A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biomedical Research

Background:

• High throughput and robustness are crucial for integrating mass spectrometry (MS)-based proteomics into clinical settings.
• Current nano-flow liquid chromatography (LC) systems offer sensitivity but lack the throughput required for widespread clinical application.
• High-flow rate systems for small molecules meet throughput needs but lack the sensitivity for complex proteomic samples.

Purpose of the Study:

• To introduce a novel LC system concept designed for high throughput and robustness in proteomics.
• To maintain the high sensitivity of nano-flow LC systems while significantly increasing sample analysis speed.
• To enable deeper proteome coverage and faster protein quantitation for biomedical and clinical research.

Main Methods:

• A simplified LC system design utilizing low-pressure pumps to elute samples from a trap column.
• Formation of a chromatographic gradient within a storage loop, combined with an auxiliary gradient for peptide re-focusing.
• Separation on an analytical column driven by a single high-pressure pump, enabling parallel processing steps between injections.

Main Results:

• The system, named Evosep One, demonstrated robust operation over thousands of sample injections.
• Analysis of fractionated HeLa cell lysates yielded deep proteomes, identifying over 130,000 unique peptides and nearly 10,000 proteins.
• Rapid quantitation of 5200 proteins was achieved in just 21 minutes using a data library.

Conclusions:

• The Evosep One system provides an extremely robust and high-throughput solution for proteomics analysis.
• This technology significantly reduces overhead time, allowing for the analysis of over 200 samples per day.
• The system achieves in-depth proteome coverage and rapid quantitation, making it suitable for demanding research and clinical applications.