Grant application resource: Using the Orbitrap Exploris 480 mass spectrometer to accelerate fundamental research

Importance of Topic

The development of highly sensitive, accurate, and high‐throughput mass spectrometry platforms is critical to advancing proteomics applications from bulk analyses to single cell experiments. Enhanced instrumentation capabilities enable deeper proteome coverage, improved quantitation precision, and novel insights into cellular heterogeneity, biomarker discovery, and translational research.

Study Objectives and Overview

This white paper describes the technology innovations of the Thermo Scientific Orbitrap Exploris 480 mass spectrometer paired with FAIMS Pro, TurboTMT, SureQuant, and BoxCar acquisition methods. The goal is to demonstrate how these features accelerate fundamental research by delivering unprecedented sensitivity, selectivity, and throughput for quantitative proteomics workflows, from untargeted discovery to targeted validation.

Methodology and Instrumentation

The Orbitrap Exploris 480 MS integrates multiple advanced components:

• Ultra‐high field Orbitrap analyzer offering up to 480,000 resolution at m/z 200 and sub‐ppm mass accuracy via EASY‐IC internal calibration.
• Advanced Quadrupole Technology (AQT) with Configuration Switch Mode for robust ion transmission and extended maintenance intervals.
• FAIMS Pro interface for differential ion mobility, enhancing signal‐to‐noise and reducing co‐isolation interferences in single cell and multiplexed workflows.
• TurboTMT acquisition powered by ΦSDM for baseline resolution of isobaric reporter ions in TMT11plex and TMTpro16plex experiments, combined with Precursor Fit filtering for optimal isolation specificity.
• SureQuant internal standard‐triggered PRM for dynamic, high‐resolution targeted quantitation without time‐scheduling constraints.
• Native BoxCar acquisition mode to expand MS1 dynamic range and improve label‐free quantitation depth in complex samples.

Main Results and Discussion

• Single cell sensitivity: 741 proteins identified in one HeLa cell and 1864 proteins from five cells using FAIMS Pro with intra‐analysis CV stepping.
• FAIMS benefits: Greater than 50 percent increase in protein and peptide identifications for low‐nanogram samples compared to no‐FAIMS acquisitions.
• TMT multiplexing: Combining TurboTMT, Precursor Fit, and FAIMS improved TMT11plex quantitation accuracy by 19 percent and identification rates by 11 percent.
• SureQuant efficiency: 80 to 90 percent of MS² scans were productive for targeted peptides vs. 10 to 15 percent in conventional PRM, enabling reliable quantification of hundreds of plasma proteins in un‐depleted samples.
• BoxCar dynamic range: Native implementation boosted protein IDs in human plasma by 40 percent over conventional DDA in a 45 min gradient.
• Instrument robustness: Continuous operation over 125 days with more than 2 mg of sample injection without performance decline; maintenance restored performance for further extended use.

Benefits and Practical Applications

• Single cell and rare cell proteomics for investigations of cellular heterogeneity and limited clinical specimens.
• High‐plex TMT11plex and TMTpro16plex workflows for large‐cohort studies and biomarker screening.
• Targeted quantitation of biomarker panels with SureQuant, supporting translational research and precision medicine assays.
• Label‐free quantitation with BoxCar for deep profiling of complex proteomes without extensive sample fractionation.
• Streamlined method templates and unified software across LC‐MS platforms for ease of use by novice and expert users.

Future Trends and Possibilities

Continued integration of ion mobility, AI‐driven acquisition strategies, and further increases in multiplexing capacity will drive deeper proteome coverage and accelerated turnaround times. Emerging microfluidic sample handling and ultrafast chromatography coupled with high‐performance MS will enable routine single‐cell multi‐omics. Cloud‐enabled data processing and intelligent method adaptation promise automated, real‐time decision making to maximize discovery and validation outputs.

Conclusion

The Orbitrap Exploris 480 MS, enhanced by FAIMS Pro, TurboTMT, SureQuant, and BoxCar acquisition modes, represents a next‐generation proteomics platform. It delivers unmatched sensitivity, accuracy, and throughput across discovery and targeted workflows, while its robust design and unified user interface ensure high uptime and simplified operation. This system empowers researchers to tackle fundamental biological questions with confidence and speed.

References

1. Cong et al. Improved Single‐Cell Proteome Coverage Using Narrow‐Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry. Anal Chem. 2020;92(3):2665–2671.
2. Budnik et al. SCoPE‐MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Genome Biol. 2018;19:61.
3. Pfammatter et al. A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements. Mol Cell Proteomics. 2018;17:2051–2067.
4. Schweppe et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High‐Field Asymmetric‐Waveform Ion Mobility Mass Spectrometry. Anal Chem. 2019;91(6):4010–4016.
5. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments. J Am Soc Mass Spectrom. 2016;27:162.
6. Gallien et al. Large‐Scale Targeted Proteomics Using Internal Standard Triggered‐Parallel Reaction Monitoring (IS‐PRM). Mol Cell Proteomics. 2015;14(6):1630–1644.
7. Meier et al. BoxCar acquisition method enables single‐shot proteomics at a depth of 10,000 proteins in 100 minutes. Nat Methods. 2018;15:440–448.
8. Bekker‐Jensen et al. A Compact Quadrupole‐Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients. Mol Cell Proteomics. 2020;19(6):1089–1099.