Multiple-buffer-additive strategies for enhanced capillary electrophoretic separation of peptides

Area of Science:

• Analytical Chemistry
• Biochemistry
• Separation Science

Background:

• Structurally similar peptides present significant separation challenges in analytical chemistry.
• Thyroid stimulating hormone (TSH) beta-subunit derived peptides serve as a model for complex mixture analysis.

Purpose of the Study:

• To develop and evaluate "multiple-buffer-additive" strategies for resolving structurally similar peptides.
• To investigate the individual and combined effects of acetonitrile (ACN), hexane sulfonic acid (HSA), and hexamethonium bromide (HxMBr) on peptide separation.

Main Methods:

• Utilized a dodeca-peptide from beta-TSH and a series of synthetic analogs, including peptides with identical amino acid compositions and alanine substitutions.
• Employed capillary electrophoresis (CE) with systematic variation of buffer additives: ACN (hydrophobicity modifier), HSA (ion-pairing agent), and HxMBr (solute-wall interaction modifier).

Main Results:

• Individual additives (ACN, HSA, HxMBr) provided only partial resolution of the peptide mixtures.
• A synergistic combination of HSA and ACN was found to be essential for achieving complete resolution of all tested peptide components.
• Buffer additive combinations significantly altered peptide selectivity, demonstrating the impact of hydrophobicity and ionic interactions.

Conclusions:

• Multiple-buffer-additive strategies are crucial for resolving challenging peptide mixtures.
• Buffer hydrophobicity and ionic strength modulation are key factors in optimizing capillary electrophoresis selectivity.
• Nearest-neighbor effects, secondary structure, and solvation likely influence peptide mobility and separation outcomes.