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Analysis of virtual two‐dimensional gels based upon affinity capillary electrophoresis hyphenated to ion trap‐mass spectrometry
Author(s) -
Machour Nadine,
Place John,
Tron François,
Charlionet Roland,
Mouchard Laurent,
Morin Christophe,
Desbène Annie,
Desbène PaulLouis
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200410213
Subject(s) - chemistry , mass spectrometry , capillary electrophoresis , electropherogram , ion trap , chromatography , analytical chemistry (journal) , analyte , electrospray ionization , electrospray , capillary electrophoresis–mass spectrometry , quadrupole ion trap , mass spectrum
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.