Gradient-Elution Nanoflow Liquid Chromatography Without a Binary Pump: Smoothed Step Gradients Enable Reproducible,...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Low flow rate liquid chromatography (LC) is crucial for mass spectrometry-based proteome profiling of trace analytes, including single cells.
• Existing methods require expensive high-pressure binary pumps and can suffer from flow inconsistencies due to gradient splitting.
• There is a need for cost-effective and reliable gradient generation methods for low-flow LC-MS.

Purpose of the Study:

• To develop a novel, cost-effective method for generating smooth liquid chromatography gradients at low flow rates.
• To demonstrate the application of this method for sensitive proteome profiling using mass spectrometry.
• To investigate differential protein expression and immune responses in single cells.

Main Methods:

• A new gradient elution technique was developed using Taylor dispersion in an open capillary to combine mobile phase segments.
• This method utilizes a single isocratic high-pressure pump, eliminating the need for binary pumps and flow splitting.
• The developed gradient profiles were applied to LC-MS/MS analysis of HeLa cell digests and single-cell proteomes using a timsTOF mass spectrometer.

Main Results:

• The Taylor dispersion-based method successfully generated smooth 10- and 20-min active gradients.
• Accurate quantification of thousands of proteins was achieved from 200 pg injections of HeLa digest.
• Significant differences in protein expression were identified between two ATG-KO HeLa cell colonies, revealing a potential mechanism for differential viral DNA immune responses.

Conclusions:

• The developed Taylor dispersion gradient method offers a cost-effective and robust alternative for low-flow LC-MS.
• This technique enables sensitive and reproducible proteome profiling of trace analytes and single cells.
• The study provides insights into cellular heterogeneity and immune response mechanisms at the proteome level.