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 wit h pI ranging from pH 3-9.5 in an ample pH interval (pH 2.5-9.0) without ads orption onto the naked silica wall. It consists of treating the capillary s urface at alkaline pH, typically 9.0, with small amounts (2-4 mM) of a quat ernarized piperazine derivative: (N-methyl-N-omega -iodobutyl)-N ' -methylp iperazine (Q-PzI). It appears that this compound is able to dock onto the w all via trifunctional links: a salt bridge via the quaternary nitrogen, a h ydrogen bond via the tertiary nitrogen, and finally, a covalent link via th e terminal iodine in the butyl. chain and a neighboring ionized silanol. Ib is last reaction seems to be completed in a few minutes of incubation of th e capillary at room temperature. Because the compound is permanently affixe d to the wall, its presence is not needed during protein/peptide separation s. 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 effec tive only at acidic pH values and are required as additives during separati ons, Q-PzI is effective in an ample pH interval (pH 2.5-9.0) and is not nee ded during the CZE analysis. A broad pI (pH 3-10) protein mix can be separa ted according to protein mobility in free phase, suggesting a strong modula ting capacity of the functionalized wall. The same separation is not obtain ed in capillaries permanently coated with neutral, hydrophilic polymers (su ch as polyacrylamide), even if the quality of a single protein/peptide prof ile in Q-PzI-conditioned capillaries is equivalent to those obtained in cap illaries permanently coated. Although there is strong indirect evidence of the ability of Q-PzI to alkylate the silica wall, to which it is then irrev ersibly bound, such an alkylation event does not occur with proteins on pot entially reacting sites, such as the free -SH of Cys or the -OH group of Ty r, as demonstrated by incubating them overnight in a large molar excess at strongly alkaline pH values and analyzing such proteins by MALDI-TOF mass s pectrometry.