High-Performance Proteomics Using Nano-, Capillary-, and Microflow Chromatographic Separations

Area of Science:

• Proteomics
• Analytical Chemistry
• Chromatography

Background:

• Mass spectrometry-based proteomics requires sensitive and high-throughput methods.
• Complex proteomes and wide dynamic ranges necessitate advanced chromatographic separations.
• Optimizing chromatography is crucial for mass spectrometry performance.

Purpose of the Study:

• To systematically evaluate proteome analysis performance across diverse chromatographic flow rates and column diameters.
• To benchmark performance using serial dilutions of HeLa cell digests.
• To provide empirical guidance for selecting optimal chromatography parameters in proteomics.

Main Methods:

• Utilized a Vanquish Neo HPLC system coupled online to a Q Exactive HF-X mass spectrometer.
• Evaluated flow rates ranging from 0.3 to 50 μL/min with various column diameters.
• Maintained a fixed total analysis time of 60 minutes per sample, enabling 24 samples per day.

Main Results:

• All tested chromatographic flow rates supported high-quality proteome analysis.
• Capillary liquid chromatography (capLC) at 1.5 μL/min proved to be a robust, sensitive, and quantitative option compared to nano liquid chromatography (nLC).
• Data on proteome, phosphoproteome, and drug proteome analysis offer practical insights for method selection.

Conclusions:

• Chromatographic flow rate and column diameter selection significantly impacts proteomic analysis outcomes.
• capLC presents a viable and efficient alternative to nLC for numerous proteomic applications.
• The study provides valuable data to guide researchers in optimizing their specific proteomic workflows.