Analysis of virtual two‐dimensional gels based upon affinity capillary electrophoresis hyphenated to ion trap‐mass spectrometry

Abstract

Affinity capillary electrophoresis (ACE) is a robust tool for the study of noncovalent biomolecular interactions and to determine the binding constants. It is advantageous due to the speed of analysis, the high and reproducible separation efficiencies, the low consumption of analytes, the ability to study several interactions at the same time, and to cover a wide range of affinity. The use of an ion trap-mass spectrometer as a sensitive and specific detector, coupled on-line with a classical UV detector, permits extracting simultaneously the electropherograms corresponding to each ionic species. The mass spectra, acquired by scanning the results of a first separation due to ACE, were assimilated into a virtual two-dimensional (2-D) gel. We developed a software application, which was designed to create and analyze these virtual 2-D gels. The validity of this new analytical tool for probing biomolecular interactions has been demonstrated on mixtures of antibiotics of the vancomycin group and several dipeptide substrates. Using the dynamic equilibrium affinity electrophoresis approach, we have shown that molecular components interacting with a low affinity are easily located on the virtual 2-D gels, and that binding constants and stoichiometry of the interactions can be assessed. As the binding constants derived from ACE-electrospray ionization-mass spectrometry (ESI-MS) are unreliable, they must only be determined with the UV detector.

Determination of binding constants by affinity capillary electrophoresis, electrospray ionization mass spectrometry and phase-distribution methods

“…One may predict that the coupling of ACE methods with MS, so far used rarely and only for model studies [112,113], will increase. In complex, multi-component equilibriums, the qualitative identification power of MS will be more important the more partners there are in the complex.…”

Recent applications of affinity interactions in capillary electrophoresis

“…Advances have recently been reviewed [115,116]. The challenge of CE-MS remains the development of suitable interfaces that allow the separation buffer conditions of CE to be compatible with the ionization process of MS. Machour et al [117] employed IT-MS as a parallel detector to UV in studying mobility shifts induced by the interaction of vancomycin derivatives with dipeptide substrates. It was concluded that CE-MS is a valuable tool by combining affinity separation with accurate mass determination, but it was found that binding constants could only be reliably calculated in this system based on the CE-UV data.…”

Determination of binding constants of vasoactive intestinal peptide to poly(amidoamine) dendrimers designed for drug delivery using ACE

“…Among the multiple methods affording the binding constant determination by CE, ACE is the most popular [33]. The broad applicability of ACE is illustrated by a variety of studies on biomolecular interactions, involving peptide-drug [34], protein-polysaccharides [35][36][37], peptide-polymers [38], drug-CDs [39,40], and protein-nucleic acid [41,42]. Although several reports have been published regarding the analysis of dendrimers [43][44][45][46][47][48] or VIP [49,50] using CE, to date, no work has reported the use of ACE to study the molecular interactions between this peptide and PAMAM dendrimers.…”