Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides

Abstract

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.

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

“…Applied LE consisted of ammonium acetate or ammonium formate in some cases added directly to the sample, whereas TE was made up of formic acid, acetic acid, or 6-aminohexanoic acid [26,50,92,232,233]. To maintain the integrity of distinct CITP zones, EOF is suppressed by shielding capillary walls with PAA, PVA, Fluorad FC-135 [231], APS [233], or by operation at extremely acidic pH [26]. In cationic tCITP leading co-ions, i.e., NH 4 1 , are added directly to the sample and pure…”

“…By on-line coupling of tCITP with CZE, these problems can be solved [96]. Contrary to dual column arrangements with CITP and CZE taking place in different capillaries [96,[229][230][231], a single capillary approach facilitates automatization [26,92,232] and even then injected sample volumes of peptides account for up to 70% of the total capillary volume [231,232]. Applied LE consisted of ammonium acetate or ammonium formate in some cases added directly to the sample, whereas TE was made up of formic acid, acetic acid, or 6-aminohexanoic acid [26,50,92,232,233].…”

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

“…5A and B might be an indication that this effect is already of relevance at 10 mmol?L 21 ammonium formate. Although the optimized LE concentration seems rather high in comparison with LE concentrations around 10 mmol?L 21 published so far [76,77], one should keep in mind that due to the low pH and the high concentration of the TE (= 1.0 mol?L 21 formic acid) H 3 O 1 concentration is rather high as well and adequately a higher counter ion concentration in the LE is needed. Comparison with the sample stacking of an identical injection volume and a reduced volume to achieve resolution comparable to tCITP clearly demonstrates the superiority of the latter one (Fig.…”

On-line sample preconcentration in capillary electrophoresis, focused on the determination of proteins and peptides

“…In the first step, the analyte is dissolved in the LE, often an ammonium solution, and introduced in the CZE-capillary using hydrodynamic pressure. This step enables the filling of about 70% of the capillary with sample [12]. Subsequently, the inlet of the CZE capillary is placed in a vial containing the TE, normally containing acetic acid.…”