Thermo Scientific UltiMate 3000 RSLCnano System

Importance of the Topic

The demand for high-resolution, high-sensitivity liquid chromatography at nano- and micro-flow rates has grown with the advancement of proteomics, biopharmaceutical characterization, and QA/QC applications. The flexibility to tune flow rates from nano to analytical scales while maintaining retention time precision and system robustness is critical for in-depth analysis of complex samples and large-scale quantitative studies.

Objectives and Overview

This application note evaluates the versatility and performance of the Thermo Scientific UltiMate 3000 RSLCnano System. Key goals include demonstrating:

• Precise flow control across 0.05–50 µL/min using ProFlow technology
• Compatibility with micro, capillary, and nano workflows
• Integration with mass spectrometers for high-confidence qualitative and quantitative analyses

Methodology

The system was assessed using:

• Peptide digests (e.g., cytochrome C, HeLa cell lysate)
• Varied column formats (75 µm × 150 mm to 75 µm × 750 mm, 2 µm particle size)
• Gradient lengths from 30 to 240 min
• Reproducibility studies over multiple days and replicates

Used Instrumentation

• UltiMate 3000 RSLCnano System with ProFlow continuous-flow pump
• WPS 3000 autosampler with µL pick-up injection and micro-fraction collection
• Thermo Scientific nanoViper low-dead-volume connections and snap-in valves
• Columns: Acclaim PepMap RSLC C18, ProSwift RP-10R, EASY-Spray
• Detectors: UV/Vis variable wavelength for nano, capillary, micro flows
• Mass spectrometers: Q Exactive HF, Orbitrap Fusion Lumos, TSQ Quantiva

Main Results and Discussion

• Retention time precision: RSD < 0.20% over five days (12 injections per day) with 75 µm × 150 mm PepMap columns
• Peak capacity up to ~900 using long nano columns (75 µm × 750 mm at 60 °C, 750 bar) and temperature control
• Label-free quantification: >40,000 protein targets with <15% peak area RSD across seven replicates of HeLa digest (90 min gradient, 300 nL/min)
• Proteome coverage: Identified up to 4,328 proteins in 90 min runs (1 µg load) with high inter-run consistency
• Sensitivity gains: ~393-fold higher signal at nano flow (75 µm ID) compared to analytical flow (2.1 mm ID) for cytochrome C digest
• Intact protein separation: Clear resolution of rituximab heavy and light chains via capillary RP chromatography with UV and MS detection
• Targeted peptide quantification: Achieved 10 amol sensitivity and robust reproducibility at 4 µL/min using 0.3 × 50 mm PepMap and HESI-II probe

Benefits and Practical Applications

• High retention time stability and reproducibility support large cohort quantitative studies
• Flexible workflow configurations enable direct injection, pre-concentration, 2D separations, offline fractionation, and tandem LC
• Robust integration with Thermo Scientific mass spectrometers via SII facilitates streamlined LC-MS/MS setups
• Enhanced sensitivity allows detection of low-abundance analytes in proteomics and biopharmaceutical quality control

Future Trends and Possibilities for Use

The ongoing evolution of nano-UHPLC will focus on:

• Further increasing system throughput through multiplexed columns and parallel analysers
• Deeper integration with AI-driven method optimization and real-time data processing
• Expanding multidimensional LC strategies to dissect complex proteomes and metabolomes
• Improved nano-ESI interfaces for extended compatibility with emerging high-resolution MS platforms

Conclusion

The UltiMate 3000 RSLCnano System delivers exceptional versatility across flow regimes, superior retention time precision, and outstanding chromatographic efficiency. Its seamless integration with high-resolution mass spectrometers and robust operation make it a powerful solution for advanced proteomics, biopharmaceutical characterization, and targeted quantification.

References

• Thermo Fisher Scientific. UltiMate 3000 RSLCnano System Application Note BR71898-EN. 2016.