Ion-interaction CZE: the presence of high concentrations of ion-pairing reagents demonstrates the complex mechanisms involved in peptide separations.
We have furthered our understanding of the separative mechanism of a novel CE approach, termed ion-interaction CZE (II-CZE), developed in our laboratory for the resolution of mixtures of cationic peptides. Thus, II-CZE and RP-HPLC were applied to the separation of peptides differing by a single amino acid substitution in 10- and 12-residue synthetic model peptide sequences. Substitutions differed by a wide range of properties or side-chain type (e.g., alkyl side-chains, polar side-chains, etc.) at the substitution site. When carried out in high concentrations (400 mM) of pentafluoropropionic acid (PFPA), II-CZE separated peptides in order of increasing hydrophobicity when the substituted side-chains were of a similar type; when II-CZE was applied to the mixtures of peptides with substitutions of side-chains that differed in the type of functional group, there was no longer a correlation of electrophoretic mobility in II-CZE with relative peptide hydrophobicity, suggesting that a third factor is involved in the separative mechanism beyond charge and hydrophobicity. Interestingly, the hydrophobic PFPA- anion is best for separating peptides that differ in hydrophobicity with hydrophobic side-chains but high concentrations of the hydrophilic H2PO4- anion are best when separating peptides that differ in polar side-chains relative to hydrophobic side-chains. We speculate that differential hydration/dehydration properties of various side-chains in the peptide and the hydration/dehydration properties of the hydrophilic/hydrophobic anions as well as the electrostatic attractions between the peptide and the anions in solution all play a critical role in these solution-based effects.
Popa
Traian V
TV
Department of Biochemistry and Molecular Genetics, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.
Mant
Colin T
CT
Hodges
Robert S
RS
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000737:chemistry; D010455:Peptides / Q000302:isolation & purification*; D015203:Reproducibility of Results
D007477:Ions; D010455:Peptides