Quantitative proteomics—the new benchmark

Importance of the topic

Quantitative proteomics transforms protein identification into dynamic measurement of abundance, enabling insights into cellular processes, disease mechanisms and biomarker discovery. The ability to accurately quantify thousands of proteins across diverse samples is essential for systems biology, translational research and precision medicine.

Objectives and overview of the article

This article presents a new benchmark for quantitative proteomics developed by Thermo Fisher Scientific. It outlines integrated workflows—from discovery to targeted analysis—leveraging high-resolution accurate-mass (HRAM) Orbitrap mass spectrometers, advanced labeling strategies and intelligent data acquisition. The goal is to achieve comprehensive protein quantitation with high precision, sensitivity and throughput.

Methodology and instrumentation

Workflows described include:

• Label-free quantitation (LFQ) using data-dependent (DDA) and data-independent acquisition (DIA) on Orbitrap Exploris instruments, optionally coupled with the FAIMS Pro interface.
• Stable isotope labeling (SILAC) for in vivo metabolic incorporation of heavy amino acids, analyzed by Orbitrap Exploris or Exploris 240 systems.
• Isobaric tagging with TMT/TMTpro reagents and SPS-MS3 on Orbitrap Eclipse Tribrid mass spectrometers, enabling multiplexed quantitation of up to 18 samples.
• Intelligent targeted assays: SureQuant for real-time guided PRM on Orbitrap Eclipse and high-resolution SRM (H-SRM) on TSQ Altis triple quadrupole systems.

Used instrumentation

• Thermo Scientific™ Orbitrap Exploris™ 480 and Exploris™ 240 mass spectrometers
• Thermo Scientific™ Orbitrap™ Eclipse Tribrid mass spectrometer
• Thermo Scientific™ TSQ™ Altis triple quadrupole mass spectrometer
• Thermo Scientific™ FAIMS Pro interface
• Thermo Scientific™ Vanquish™ and Dionex UltiMate™ UHPLC systems

Main results and discussion

• Orbitrap analyzers deliver up to 500,000 resolution at m/z 200, attomole-level sensitivity, five orders of linear dynamic range and run-to-run CVs below 2% for HeLa digest replicates.
• SPS-MS3 on Eclipse Tribrid improves TMT quantitation by reducing ratio compression and increasing reporter ion signal intensity, enabling accurate multiplexed analysis.
• FAIMS Pro interface enhances selectivity by reducing co-eluting interference, boosting proteome coverage and quantitative accuracy across six orders of magnitude.
• SureQuant assays increase sensitivity for hundreds of targets by using internal standards for real-time MS2 acquisition and optimized high-resolution scans.
• H-SRM on TSQ Altis achieves superior specificity with 0.2 Da resolution and ultrafast scan rates, enabling robust quantitation in complex matrices.

Benefits and practical applications of the method

• High reproducibility and precision across workflows support large-scale studies and clinical sample cohorts.
• Deep proteome profiling by LFQ or SILAC enables discovery of novel pathways and differential expression.
• Multiplexed TMT/TMTpro workflows accelerate comparisons across multiple conditions, time points and single cells.
• Targeted assays (SureQuant, PRM, SRM) provide sensitive and accurate quantitation for biomarker verification and validation.
• Integrated software (Proteome Discoverer, TraceFinder, Skyline) streamlines data processing, analysis and reporting.

Future trends and applications

• Expansion of single-cell proteomics with higher multiplexing and improved real-time acquisition strategies.
• Greater integration of ion mobility (FAIMS) and AI-driven workflows for enhanced selectivity and throughput.
• Automated end-to-end platforms from sample prep to data analysis to support clinical proteomics and precision medicine.
• Advances in instrumentation delivering even higher resolution, speed and sensitivity for low-abundance targets.
• Broader adoption of quantitative proteomics in quality control, industrial analytics and pharmacoproteomics.

Conclusion

By combining robust sample preparation, HRAM Orbitrap detection, advanced labeling and intelligent acquisition on dedicated mass spectrometers, Thermo Fisher Scientific establishes a new benchmark in quantitative proteomics. These integrated workflows deliver confidence, rigor and unprecedented depth—empowering researchers to transform proteome snapshots into dynamic biological insights.