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

Significance of High-Throughput DIA Proteomics

Modern biological research and clinical applications increasingly rely on comprehensive, quantitative proteome analysis to decode complex cellular processes and identify biomarkers. High-throughput data-independent acquisition (HT-DIA) workflows enable rapid, reproducible, and accurate quantitation across large sample cohorts, addressing demands in drug discovery, precision medicine, and systems biology.

Objectives and Study Overview

This study evaluates the qualitative and quantitative performance of fast HT-DIA gradients on the Orbitrap Exploris 240 mass spectrometer combined with a Vanquish Neo UHPLC system and a μPAC Neo high-throughput column. Three gradient lengths—3.5 min (260 samples/day), 5.5 min (170 samples/day), and 11 min (100 samples/day)—were benchmarked for proteome coverage, quantitation precision, robustness over 1000 consecutive injections, and inter-laboratory reproducibility across three European sites.

Methodology

HeLa peptides and a three-species mixture (human, yeast, E. coli) were analyzed to assess depth and accuracy. Key workflow parameters included:

• LC gradients: 3.5 min at 3 μL/min, 5.5 min at 1.25 μL/min, 11 min at 1 μL/min;
• Column temperature: 50–60 °C;
• MS settings: MS1/MS2 resolution 30k/15k, scan range m/z 525–800 or 825, DIA isolation windows 8–16 m/z, NCE 28%;
• Data analysis: Spectronaut 18 (DirectDIA+), DIA-NN 1.8.1, Proteome Discoverer 3.1 with CHIMERYS 2.0.

Used Instrumentation

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

Main Results and Discussion

All three HT-DIA methods delivered deep proteome coverage and precise quantitation:

• Protein group identifications ranged from ~3,900 (170 SPD) to ~4,600 (260 SPD) with Spectronaut and up to ~6,000 at 100 SPD using directDIA workflows.
• Quantitative accuracy for three-species mixtures matched theoretical ratios (1:1, 1:0.5, 1:4) with narrow distributions.
• Median data points per chromatographic peak remained at 5–6 across fast gradients, supporting robust peak integration.
• Coefficients of variation were consistently below 5% at the protein level and below 10% at the peptide level.
• Workflow robustness demonstrated by >1,000 consecutive runs at 100 SPD with 1.37% RSD in proteome coverage.
• Inter-laboratory reproducibility across Belgium, Switzerland, and Germany showed RSDs of 4.9–6.7% for protein identifications.

Benefits and Practical Applications

This HT-DIA workflow offers:

• High sample throughput (up to 260 SPD) with minimal compromise on depth.
• Excellent quantitation accuracy for low-abundance proteins in complex matrices.
• Outstanding instrument productivity for large cohort studies.
• Robust performance enabling multi-site and long-term projects in clinical and industrial laboratories.

Future Trends and Potential Applications

Next-generation developments may include:

• Shorter columns and faster chromatography for >300 SPD throughput.
• Integration of real-time quality control and machine-learning-driven acquisition strategies.
• Expansion to single-cell proteomics and clinical proteome screening.
• Enhanced multiplexing and deeper coverage via advanced ion mobility or parallel fragmentation.

Conclusion

The combination of Orbitrap Exploris 240, Vanquish Neo UHPLC, and μPAC Neo HPLC column establishes a robust, high-throughput DIA proteomics platform. It achieves deep, accurate quantitation across rapid gradients, demonstrating exceptional reproducibility, accuracy, and instrument productivity essential for large-scale proteomic studies.

Reference

• No formal literature references provided in the source document.