Unleashing the power of HT-DIA acquisition on Orbitrap Exploris 240 MS – Precise and accurate quantitation at 260 SPD

Importance of the Topic

The ability to rapidly generate deep and reproducible proteome profiles is critical for large-scale studies in clinical research, biomarker discovery, and mechanistic investigations. High-throughput label-free quantitation using data-independent acquisition (DIA) maximizes sample throughput while preserving quantitative accuracy and depth, addressing the growing demand for processing hundreds to thousands of samples with consistent performance.

Objectives and Study Overview

This study benchmarks three ultrafast DIA workflows on the Thermo Scientific Orbitrap Exploris 240 mass spectrometer coupled with a Vanquish Neo UHPLC system and a 5.5 cm µPAC Neo high-throughput column. Gradient lengths corresponding to 260, 170, and 100 samples per day (SPD) were compared in terms of proteome coverage, quantitative precision, accuracy, and long-term robustness. Interlaboratory reproducibility across three sites further validates the workflow.

Methodology and Instrumentation

The workflows employ directDIA+ acquisition at a fixed MS1 resolution of 30 k and MS2 at 15 k with overlapping isolation windows. Three LC gradients were tested: 3.5 min at 3 µL/min (260 SPD), 5.5 min at 1.25 µL/min (170 SPD), and 11 min at 1 µL/min (100 SPD). Sample loading and pressure control were standardized. A mixed-species digest (human, yeast, E. coli) assessed quantitative accuracy, while HeLa digests measured depth and precision. Data analysis used Spectronaut 18 (DirectDIA+), DIA-NN 1.8.1, and Proteome Discoverer 3.1 with the CHIMERYS search algorithm, all filtered at 1% false discovery rate.

Used Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system with Binary Pump, Split Sampler NT, and Column Compartment N
• Thermo Scientific µPAC Neo high-throughput HPLC column, 5.5 cm
• Stainless steel 30 µm emitter and Nanospray Flex ion source
• Orbitrap Exploris 240 mass spectrometer (Tune v4.2 SP1)
• Spectronaut 18, DIA-NN 1.8.1, Proteome Discoverer 3.1 with CHIMERYS 2.0

Main Results and Discussion

All three methods delivered high proteome coverage, identifying nearly 6,000 protein groups at 260 SPD, over 5,500 at 170 SPD, and around 4,900 at 100 SPD. Quantitative precision was excellent, with coefficients of variation below 10% at both peptide and protein levels. Experimental abundance ratios in the three-species mixture matched theoretical values closely across gradients, demonstrating accurate quantification. The workflow sustained more than 1,000 consecutive runs at 100 SPD over 11 days without hardware changes, showing a proteome coverage RSD of ~1.37%. Interlaboratory tests across three European sites yielded RSDs of 4.9–6.7% for protein identifications, confirming robustness across laboratories.

Benefits and Practical Applications

• Throughput up to 260 samples per day with deep proteome coverage
• High quantitative accuracy and precision for complex and mixed-species samples
• Minimal missing data with consistent identification rates across replicates
• Long-term stability and reproducibility, suitable for large clinical cohorts
• Validated across multiple laboratories for multi-center studies

Future Trends and Applications

Ongoing enhancements in column design, acquisition speed, and AI-driven data analysis promise further gains in throughput and depth. Integration with automated sample preparation and cloud-based informatics could enable real-time monitoring in clinical workflows. Expansion to post-translational modification mapping and single-cell proteomics is anticipated as instrument sensitivity and data analysis tools continue to evolve.

Conclusion

This work demonstrates a robust, high-throughput DIA strategy on the Orbitrap Exploris 240 with Vanquish Neo UHPLC and µPAC Neo column, achieving exceptional proteome depth, quantitative reliability, and long-term stability. The validated workflows support large-scale and multi-center proteomic studies with minimal hardware intervention.