C. Gelfi et al., Protein analysis by capillary zone electrophoresis utilizing a trifunctional diamine for silica coating, ANALYT CHEM, 73(16), 2001, pp. 3862-3868
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.