Tandem nanoLC-MS: maximum MS utilization for deep-dive proteomic analysis

Significance of the Topic

Time-intensive nanoLC-MS workflows for deep proteome profiling suffer from long idle times due to column washing, equilibration and sample loading. Tandem nano-flow LC strategies can dramatically improve mass spectrometer utilization by overlapping sample elution on one separation channel with column cleaning and sample loading on a second channel. This approach enhances throughput and sensitivity in discovery proteomics and industrial QC contexts.

Objectives and Study Overview

This study describes the design and implementation of a tandem nano-flow LC-MS platform using the Thermo Scientific UltiMate 3000 RSLCnano system. The aims are to double instrument throughput, achieve over 97 percent continuous MS data acquisition, minimize idle time, reduce carryover, and demonstrate reproducible protein identification in deep-dive proteomic experiments.

Methodology and Instrumentation

• System configuration: An UltiMate 3000 RSLCnano equipped with two high-pressure gradient pumps, dual columns and trap setup, nano switching valves and WPS-3000TFC autosampler. Post-column flow diversion directs wash eluent to waste.
• Columns and consumables: Two 75 µm × 50 cm Acclaim PepMap RSLC C18 columns with 2 µm particles, trap cartridges, nanoViper capillaries and fittings.
• Sample: Thermo Scientific HeLa protein digest at 200 ng/µL.
• LC conditions: 90-minute shallow gradient at 300 nL/min; parallel saw-tooth wash cycles for column cleaning and equilibration.
• MS settings: Orbitrap analysis with 60 k resolution MS1, top 20 data-dependent acquisition, 15 k resolution MS2, 375–1500 m/z range.
• Data processing: Thermo Scientific Proteome Discoverer 2.2 with Sequest HT search below 1 percent FDR.

Main Results and Discussion

• Continuous MS utilization above 97 percent was achieved by alternating elution and wash steps on two separation channels.
• Retention time reproducibility within and between columns was maintained with relative standard deviations of 1.8 to 7.1 percent for selected peptides.
• Carryover in blank runs was below 0.2 percent for targeted HeLa peptides.
• Reproducible protein identification with over 4200 protein groups per injection and inter-channel RSD of 1.6 to 4.0 percent for PSMs, peptide and protein counts across 22 injections.
• Minor increase in peak width due to post-column nano valve dead volume.

Benefits and Practical Applications

The tandem nanoLC-MS workflow offers:

• Near full utilization of MS acquisition time for higher throughput in discovery and QA/QC proteomics.
• Enhanced cleaning and equilibration without extending total cycle time.
• Flexibility to adapt gradient length and column dimensions to experimental needs.

Future Trends and Applications

• Integration with ultrahigh resolution instruments and ion mobility for deeper proteome coverage.
• Automation of valve switching and method setup through advanced software scripting.
• Application to glycoproteomics, metabolomics or single-cell proteomics requiring high sensitivity and throughput.
• Scaling to multiplexed tandem configurations for further throughput gains.

Conclusion

The tandem nano-flow LC-MS configuration on the UltiMate 3000 RSLCnano platform significantly increases mass spectrometer productivity for deep-dive proteomic analyses. By overlapping sample elution, column cleaning and loading, this approach achieves continuous MS acquisition, robust chromatography and reproducible protein identification with minimal carryover.

References

• Thermo Scientific UltiMate 3000 RSLCnano System – Versatility and Performance – the Ultimate Solution for All Separation Workflows, BR-71898; 2016.
• Thermo Scientific UltiMate 3000 RSLCnano Standard Applications Guide v3.0; 2018.
• LC-MS methods available in AppsLab https://appslab.thermofisher.com/App/4213/tandemnanolcms