Capillary zone electrophoresis of metal‐binding proteins in formic acid with UV‐ and mass spectrometric detection using cationic transient capillary isotachophoresis for preconcentration

Abstract

A capillary zone electrophoresis (CZE) method with preceding cationic transient capillary isotachophoresis (tCITP-CZE) was developed for uncoated fused-silica capillaries to analyze metal-binding proteins (MBPs) of clinical relevance. UV detection was followed by mass spectrometry (MS). Optimization was done with model proteins of properties similar to relevant human MBPs. Using 1.0 mol x L(-1) formic acid (pH 1.78) as electrolyte resulted in up to 165000 plates m(-1) in CZE and 230000 plates m(-1) in combination with tCITP and analysis time was less than 5 min in uncoupled mode. Cationic tCITP with 125 mmol x L(-1) ammonium formate, buffered to pH 4.00, as leading electrolyte improved sample loadability considerably in comparison with sample stacking without impairing resolution. Following systematic optimization of the electrospray ionization process (ESI) the coupled system ((tCITP)-CZE-UV-ESI-MS) was tested with protein model mixtures and human MBPs. Repeatability of migration times was < 0.64% in pure CZE mode and in tCITP-CZE mode and < 0.83% in CZE-ESI-MS coupled mode. Mass accuracy was < 0.015%. Limits of detection were found to be in the range 50-160 fmol.

Detection of coexisting protein conformations in capillary zone electrophoresis subsequent to transient contact with sodium dodecyl sulfate solutions

“…Both approaches aim to minimize protein adsorption to the capillary wall and to avoid fluctuations in the electroosmotic flow (EOF) or even suppress it completely. Anyhow, at low pH, proteins cannot be addressed a priori as true native conformations [3] since unfolding is introduced, releasing prosthetic groups as in the case of Mb and Hb (data not shown) [55] and consequently MGs exist [18,56]. In the case of dynamic coating there is evidence that coating agents compete with the polypeptide backbone for SDS adsorption, thus probably quenching the SDS effect [51].…”

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

“…MS data in general are highly desirable not only in stability and purity studies [24,25] but also for revealing and monitoring diagnostic marker peptides and proteins. In capillary zone electrophoresis (CZE)-ESI-MS, mass accuracies down to 0.0008% have been realized for model mixtures of proteins [26].The high number of reviews published in the recent years, dealing not only with CE separations of proteins and peptides [27,28], but also with CE-ESI-MS coupling in general [19,20,23,29,30] …”

Capillary electrophoresis of proteins 2003–2005

“…Metal-binding proteins of clinical relevance were concentrated in uncoated fusedsilica capillaries by cationic transient ITP [17]. 125 mM ammonium formate pH 4.0 was used as leading electrolyte in transient ITP followed by CZE in 1 M formic acid, pH 1.8.…”