Ion-interaction–capillary zone electrophoresis of cationic proteomic peptide standards

Abstract

We have employed a novel capillary electrophoresis (CE) approach recently developed in our laboratory, termed ion-interaction-capillary zone electrophoresis (II-CZE), to the resolution of a mixture of 27 synthetic cationic proteomic peptide standards. These peptides were comprised of three groups of nine peptides (with net charges of +1, +2 and +3 for all nine peptides within a group), the hydrophobicity of the nine peptides within a group varying only subtly between adjacent peptides. This bidimensional CE approach achieved excellent resolution of the peptides with high peak capacity by combining the powerful CZE mechanism located in the background electrolyte (BGE) with an hydrophobicity-based mechanism also located in the BGE, the latter consisting of high concentrations (up to 0.4 M) of aqueous perfluorinated acids ( trifluoroacetic acid, pentafluoropropionic acid and heptafluorobutyric acid). Thus, concomitant with a CZE separation of the three differently charged groups of peptides, there is an hydrophobically-mediated separation of the peptides within these groups effected through interaction of the hydrophobic anions of the perfluorinated acids with hydrophobic amino acid side-chains in the peptides. This methodology is dramatically different from other CE methods that have used complexing agents such as micelles or cyclodextrins in MEKC. Overall, the results presented here demonstrate the value of CE as a peptide separative tool in its own right, including its use for proteomic applications, and not merely as a complementary technique to reversed-phase high-performance liquid chromatography (RP-HPLC).

Ion‐interaction CZE: The presence of high concentrations of ion‐pairing reagents demonstrates the complex mechanisms involved in peptide separations

“…The migration order in the presence of all three hydrophobic counterions is the same for both the +1 and +2 peptide groups. These results confirm our previous findings that the phosphate counterion performs poorly in the separation of peptides differing subtly in hydrophobicity of alkyl side-chains [20]. All three of the +2 peptides (K, R, H) were baseline resolved under all conditions, albeit the separation improved with increasing hydrophobicity of perfluorinated acid (TFA<PFPA<HFBA).…”

“…The results of Figs. 5 and 6 would suggest that the hydrophobically mediated mechanism proposed by Popa et al [20] is in fact occurring for a wide range of side-chain types.…”

“…The addition of such perfluorinated anionic ion-pairing reagents more commonly employed in (RP-HPLC) mobile phases at low pH [9][10][11][12][13][14][15][16][17][18] to the cationic peptides allowed resolution of the peptides through a mechanism based on differences in peptide hydrophobicity [14,19,20]. Migration times of the peptides increased with increasing acid concentration and increasing hydrophobicity of the acidic counterion (TFA − < PFPA − <HFBA − ).…”

“…The separations at these high perfluorinated acid concentrations represent a monodimensional separation process based on two unrelated parameters: first, peptides of the same length but of different nominal charge are separated in order of decreasing charge (i.e., +3 peptides migrate the earliest and +1 peptides migrate last) according to a CZE mechanism; and second, peptides within each group of peptides with the same nominal charge are separated in order of increasing hydrophobicity according to an hydrophobically mediated mechanism introduced by the anionic ion-pairing reagent. Thus, we termed this CE method as ion-interaction CZE (II-CZE) [14,[19][20][21]. In our most recent study, peptides of identical charge-to-mass ratio were resolved using II-CZE [21].…”

Recent developments in CE and CEC of peptides

“…A new CZE approach, the so-called ion-interaction CZE (II-CZE), has been employed to the separation of 27 synthetic cationic proteomic peptide standards comprising of three groups of nine decapeptides with net charges of 11, 12, and 13 for all nine peptides within a group and with subtle changes of hydrophobicity within each group of nine peptides [97]. This bidimensional CE approach provided an excellent resolution of the peptides with high peak capacity by combining the powerful inherent ZE separation mechanism (different electrophoretic mobilities due to different charge/size ratios) with an hydrophobicity-based mechanism consisting in differences of peptide interactions with BGE constituents, perfluorinated carboxylic acids (trifluoroacetic, pentafluoropropionic, heptafluorobutyric) present in high concentrations (up to 400 mM) and adjusted by LiOH to pH 2.0.…”