LC-MS: Vanquish Neo UHPLC system-to-system reproducibility ensures consistent and reliable results in nanoLC-MS proteomics

Importance of Topic

High reproducibility in nanoLC-MS proteomics is critical to ensure consistent, reliable data across multiple systems and over time. As nanoLC-MS remains the gold standard for bottom-up discovery proteomics, consistent retention times, peak shapes, and identification metrics are essential for large cohort studies, biomarker discovery, and quantitative analyses in clinical and industrial settings.

Objectives and Study Overview

This study evaluates intra- and inter-system reproducibility of the Thermo Scientific Vanquish Neo UHPLC system in combination with Thermo Scientific Orbitrap Exploris 240 mass spectrometers and EASY-Spray PepMap Neo columns. Two nanoLC methods were compared: a rapid 20-minute gradient and a standard 100-minute gradient. Metrics included retention time stability, peak area, peak height, full width at half maximum (FWHM), and the number of peptide and protein identifications across six independent systems.

Methodology

Sample preparation protocols were standardized:

• Cytochrome C digest diluted to 1 pmol/µL for retention time studies (20 min method).
• HeLa digest spiked with PRTC standard (200 ng/µL HeLa, 100 fmol/µL PRTC) for identification metrics (100 min method).

Chromatographic conditions included direct injection onto a 75 µm × 50 cm, 2 µm PepMap Neo column with 1500 bar loading and equilibration pressure. Two gradients were employed: a 20 min separation with 1–50% acetonitrile and a 100 min separation with 1–35% acetonitrile, both at sub-microliter flow rates.

Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system (VN-S10-A-01) with Split Sampler NT and Binary Pump N.
• Thermo Scientific EASY-Spray PepMap Neo column (ES75500PN), 75 µm × 500 mm, 2 µm particles.
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer controlled via Chromeleon 7.2.10 MUd.
• Data-dependent acquisition (DDA) and full-scan methods with resolution up to 120,000, normalized HCD at 26%, and dynamic exclusion for 100 min runs.

Key Results and Discussion

Intra-system reproducibility using the 20 min method showed peptide retention time RSDs below 0.2% across four replicates. Inter-system retention time standard deviations for Cytochrome C peptides remained between 0.1–0.25 min, while PRTC peptides in the 100 min method exhibited RSDs ≤ 2 min. Peak property variation (area, height, FWHM) for PRTC peptides across six systems was ≤ 15% RSD. Proteome profiling of 200 ng HeLa digest generated on average 33,000 peptides and 4,400 protein groups per run, with inter-system variability limited to 4.1% for peptides and 2.2% for proteins.

Benefits and Practical Applications

The demonstrated reproducibility supports robust label-free quantitation across multiple instruments and sites. Consistent retention times facilitate peak matching and library-based identification, while stable peak areas and shapes ensure reliable quantitation in biomarker discovery, pharmaceutical QC, and large cohort studies.

Future Trends and Opportunities

Further integration of high-throughput nanoLC workflows with advanced data processing—such as real-time database matching and machine-learning–driven peak picking—will enhance quantitation accuracy. Expansion to automated sample handling and microflow-LC formats may improve robustness and ease of use in regulated environments.

Conclusion

The Vanquish Neo UHPLC system combined with PepMap Neo columns and Orbitrap Exploris 240 MS delivers exceptional intra- and inter-system reproducibility for nanoLC-MS proteomics. This performance enables reliable, high-sensitivity profiling for large-scale and multi-center studies, supporting the transition of nanoLC-MS applications into routine clinical and industrial workflows.