Fast, sensitive, and reproducible nano- and capillary-flow LCMS methods for high− throughput proteome profiling using the Vanquish Neo UHPLC system hyphenated with the Orbitrap Exploris 480 MS

Significance of the Topic

Rapid and reproducible proteome profiling is essential for biomarker discovery, precision medicine, and large‐scale biological studies. High‐throughput LC-MS methods balance depth of coverage with sample throughput, addressing limitations of traditional nano-flow approaches that restrict daily sample capacity. The integration of ultra-high-pressure UHPLC systems with high‐resolution mass spectrometers enables accelerated analyses while maintaining sensitivity and chromatographic resolution.

Objectives and Study Overview

This study aims to evaluate the Thermo Scientific™ Vanquish™ Neo UHPLC system coupled with an Orbitrap Exploris™ 480 mass spectrometer for high‐throughput, bottom-up proteome profiling. Five trap-and-elute methods were developed on a 75 µm I.D. × 150 mm PepMap™ Neo column to achieve throughputs of 24, 30, 60, 100, and 180 samples per 24 h. Key goals include maximizing MS utilization, assessing proteome depth, and verifying reproducibility within and across laboratories.

Materials and Methods

Sample preparation used Thermo Scientific™ HeLa Digest spiked with PRTC standard at 200 ng/µL HeLa and 100 fmol/µL PRTC in 0.1% formic acid. LC solvents comprised water/0.1% FA (mobile phase A) and 80/20 ACN/water with 0.1% FA (mobile phase B). The trap-and-elute workflow employed a 300 µm × 5 mm PepMap Neo trap and a 75 µm × 150 mm, 2 µm PepMap Neo separation column. Five gradients (8, 14.4, 24, 48, 60 min cycle times) were optimized for each throughput level. MS acquisition in data-dependent mode used full scans (m/z 375–1200) at 45,000–60,000 resolution and HCD MS2 scans at 7,500–15,000 resolution. Data processing utilized Proteome Discoverer™ 2.5 (Sequest HT, INFERYS) with FDR <1% at peptide and protein levels, supplemented by Skyline for extracted ion chromatogram analysis.

Instrumentation

• Vanquish Neo UHPLC System (binary pump N, split sampler NT, solvent rack, system controller)
• EASY-Spray PepMap Neo separation column: 75 µm × 150 mm, 2 µm, 1500 bar
• PepMap Neo trap column: 300 µm × 5 mm, 5 µm, 1500 bar
• Orbitrap Exploris 480 mass spectrometer

Key Results and Discussion

MS utilization ranged from 68% for the 8 min method to 95% for the 60 min method, driven by parallel column equilibration and sampler wash routines. Protein identifications increased from ~1,200 (8 min) to ~4,050 (60 min), with peptide groups rising from ~5,670 to ~29,000. Intra-run RSD for peptide and protein IDs was below 2%, and inter-column variability across three column sets stayed under 6%. A 100-injection longevity study with the 14.4 min method showed stable PRTC retention times (RSD ≤5.5%), consistent identifications (~11,800 peptides, ~2,000 proteins per run), and 2,800 proteins quantified after match-between-runs. Inter-laboratory tests in Germany and the USA demonstrated 72.5%–88.5% overlap in protein IDs across sites.

Benefits and Practical Applications

These methods deliver scalable sample throughput with minimal hardware changes, high sensitivity, and robust quantitative performance. The high MS utilization minimizes instrument idle time, making the approach ideal for large cohort studies in translational proteomics, biomarker validation, quality control, and industrial analytics.

Future Trends and Applications

Emerging trends include further automation of sample preparation, real-time method optimization driven by AI, and integration of micro- and capillary-flow LC with next-generation high-pressure systems. Applications may expand to single-cell proteomics, clinical diagnostics, and multi-omics workflows, supported by standardized protocols for cross-laboratory consistency.

Conclusion

The Vanquish Neo UHPLC system paired with Orbitrap Exploris 480 and PepMap Neo columns provides a versatile platform for high-throughput, sensitive, and reproducible proteome profiling. Five standardized methods achieve up to 180 samples per day with deep proteome coverage and robust inter-run and inter-lab performance.

References

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