Deep Single-Shot NanoLC-MS Proteome Profiling with a 1500 Bar UHPLC System, Long Fully Porous Columns, and HRAM MS

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Bottom-up proteomics analysis using nanoflow liquid chromatography-mass spectrometry (nanoLC-MS) is crucial for deep proteome profiling.
• Existing nanoLC-MS methods face limitations in throughput and the number of protein identifications per experiment.

Purpose of the Study:

• To demonstrate the integration of ultrahigh-performance liquid chromatography (UHPLC) with high-resolution accurate-mass (HRAM) mass spectrometry (MS) for improved bottom-up proteomics.
• To leverage UHPLC's high back pressure capabilities for enhanced nanoLC-MS performance.

Main Methods:

• Utilized UHPLC technology with long columns (75 cm) packed with 2 µm fully porous particles.
• Employed a constant pressure pump operation at 1500 bar to enable various flow rates (300 nL/min, elevated, and reduced).
• Integrated HRAM mass spectrometry for sensitive and accurate peptide and protein identification.

Main Results:

• Achieved routine proteome profiling and precise quantification of over 7000 proteins in single-shot nanoLC-MS experiments.
• Demonstrated reduced sample loading, washing, and equilibration times, maximizing mass spectrometry utilization.
• Showcased the ability to optimize flow rates for balancing sensitivity, throughput, and sample loading.

Conclusions:

• The combination of UHPLC, HRAM-MS, and long columns significantly enhances bottom-up proteomics analysis.
• This approach enables deeper proteome profiling and precise quantification, facilitating large-scale discovery studies.
• Optimized nanoLC-MS workflows offer greater efficiency and a higher number of protein identifications.