Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Microflow liquid chromatography tandem mass spectrometry (microLC-MS/MS) is emerging as a practical alternative to nanoflow LC-MS/MS for proteomic analyses.
• Previous work highlighted the potential of microLC-MS/MS systems for high-throughput applications using specific column dimensions and flow rates.

Purpose of the Study:

• To evaluate the robustness and performance of a microLC-MS/MS system for large-scale proteomic analysis.
• To demonstrate the system's capability for both in-depth and high-throughput clinical applications.

Main Methods:

• Analysis of approximately 38,000 samples over two years using two microLC-MS/MS instruments.
• Utilized a 1 mm i.d. × 150 mm column operating at a flow rate of 50 microL/min.
• Analyzed diverse proteomes (cell lines, tissues, human body fluids) with and without peptide fractionation or stable isotope labeling.

Main Results:

• The microLC-MS/MS approach proved extremely robust, with up to 1500 analyses performed monthly.
• A single column supported over 14,000 sample analyses without compromising chromatographic performance.
• The system quantified ~2600 proteins from undepleted human plasma and ~5000 proteins from crude human urine within a single day.

Conclusions:

• MicroLC-MS/MS is a highly robust and efficient technique for large-scale proteomic studies.
• The system demonstrates significant potential for both in-depth and high-throughput clinical proteomic applications.
• Exceptional column stability and high sample throughput make microLC-MS/MS suitable for demanding analytical workflows.