Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic...

Area of Science:

• Proteomics and Analytical Chemistry

Background:

• Hydrophilic interaction chromatography (HILIC) retention of charged analytes is significantly increased by well-hydrated counterions.
• Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) combines charged columns with HILIC for enhanced selectivity.
• ERLIC has been explored for isolating peptides with specific post-translational modifications (PTMs).

Purpose of the Study:

• To investigate the use of magnesium trifluoroacetate as a salt additive in ERLIC for enhanced peptide and PTM isolation.
• To evaluate the selectivity and efficiency of ERLIC for enriching peptides with multiple or complex PTMs.
• To compare the performance of ERLIC with other established methods for PTM-enriched peptide isolation.

Main Methods:

• Utilized ERLIC with magnesium trifluoroacetate as the optimal salt additive.
• Applied the optimized ERLIC conditions to a tryptic digest of mouse cortex.
• Employed gradient elution to generate fractions enriched in peptides with various PTMs.
• Analyzed fractions using C-18 reversed-phase liquid chromatography-tandem mass spectrometry after desalting.

Main Results:

• Magnesium trifluoroacetate significantly enhanced peptide retention and selectivity (6- to 66-fold) by interacting with charged functional groups.
• ERLIC successfully enriched peptides containing phosphate, mannose-6-phosphate, and N- and O-linked glycans.
• The number of identified PTM-containing peptides using ERLIC equaled or exceeded those from alternative methods.
• A significant proportion (47-100%) of multiply phosphorylated or sialylated peptides were unretained on C-18 but retained on porous graphitic carbon, suggesting prior underestimation.

Conclusions:

• ERLIC with magnesium trifluoroacetate is a powerful and convenient method for simultaneously isolating peptides with diverse PTMs.
• This technique facilitates the study of complex protein modifications and crosstalk.
• Findings suggest that previous proteomic studies may have underestimated the abundance of certain PTM-containing peptides due to analytical method limitations.