Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings

Abstract

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

“…Previously this coating was applied to the separation of glycoforms and isoforms of other proteins such as a1-antitrypsin, BSA and human transferrin by CZE-UV [32]. The LN coating is strongly adsorbing and can be applied in a precoating mode.…”

Capillary electrophoresis‐based separation techniques for the analysis of proteins

“…Common polymers that are used to suppress protein adsorption and EOF include celluloses [84], dextran [85], PA [86,87], poly(vinyl alcohol) [88,89], PVP [90][91][92], and poly(ethylene oxide) (PEO) [93]. New dynamic coating materials such as EOTrol and UltraTrol have proved to be useful for the separation of protein isoforms by CZE [94]. The coatings allow reproducible EOF modulation in the cathodal direction to a level of…”

Capillary electrophoresis of proteins 2003–2005

“…In 2-D CE, in which both separation methods were based on CE, the capillaries for the first and second dimension have been interfaced with a microdialysis hollow fiber interface [118,[164][165][166][167][168] and etched porous junction [169]. A fully automated 2-D CE system combining capillary gel electrophoresis (CGE) with MEKC has been constructed [170,171].…”

“…CZE was used to analyze protein isoforms including a 1 -antitrypsin (Fig. 4) [168], metallothionein isoforms in rat liver [76] and in Synecho- Figure 4. Four replicate CZE separations of six major a 1 -antitrypsin isoforms.…”

“…Four replicate CZE separations of six major a 1 -antitrypsin isoforms. Reprinted from [168], with permission.…”