Novel On-Line Multidimensional Low-Flow LCMS Setups for Comprehensive and Fast Proteome Profiling

Importance of the Topic

Comprehensive proteome profiling is essential for understanding biological systems, disease mechanisms and biomarker discovery. Traditional one-dimensional nano-LC-MS workflows force a compromise between depth of coverage and sample throughput. Online multidimensional separations offer a route to improve peptide identification rates while retaining high throughput and robustness required for routine analysis of complex biological matrices.

Objectives and Study Overview

This work demonstrates a simple online two-dimensional low-flow reversed-phase × reversed-phase (RP × RP) LC-MS/MS setup. The study aims to split a peptide digest into multiple fractions within a conventional nano-LC time frame (~2 h), achieve minimal fraction overlap, and deliver high reproducibility and proteome depth using industry-standard hardware.

Methodology

The sample (HeLa protein digest) was separated in the first dimension at high pH on a PepSwift monolithic capillary column. Automated fractionation onto trap cartridges was achieved via a two-position switching valve. Eluate was acidified online, transferred to one of two traps and then resolved in the second dimension at low pH on an EASY-Spray nano column. Gradients were staggered to distribute peptides evenly across fractions and maximize MS utilization.

Used Instrumentation

The online 2D setup employed a Thermo Scientific UltiMate 3000 RSLCnano system, configured with NCS-3500RS, NCP-3200RS, WPS-3000RS and VWD-3400RS modules. A Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer acquired data under Full MS mode. Control and data processing were managed via Xcalibur 4.1, SII and FreeStyle 1.5 software.

Main Results and Discussion

Peptide peaks were symmetrical in each fraction with negligible overlap, confirming the orthogonality of high-pH and low-pH RP separations. Four fractions were collected and analyzed in approximately four hours, enabling deep coverage without extending the total run time significantly. Second-dimension throughput supports up to 180 analyses per day while preserving high MS utilization and retention time precision comparable to one-dimensional methods.

Benefits and Practical Applications

• High reproducibility across both dimensions and minimal manual handling of fractions.
• Compatibility with standard nano-LC workflows and minimal hardware modifications.
• Enhanced peptide and protein identifications in challenging matrices.
• Potential for scheduled targeted assays in complex samples.

Future Trends and Potential Applications

Further developments may include integration of ion mobility separation, optimization of gradient shapes with machine learning, scaling to more fractions for ultra-deep profiling and deployment in clinical proteomics for high-throughput biomarker discovery. Automation of valve switching and data analysis pipelines will broaden accessibility.

Conclusion

The proposed online 2D low-flow RP × RP LC-MS approach offers a balanced solution between speed and depth for proteome profiling. It is robust, easy to implement on existing nano-LC systems and capable of high reproducibility, making it a valuable alternative to long one-dimensional methods.

Reference

1. RSLCnano pre-concentration nano LC kit, Ultimate 3000 RSLCnano Standard Applications Guide, Thermo Fisher Scientific.
2. Boychenko A., Pynn C., van den Berg B., et al. High-throughput capillary-flow LC-MS proteomics with maximum MS utilization. Thermo Fisher Scientific TN 72227.