Thermo Scientific™ UltiMate™ 3000 RSLCnano system with ProFlow™ technology

Importance of the topic

Low-flow liquid chromatography–mass spectrometry (LC-MS) has become a cornerstone in high-resolution proteomics and bioanalysis. By coupling sub-microliter flow rates with advanced mass spectrometers, researchers achieve enhanced sensitivity, reduced solvent consumption, and improved separation efficiency. Such performance is crucial for profiling complex biological matrices, including cell digests and plasma extracts, where trace-level detection and high throughput are both required.

Objectives and study overview

This application note evaluates the Thermo Scientific UltiMate 3000 RSLCnano system equipped with ProFlow technology to:

• Demonstrate robust, quantitative low-flow LC-MS profiling across diverse gradients (8–60 min).
• Assess throughput, peak performance, and MS utilization under different flow rates.
• Compare peptide and protein identifications in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes using standard HeLa digests.
• Illustrate reproducibility and carryover performance in crude plasma analyses over extended sample sets.

Methodology

Gradient lengths from 60 to 8 minutes were run at flow rates of 0.3 to 1.5 µL/min. Key performance metrics recorded per 24 h included sample throughput (24–180 samples), MS utilization (95–75 %), peak width at half-height (PWHM: 10–3 s) and tailing/asymmetry factors (~1.2). DDA experiments employed a Thermo Orbitrap Exploris 480 analyzing 200 ng HeLa digest on a 75 µm × 15 cm EASY-Spray column. DIA workflows compared protein group quantification at CV < 10 % and CV < 20 % across short gradients.

Instrumentation used

• Thermo Scientific UltiMate 3000 RSLCnano system with ProFlow technology
• Thermo Scientific EASY-Spray 75 µm × 15 cm column
• Thermo Scientific Orbitrap Exploris 480 mass spectrometer

Main results and discussion

Shorter gradients increased throughput up to 180 samples per day while maintaining MS utilization above 75 %. Peak capacity and shape remained satisfactory even at 1.5 µL/min. In DDA mode, unique peptide identifications scaled with gradient length, reaching over 25 000 peptides at 48 min. Protein group counts in DIA rose from ~2 525 (8 min) to ~3 164 (48 min) at CV < 20 %, supporting quantitative precision. Crude plasma digest analyses yielded ~204–265 protein groups across 8–60 min methods, with negligible carryover and consistent total ion chromatogram profiles over hundreds of injections.

Benefits and practical applications

• High-throughput workflows with robust run-to-run consistency.
• Versatility to adapt gradient length and flow to diverse sample types.
• Enhanced sensitivity for low-abundance analytes in complex matrices.
• Reduced solvent usage and operational costs at low flow rates.

Future trends and opportunities

Advances may include microfluidic integration for single-cell proteomics, AI-driven method optimization, ultrafast scanning mass analyzers for sub-minute gradients, and expanded compatibility with emerging sample preparation platforms. Continued development of column chemistries and higher-order multiplexing will drive deeper proteome coverage in routine analyses.

Conclusion

The UltiMate 3000 RSLCnano with ProFlow technology delivers a powerful solution for high-throughput, low-flow LC-MS applications. Its combination of speed, sensitivity, and robustness enables both discovery proteomics and quality-control environments to achieve precise, reproducible results across a wide range of sample types.

Reference

• Thermo Fisher Scientific Application Note PlanetOrbitrapA2336
• Thermo Fisher Scientific Application Note PlanetOrbitrapA2357