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Updated: May 10, 2026
10:23
Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid
Published on: December 4, 2017
High-resolution peptide mapping separations with MS-friendly mobile phases and charge-surface-modified C18.
Matthew A Lauber1, Stephan M Koza, Scott A McCall
1Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757-3696, United States. Matthew_Lauber@Waters.com
Analytical Chemistry
|June 19, 2013
Summary
A new charged-surface C18 stationary phase (CSH C18) enhances peptide separation efficiency in chromatography. This advanced column chemistry offers improved performance for mass spectrometry applications, aiding complex analyses like peptide mapping.
Area of Science:
- Analytical Chemistry
- Chromatography
- Biochemistry
Background:
- Ionic analytes, like peptides, present separation challenges in reverse-phase chromatography.
- Poor peak shapes and mobile phase compatibility issues hinder optimal separation efficiency, especially for mass spectrometry (MS).
Purpose of the Study:
- To evaluate a novel charged-surface hybrid (CSH) C18 stationary phase for reverse-phase peptide separations.
- To compare the performance of CSH C18 against the established ethylene-bridged hybrid (BEH) C18 column chemistry.
Main Methods:
- Separation of a nine-peptide standard using CSH C18 and BEH C18 columns.
- Evaluation of peak shape, loadability, peak capacity, and selectivity.
- Application to peptide mapping of the therapeutic antibody trastuzumab using LC-UV-MS.
Main Results:
- CSH C18 demonstrated improved loadability, higher peak capacities, and unique selectivity compared to BEH C18.
- The CSH C18 phase showed reduced dependence on trifluoroacetic acid (TFA) ion pairing, beneficial for MS sensitivity.
- CSH C18 facilitated the development of a high-resolution, high-sensitivity LC-UV-MS peptide mapping method for trastuzumab.
Conclusions:
- The charged-surface C18 stationary phase (CSH C18) effectively addresses challenges in reverse-phase peptide separations.
- CSH C18 offers significant advantages for MS-based peptide analysis, including improved performance and reduced mobile phase optimization needs.
- This novel column chemistry shows promise for advancing challenging peptide analyses and biopharmaceutical characterization.
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