Routine Single-Shot Identification of >9K Proteins and >100K Peptides with the Next-Generation Low-Flow UHPLC Coupled to HRAM MS

Significance of the Topic

The ability to identify and quantify thousands of proteins in a single liquid chromatography–mass spectrometry (LC-MS) run drives advances in biomarker discovery, systems biology and precision medicine. High-efficiency separations and sensitive detection are essential to overcome complex proteome dynamics and deepen coverage in bottom-up proteomics.

Objectives and Study Overview

This study evaluates the next-generation low-flow Vanquish Neo UHPLC system coupled to an Orbitrap Exploris 480 mass spectrometer using a 75 μm I.D.×75 cm EASY-Spray PepMap Neo column. Key aims include testing long gradients (up to 240 min), assessing flow-rate effects, and demonstrating single-shot identification of >9 000 proteins and >100 000 peptides in HeLa digest.

Materials and Methods

A standardized HeLa digest spiked with PRTC peptides (400 ng/μL HeLa, 200 fmol/μL PRTC) was analyzed by direct injection. Gradients from 90 to 240 min at flow rates of 200–500 nL/min were applied. Data-dependent acquisition (DDA) on the Orbitrap Exploris 480 generated high-resolution MS1 and MS2 spectra. Raw files were processed in Thermo Scientific Proteome Discoverer 2.5/3.0 using Sequest HT, INFERYS rescoring, and chimeric spectrum resolution with a 1% FDR threshold.

Instrumentation Used

• Vanquish Neo UHPLC system with ultra-high pressure up to 1500 bar
• Thermo Scientific EASY-Spray PepMap Neo column (75 μm I.D. × 75 cm)
• Orbitrap Exploris 480 mass spectrometer
• Proteome Discoverer software with Sequest HT and INFERYS

Results and Discussion

Long 75 cm columns improved peak capacity, reducing full width at half maximum (FWHM) by ~2 s versus 50 cm columns in 90 min gradients. Lower flow rates (200 nL/min) enhanced MS1 intensities by up to 40% without affecting the number of MS/MS events or identifications. Using 240 min gradients and 1 μg HeLa load, >7 100 proteins and ~80 000 peptides were quantified (1% FDR). Reprocessing with chimeric spectrum resolution and spectral libraries enabled single-shot identification of >9 000 proteins and >100 000 peptides in 180–240 min runs. Column-to-column reproducibility was excellent, with <5% variation in peptide IDs and <1% in protein IDs across four columns.

Benefits and Practical Applications

• Deep proteome coverage in a single run supports discovery workflows and high-throughput studies
• Reproducible performance across columns enables robust quality control and large cohort analyses
• Flexible operation at varied flow rates optimizes electrospray ionization sensitivity
• Direct injection and trap-and-elute workflows maximize MS utilization and sample throughput

Future Trends and Potential Applications

Continued exploration of ultra-long columns and extended gradients promises even deeper proteome profiling. Integration with advanced data analysis (e.g., AI-driven spectrum deconvolution) and clinical sample workflows may expand applications in biomarker validation and personalized medicine. Emerging nanoLC-MS platforms will likely focus on further reducing sample consumption while maintaining throughput and depth.

Conclusion

The Vanquish Neo UHPLC system combined with a 75 μm I.D.×75 cm PepMap Neo column and Orbitrap Exploris 480 enables routine single-shot bottom-up proteomics with unprecedented depth (>9 000 proteins, >100 000 peptides), high reproducibility, and flexible workflow options.

Reference

1. Zheng R et al. Vanquish Neo UHPLC system sets new performance standards for single-shot nanoLC-MS bottom-up proteomics. TN74152 (2021).