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Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry
Published on: October 24, 2018
Ultrahigh pressure fast size exclusion chromatography for top-down proteomics
1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA; Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA.
Proteomics
|June 25, 2013
Summary
Ultrahigh pressure size exclusion chromatography (SEC) rapidly separates intact proteins for top-down proteomics. This method enhances signal-to-noise ratios and allows direct mass spectrometry analysis without desalting.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Top-down mass spectrometry (MS)-based proteomics requires efficient separation of intact proteins.
- Size-based separation is crucial due to signal-to-noise ratio decay with increasing protein mass.
- Conventional size exclusion chromatography (SEC) offers low resolution and protein dilution.
Purpose of the Study:
- To introduce ultrahigh pressure (UHP) SEC for rapid and high-resolution intact protein separation.
- To enable direct top-down MS analysis of SEC-eluted proteins.
- To overcome limitations of traditional SEC in proteomics.
Main Methods:
- Utilized ultrahigh pressure (UHP) size exclusion chromatography (SEC).
- Separated intact proteins ranging from 6 to 669 kDa.
- Employed a mass spectrometry (MS)-friendly volatile solvent system.
Main Results:
- Achieved fast separation of intact proteins (< 7 min) with high resolution and efficiency.
- Demonstrated high-resolution separation of intact proteins using a volatile solvent system.
- Enabled direct top-down MS analysis of SEC-eluted proteins, eliminating the need for desalting.
Conclusions:
- UHP-SEC is a highly effective LC strategy for size-based protein separation.
- This method significantly improves intact protein separation for top-down proteomics.
- UHP-SEC offers a promising approach for advancing top-down proteomics workflows.
