Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants†

Abstract

One pressure and three chemical mobilization strategies have been optimized and tested for two-step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1-10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3-10 with Pharmalyte 5-6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5-6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. l-Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. l-Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed.

Determination of protein charge variants with (imaged) capillary isoelectric focusing and capillary zone electrophoresis

“…Kristl et al. also varied H 3 PO 4 and NaOH concentrations and could not observe significant differences in the measurement of model proteins. Therefore, the highest tested concentrations, 200 mM H 3 PO 4 and 300 mM NaOH, were chosen due to the stated advantages of Mack et al.…”

“… changed buffers after every third injection and Kristl et al. changed anolyte and catholyte after two or three runs, for example.…”

“…Moreover, it is common to mix broad CAs of p I 3–10 with narrow ones that comprise the p I range of the sample, allowing a higher resolution within the requested p I range. Caution is required to avoid peak splitting and therefore an unnoticed distortion of the results. A thoroughly optimization of the CAs’ concentrations and mixing ratios must be an integral part of method development.…”

“…A comprehensive comparison of different mobilization techniques was done by Kristl et al. . In particular, compositions and concentrations of the CAs were adapted individually.…”

“…This method was validated according to ICH Q2(R1) and did not include urea or salt. Kristl et al [58] are one of the few in the last years who intensively studied different kinds of mobilization. One pressure and three chemical mobilization strategies of ovalbumin variants were compared.…”