Identifying kinetically stable proteins with capillary electrophoresis

Abstract

Unlike most proteins, which are in equilibrium with partially and globally unfolded conformations, kinetically stable proteins (KSPs) are trapped in their native conformations and are often resistant to harsh environment. Based on a previous correlation between kinetic stability (KS) and a protein's resistance to sodium dodecyl sulfate (SDS), we show here a simple method to identify KSPs by SDS-capillary electrophoresis (CE). Control non-KSPs were fully denatured by SDS and formed protein:SDS complexes that exhibited similar mobility in CE. In contrast, KSPs bound fewer SDS molecules, and showed a very different migration time and peak pattern in CE, thereby providing some insight about the structural heterogeneity of SDS:protein complexes and the relative KS of the various proteins.

Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking

“…Some of these proteins do not properly migrate in SDS CSE either [46,58–60]. Probably, excessive β-sheet structure is the cause of the anomalous behavior and denaturation [58–61].…”

“…Nevertheless, some proteins do not denature on the capillary and require a heat denaturation prior the analysis. In this respect, the method does not differ significantly from SDS electrophoresis [59,60]. …”

The potential of the capillary electrophoresis techniques for quality control of biopharmaceuticals—A review

“…The simplest and most commonly used CE mode is CZE that separates analytes based on the differences in their electrophoretic mobility . CZE has been applied to determine acetic acid as a counterion in bradykinin acetate products ; assay the enzymatic activity of β1,4‐galactosyltranferase ; separate mixture of therapeutic peptide hormones ; quantify net charge of proteins ; identify kinetically stable proteins ; and characterize the electrical properties of antihuman vascular endothelial growth factor proteins to develop their iontophoretic delivery systems .…”

Proteomics Analysis of Kinetically Stable Proteins

“…Based on the previous correlation between KS and a protein's SDS-resistance, we set out to explored whether SDS-capillary electrophoresis (CE) would be suitable for identifying KSPs (Zhang, Xia, Chung, Cramer, & Colon, 2010). We used eight control proteins, including four KSPs and four non-KSPs.…”