Robust Capillary- and Micro-Flow Liquid Chromatography-Tandem Mass Spectrometry Methods for High-Throughput Proteome...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Capillary-flow liquid chromatography-tandem mass spectrometry (capLC-MS/MS) and micro-flow LC-MS/MS are emerging as robust, high-throughput alternatives to nano-flow LC-MS/MS.
• A systematic comparison of these systems for global proteome profiling is lacking.

Purpose of the Study:

• To systematically compare capLC-MS/MS and μLC-MS/MS systems for global proteome profiling.
• To evaluate their performance in terms of sensitivity, throughput, separation efficiency, and robustness.

Main Methods:

• Established capLC-MS/MS (150 μm i.d. column, 1 μL/min) and μLC-MS/MS (1 mm i.d. column, 50 μL/min) systems.
• Utilized an UltiMate 3000 RSLCnano coupled to an Orbitrap Exploris 240.
• Conducted a 7-day long-term performance test.

Main Results:

• capLC-MS/MS demonstrated ~10 times higher sensitivity than μLC-MS/MS.
• μLC-MS/MS achieved higher analysis throughput and separation efficiency.
• Both systems exhibited excellent reproducibility in chromatographic width, retention time, and protein identification (RSD < 3%).

Conclusions:

• capLC-MS/MS is ideal for high-throughput clinical proteomic analysis with limited sample material.
• μLC-MS/MS is preferred when larger sample quantities are available.
• Both systems, when coupled with the Orbitrap Exploris 240, are suitable for large-cohort clinical proteomic studies.