Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer - Quantitation–widest range of quantitative strategies that deliver biological insights

Importance of Accurate Quantitative Proteomics

Quantitative proteomics is essential for deciphering protein abundance, dynamics and interactions in complex biological systems. Accurate measurement of protein levels enables deeper insights into cellular processes, biomarker discovery and therapeutic development.

Study Objectives and Overview

This work evaluates the Thermo Scientific Orbitrap Exploris 240 mass spectrometer across three major quantitative strategies: label-free quantitation (LFQ), Tandem Mass Tag (TMT) multiplexing and parallel reaction monitoring (PRM). Key goals include assessing sensitivity, dynamic range, accuracy and reproducibility for each workflow.

Used Instrumentation

• Thermo Scientific Orbitrap Exploris 240 MS with FAIMS Pro interface
• Thermo Scientific EASY-nLC 1200 system
• Thermo Scientific Nanospray Flex and EASY-Spray ion sources
• Sonation Column Oven
• Proteome Discoverer software version 2.4
• Skyline software for PRM data analysis

Methodology and Workflow

Label-Free Quantitation (LFQ):

• High-resolution accurate-mass (HRAM) detection of peptides.
• Two-proteome mixture (yeast spiked at 1:2, 1:5, 1:10 into HeLa digest) analyzed in quadruplicate.
• 25 cm IonOpticks UHPLC column, 120 min gradient at 300 nL/min.

TMT Multiplexing:

• Use of TMT11plex and TMTpro16plex reagents on FAIMS Pro to reduce co-isolation interference.
• Analysis of Pierce TMT11plex Yeast Digest Standard over three replicates.
• EASY-Spray HPLC column, 120 min gradient.

Parallel Reaction Monitoring (PRM):

• Targeted analysis of 30 peptides from the AKT pathway using retention time scheduling.
• Dilution series covering five orders of magnitude in concentration.
• 150 mm EASY-Spray column, 40 min gradient at 300 nL/min.

Main Results and Discussion

• LFQ demonstrated a linear response across the yeast spike-in range with consistent quantitation and high precision of precursor ratios.
• TMT workflows delivered >10 800 peptides and ~2 350 proteins per run at 1% FDR. FAIMS Pro usage yielded an average interference-free index (IFI) of 0.89, indicating minimal ratio distortion.
• PRM assays achieved an average coefficient of variation (CV) of 1.36% across all peptides, with over 80% of peptides at the lower limit of quantitation showing CVs below 10%. Dynamic range exceeded five orders of magnitude.

Practical Benefits and Applications

The combined high resolution, mass accuracy and FAIMS-enhanced selectivity enable deep proteome coverage in label-free experiments, reliable multiplexed quantitation for large sample cohorts and highly sensitive targeted assays. This versatility supports discovery studies, biomarker validation, QA/QC and clinical research workflows.

Future Trends and Opportunities

• Integration of single-cell proteomics and automated high-throughput screening.
• Real-time, AI-driven data acquisition strategies to maximize proteome depth.
• Expansion of multiplexing capacity beyond 16-plex with improved interference removal.
• Combined multi-omics platforms for comprehensive systems biology analyses.

Conclusion

The Orbitrap Exploris 240 MS sets a new benchmark in proteomics by delivering exceptional sensitivity, accuracy and precision across label-free, isobaric labeling and targeted quantitation workflows. The FAIMS Pro interface further enhances performance, enabling confident, high-throughput protein profiling and precise biomarker measurement.