LC-MS: Vanquish Neo UHPLC system sets new performance standards for single-shot nanoLCMS bottom-up proteomics

Significance of the Topic

High-performance liquid chromatography coupled with high-resolution mass spectrometry (nanoLC-HRMS) has become a cornerstone in bottom-up proteomics, enabling deep coverage of complex proteomes in fields such as biomarker discovery, precision medicine and systems biology. Achieving high sensitivity, specificity, and reproducibility demands innovations in chromatography hardware, column design and fluidic control to resolve thousands of peptides in single-shot analyses.

Objectives and Study Overview

This study evaluates the Thermo Fisher Vanquish Neo UHPLC system in combination with the Orbitrap Exploris 480 mass spectrometer and the new PepMap Neo and double nanoViper Neo columns. Key aims include demonstrating: ultrahigh pressure operation for fast sample loading and equilibration; improved chromatographic peak capacity with 75 μm × 75 cm separations; flow-rate versatility for enhanced ionization; reproducibility across multiple columns; and proof-of-principle column coupling for extended separations.

Methodology

Sample Preparation and Workflow

• HeLa protein digest spiked with PRTC standard at 400 ng/μL digest and 200 fmol/μL PRTC.
• Direct injection workflow using 0.2 μL/min to 0.5 μL/min flow rates, gradients of 90 min to 240 min.
• Fast loading and equilibration at 1500 bar enabled by binary pump and split sampler design.

Chromatographic Conditions

• PepMap Neo columns (75 μm × 50 cm and 75 μm × 75 cm, 2 μm particles) operated at 60 °C.
• Gradients ramped from 5% to 28% organic over separation phase, followed by wash and re-equilibration.

Data Acquisition and Processing

• Orbitrap Exploris 480 in data-dependent acquisition mode, MS1 at 60 000 resolution.
• Proteome Discoverer 2.5 with Sequest HT search, FDR below 1% at peptide and protein levels.

Instrumentation

• Thermo Fisher Vanquish Neo UHPLC system (binary pump, split sampler, low-volume fluidics).
• Thermo Fisher Orbitrap Exploris 480 mass spectrometer.
• Thermo Fisher EASY-Spray PepMap Neo and double nanoViper PepMap Neo columns.
• HeLa Digest/PRTC standard and LC-MS grade solvents.

Main Results and Discussion

Fast Loading and High Pressure Capability

• 1500 bar loading and equilibration reduced cycle overhead, increasing MS utilization to over 80% in 4 h analyses.

Enhanced Peak Capacity and Resolution

• 75 cm vs. 50 cm columns in 90 min gradients reduced peptide peak widths by 2 s on average and up to 4 s in best cases, boosting identifications.

Gradient Length and Sample Load Effects

• Extending gradients to 240 min and loading up to 4 μg improved peak height and identification numbers; 1 μg load yielded over 80 000 peptides and 7 100 proteins at 1% FDR.
• Quantification precision remained robust, with >80% peptides and >90% proteins showing RSD <25% across conditions.

Column-to-Column Reproducibility

• Four independent 75 cm PepMap Neo columns produced <5% variation in peptide IDs and <1% in protein IDs under identical 240 min gradients.

Flow Rate Versatility

• Operating flow rates from 200 to 500 nL/min showed up to 40% MS1 sensitivity gain at lower flows without changing identification depth, demonstrating flexibility for sample-limited analyses.

Ultra-Long Column Coupling

• Coupling a double nanoViper 75 cm column to a second 75 cm EASY-Spray column yielded a 150 cm separation (1350 bar backpressure) with ~3 s FWHM improvement in a 240 min gradient, illustrating potential for unparalleled peak capacity.

Benefits and Practical Applications

• Accelerated throughput via rapid loading/equilibration enhances MS duty cycle.
• High reproducibility across columns and runs suitable for large-scale cohort studies and QC.
• Deep proteome coverage in single-shot analyses enables discovery of low-abundance biomarkers.
• Flow-rate flexibility supports sensitivity optimization for limited samples.

Future Trends and Applications

Advances in ultrahigh-pressure nanoLC open avenues for routine use of coupled columns exceeding one meter in length for maximum peak capacity. Integrating real-time feedback and AI-driven method optimization could further enhance throughput and data quality. Continued miniaturization and automation promise broader adoption in clinical and high-throughput environments.

Conclusion

The Vanquish Neo UHPLC system paired with Orbitrap Exploris 480 and PepMap Neo columns delivers robust, high-pressure nanoLC separations with superior peak capacity, throughput and reproducibility. Flow-rate versatility and proof-of-principle column coupling demonstrate its potential to push single-shot bottom-up proteomics to new depths, supporting complex biological investigations and large-scale studies.

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