CZE-MS peptide mapping: To desalt or not to desalt?

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Matrix removal is crucial in shotgun proteomics using HPLC or CZE-MS.
• CZE has high salt tolerance, yet desalting is commonly reported.
• Lack of comparative studies on desalted vs. non-desalted digests creates ambiguity in CZE-MS protocols.

Purpose of the Study:

• To investigate the impact of desalting on CZE-MS analysis of protein digests.
• To compare desalted and non-desalted samples across varying complexities and matrices.
• To establish optimal sample preparation for CZE-MS.

Main Methods:

• Solid-phase extraction (SPE) pipette tips for sample cleanup.
• Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS).
• Analysis of standard protein digests and complex yeast cell lysate.

Main Results:

• Desalting altered peptide profiles, primarily affecting hydrophilic peptides.
• Desalting enhanced field-amplified sample stacking due to low conductivity.
• Non-desalted samples showed stacking via transient-isotachophoresis; adding ions/acetonitrile improved this.
• Complex samples exhibited similar trends.

Conclusions:

• Sample cleanup, specifically desalting, can be omitted in bottom-up CZE-MS.
• Eliminating desalting may improve detection sensitivity, separation efficiency, and sequence coverage.
• Non-desalted CZE-MS offers a potentially more sensitive and efficient proteomic analysis method.