400 proteins) across a 70-minute gradient with high precision.

Benefits and Practical Applications

• Superior quantitative performance with low‐amol detection limits and 60% of peptides showing CV<5%.
• Turnkey usability via embedded method templates and “Group ID” for streamlined assay setup and transfer between laboratories.
• Applicability to signaling pathway studies, biomarker verification, QA/QC workflows, and large‐scale proteomic screens.

Future Trends and Opportunities

• Expansion of preconfigured internal standard panels targeting additional pathways and biofluids.
• Integration with automated data analysis platforms for real‐time survey, method refinement, and report generation.
• Application in clinical proteomics for high‐throughput biomarker validation and personalized medicine studies.

Conclusion

The native SureQuant IS‐PRM implementation on modified Orbitrap platforms provides a robust, sensitive, and user‐friendly solution for large‐scale targeted proteomics. By leveraging internal standards to dynamically control acquisition parameters, it ensures high throughput, reproducibility, and quantitative performance across diverse sample types.

Reference

• Gallien S, Gajadhar AS, Patel B, Kellmann M, Arrey TN, Thoeing C, Harder A, Huguet R, McAlister G, Bailey D, Eliuk S, Chen EL, Xuan Y, Huhmer A. Towards Turnkey Targeted Proteomics Solutions Using Internal Standard Triggered Acquisitions on Modified Orbitrap MS. Thermo Fisher Scientific; 2019.