ZIC-cHILIC as a fractionation method for sensitive and powerful shotgun proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Multidimensional liquid chromatography (LC) coupled with mass spectrometry (MS) is crucial for complex biological samples in proteomics.
• Effective fractionation is essential for comprehensive proteomic analysis but often leads to significant sample loss.
• Analyzing limited biological materials like cell lines and sorted cells presents challenges due to sample loss during extensive fractionation.

Purpose of the Study:

• To develop and present a two-dimensional LC protocol for proteomics that minimizes sample loss.
• To optimize sample preparation for maximum recovery in a Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-cHILIC) and reversed-phase LC system.
• To demonstrate the sensitivity and versatility of the developed protocol using various biological samples.

Main Methods:

• A two-dimensional LC strategy combining Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-cHILIC) with reversed-phase chromatography.
• Detailed discussion of experimental aspects for optimizing sample recovery.
• Application of the protocol to protein lysates from cultured cell lines and flow cytometry-sorted cells.

Main Results:

• The ZIC-cHILIC and reversed-phase LC combination enables proteomic analysis with minimal sample loss.
• The protocol is effective for analyzing complex biological samples, including those from limited sources.
• Demonstrated power, sensitivity, and versatility across different sample types.

Conclusions:

• This two-dimensional LC protocol offers a solution to sample loss in proteomics, particularly for limited material analysis.
• The ZIC-cHILIC based fractionation system provides a robust and sensitive approach for comprehensive proteomic studies.
• The protocol, once optimized, requires approximately 5-6 days for completion, including sample preparation and analysis.