Protein analysis by capillary zone electrophoresis utilizing a trifunctional diamine for silica coating

A novel method is here reported for the analysis of mixture of proteins with pI ranging from pH 3−9.5 in an ample pH interval (pH 2.5−9.0) without adsorption onto the naked silica wall. It consists of treating the capillary surface at alkaline pH, typically 9.0, with small amounts (2−4 mM) of a quaternarized piperazine derivative:  (N-methyl-N-ω-iodobutyl)-N‘-methylpiperazine (Q-PzI). It appears that this compound is able to dock onto the wall via trifunctional links:  a salt bridge via the quaternary nitrogen, a hydrogen bond via the tertiary nitrogen, and finally, a covalent link via the terminal iodine in the butyl chain and a neighboring ionized silanol. This last reaction seems to be completed in a few minutes of incubation of the capillary at room temperature. Because the compound is permanently affixed to the wall, its presence is not needed during protein/peptide separations. By properly dosing the level of Q-PzI in the preconditioning step, it is possible to strongly reduce the electroendoosmotic flow (EOF), zero it, or reverse it. Unlike dynamic coatings with oligoamines, which are most effective only at acidic pH values and are required as additives during separations, Q-PzI is effective in an ample pH interval (pH 2.5−9.0) and is not needed during the CZE analysis. A broad pI (pH 3−10) protein mix can be separated according to protein mobility in free phase, suggesting a strong modulating capacity of the functionalized wall. The same separation is not obtained in capillaries permanently coated with neutral, hydrophilic polymers (such as polyacrylamide), even if the quality of a single protein/peptide profile in Q-PzI-conditioned capillaries is equivalent to those obtained in capillaries permanently coated. Although there is strong indirect evidence of the ability of Q-PzI to alkylate the silica wall, to which it is then irreversibly bound, such an alkylation event does not occur with proteins on potentially reacting sites, such as the free −SH of Cys or the −OH group of Tyr, as demonstrated by incubating them overnight in a large molar excess at strongly alkaline pH values and analyzing such proteins by MALDI-TOF mass spectrometry