Selective Comprehensive Online NanoLCxCZE-MS Platform for Top-Down Proteoform Analysis.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Efficient separation techniques are essential for comprehensive proteoform analysis in complex biological samples.
• Two-dimensional (2D) separation methods offer superior selectivity and peak capacity over one-dimensional (1D) approaches.

Purpose of the Study:

• To develop and present a novel online selective comprehensive 2D platform coupling reversed-phase nanoLC with capillary zone electrophoresis-mass spectrometry (CZE-MS).
• To apply this platform for intact top-down (TD) proteoform analysis.
• To enhance the detection and characterization of proteoforms in complex biological samples.

Main Methods:

• Development of an online selective comprehensive 2D platform integrating reversed-phase nanoLC (1st dimension) and capillary zone electrophoresis-mass spectrometry (CZE-MS) (2nd dimension).
• Utilized successive multiple ionic-polymer layers for enhanced CZE-MS separation.
• Analyzed a human cell lysate to evaluate the platform's performance for intact TD proteoform characterization.

Main Results:

• The 2D platform detected two to four times more proteoforms compared to 1D nanoLC-MS within the same nanoLC time window.
• Targeted analysis of Histone H4 identified additional proteoforms with variations in acetylation and methylation previously undetected by 1D nanoLC-MS.
• Demonstrated the platform's capability for advanced intact proteoform analysis.

Conclusions:

• The coupled nanoLC and CZE-MS platform provides significantly improved proteoform detection and characterization at the intact protein level.
• This 2D approach advances targeted proteoform analysis in complex biological matrices.
• The developed platform holds promise for future in-depth proteomic studies.