Unleashing the power of DIA acquisition on an Orbitrap Exploris 240 mass spectrometer – precise and accurate quantitation at 260 SPD

Significance of the topic

The integration of data‐independent acquisition (DIA) strategies with high‐throughput liquid chromatography–mass spectrometry (LC‐MS) platforms enables comprehensive, reproducible, and accurate proteome quantification across large sample cohorts. Advances in mass spectrometer speed and chromatographic media stability are crucial to meet the demands of clinical and industrial proteomic studies where hundreds of samples must be analyzed with minimal downtime and consistent data quality.

Study objectives and overview

This work aimed to evaluate the qualitative and quantitative performance of ultrafast DIA workflows on the Orbitrap Exploris 240 mass spectrometer coupled to a Thermo Scientific Vanquish Neo UHPLC system and a 5.5 cm µPAC Neo high‐throughput column. Three gradient lengths—3.5, 5.5, and 10 minutes—yielding sample throughputs of approximately 260, 170, and 100 samples per day (SPD) were benchmarked in terms of proteome coverage, quantitation precision, accuracy, and long‐term robustness. Interlaboratory reproducibility was assessed by running identical HeLa digest standards at three distinct sites in Europe.

Methodology and Instrumentation

Samples comprised a HeLa peptide digest and a controlled three‐species mixture (HeLa background spiked with yeast and E. coli digests at defined ratios). Peptides (200 ng load) were separated by direct injection on a Vanquish Neo UHPLC system equipped with a 5.5 cm µPAC Neo high‐throughput column and a stainless steel nanospray emitter. Three LC gradients delivered 0.1% formic acid in water (solvent A) and 80% acetonitrile with 0.1% formic acid (solvent B) at 400 bar loading pressure. DIA acquisition employed 30,000/15,000 resolution for MS1/MS2, fixed isolation windows (8–16 m/z), and optimized collision energy (28% NCE). Data were processed with Spectronaut 18 (directDIA), DIA-NN, and Proteome Discoverer 3.1 with CHIMERYS, applying 1% FDR at peptide and protein levels.

Instrumentation used

• Vanquish Neo UHPLC system (binary pump, split sampler, column compartment, temperature control)
• µPAC Neo high‐throughput HPLC column, 5.5 cm
• Orbitrap Exploris 240 mass spectrometer with Nanospray Flex ion source
• Software: Spectronaut 18, DIA-NN 1.8.1, Proteome Discoverer 3.1 with CHIMERYS 2.0

Main results and discussion

Proteome coverage: In 200 ng HeLa runs, all three gradient lengths yielded deep proteome coverage: ~3,000 proteins at 260 SPD, ~3,900 proteins at 170 SPD, and ~5,000 proteins at 100 SPD, with corresponding peptide counts of ~23,000, ~32,000, and ~49,000. Spectronaut and DIA-NN outperformed library‐free CHIMERYS searches in identifications.

Quantitation precision and accuracy: Six PRTC peptides spiked into each run showed a median of 5–6 data points per peak even at the shortest gradient. Technical replicates produced median coefficients of variation (CVs) below 5% for proteins and below 10% for peptides. In the three‐species mixture, measured abundance ratios (1:1 for human, 0.5:1 yeast, and 4:1 E. coli) closely matched theoretical values across all gradient lengths, demonstrating high quantitative fidelity.

Robustness and interlaboratory reproducibility: Over 1,000 consecutive injections at 100 SPD exhibited minimal decline in identifications (RSD ~1.4%), confirming column stability and system ruggedness. Cross‐site runs of HeLa digest at three European laboratories yielded proteome coverage RSDs of 5–7% across all methods, underlining method transferability.

Benefits and practical applications

• High sample throughput (up to 260 SPD) without sacrificing data depth or reproducibility
• Accurate and precise LFQ suitable for large clinical and biomarker discovery studies
• Robust system performance over prolonged operation and across multiple laboratories

Future trends and potential applications

Ongoing improvements in mass spectrometer duty cycles and chromatographic media will push throughput beyond 300 SPD while further reducing missing data in large cohorts. Integration of real‐time data analysis, AI‐driven peak detection, and automated quality control will streamline biomarker validation pipelines. Emerging microfluidic LC‐MS platforms may enable single‐cell DIA proteomics at similar throughputs.

Conclusion

The described high‐throughput DIA workflow combining Orbitrap Exploris 240 and µPAC Neo chromatography delivers exceptional proteome coverage, quantitation accuracy, and ruggedness. Its reproducibility across sites and sustained performance over thousands of injections make it an ideal solution for large‐scale, label‐free quantitative proteomics in clinical research and industrial quality control.