Determination of acidity constants and prediction of electrophoretic separation of amyloid beta peptides

In this paper we describe a strategy to estimate by CE the acidity constants (pK_a) of complex polyprotic Peptides from their building peptide fragments. CE has been used for the determination of the pK_as of five short polyprotic Peptides that cover all the sequence of amyloid beta (Aβ) Peptides 1-40 and 1-42 (Aβ fragments 1-15, 10-20, 20-29, 25-35 and 33-42). First, the electrophoretic mobility (m_e) was measured as a function of pH of the background electrolyte (BGE) in the pH range 2-12 (bare fused silica capillary, I=25mM and T=25°C). Second, the m_es were fitted to equations modelling the ionisable behaviour of the different fragments as a function of pH to determine their pK_as. The accuracy of the pK_as was demonstrated predicting the electrophoretic behaviour of the studied fragments using the classical semiempirical relationships between m_e and peptide charge-to-mass ratio (m_e vs. q/M_r^1/2, classical polymer model, q=charge and M_r=relative molecular mass). Separation selectivity in a mixture of the fragments as a function of pH was evaluated, taking into account the influence of the electroosmotic flow (EOF) at each pH value, and a method for the simple and rapid simulation of the electropherograms at the optimum separation pH was described. Finally, the pK_as of the fragments were used to estimate the pK_as of the Aβ Peptides 1-40 and 1-42 (^tC and D 3.1, E 4.6 and Y 10.8 for acidic Amino acids and ^tN-D 8.6, H 6.0, K 10.6 and R 12.5 for basic Amino acids), which were used to predict their behaviour and simulate their electropherograms with excellent results. However, as expected due to the very small differences on q/M_r^1/2 values, separation resolution of their mixtures was poor over the whole pH range. The use of poly(vinyl alcohol) (PVA) coated capillaries allowed reducing the EOF and a slight improvement of resolution.