High-throughput capillary-flow LC-MS proteomics with maximum MS utilization

Significance of the topic

Objectives and study overview

• Establish a robust capillary flow LC MS method with an 8 minute run to run cycle
• Maximize mass spectrometer utilization to 75 percent of cycle time
• Maintain high reproducibility and minimal carryover during extended runs

Methodology

• Sample digests of HeLa and Cytochrome C proteins with trap based pre concentration and desalting
• Thermo Scientific UltiMate 3000 RSLCnano system with ProFlow meter and EASY Spray ES800 column 15 cm by 75 micrometer
• Gradient at 1.5 microliter per minute for a total cycle of eight minutes including wash and equilibration
• Mass spectrometry on Q Exactive HF X in Full MS and data dependent acquisition modes Top 40 with high resolution Orbitrap detection
• Data processing with Xcalibur Chromeleon and Proteome Discoverer using SEQUEST HT at sub one percent FDR

Instrumentation

• UltiMate 3000 RSLCnano chromatography system and ProFlow flow meter
• EASY Spray ES800 capillary column and nanoViper fluidic connections
• Q Exactive HF X Orbitrap mass spectrometer

Main results and discussion

• Eight minute sample to sample cycle with six minutes of peptide data acquisition
• Retention time stability under 0.1 minute standard deviation and peak widths below three seconds
• Carryover below 0.2 percent across 180 injections in a 24 hour period
• More than ten thousand MS MS spectra per run and over six thousand peptide spectrum matches for 200 nanograms of digest
• Throughput of 180 samples per day compared to 100 samples per day on an alternative system

Benefits and practical applications

• High throughput proteomic analysis for biomarker validation and quality control
• Reduced solvent consumption and environmental impact
• Minimal downtime for column washing and equilibration
• Customizable methods for diverse applications including metabolomics

Future trends and potential applications

• Integration of capillary flow LC MS in large cohort clinical studies
• Expansion to hydrophilic interaction chromatography metabolomics assays
• Automation of sample preparation and AI assisted data interpretation
• Adoption of microflow methods for higher throughput liquid chromatography separations

Conclusion

References

• Meding S Boychenko A Chromatography Today 2016
• Steinhilber AE et al Anal Chem 2018
• Boychenko A Meding S et al Poster PN64787
• Thermo Fisher Scientific UltiMate 3000 RSLCnano Standard Applications Guide 2016