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

Abstract

In this paper we describe a strategy to estimate by CE the acidity constants (pK) of complex polyprotic peptides from their building peptide fragments. CE has been used for the determination of the pKs 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) 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 ms were fitted to equations modelling the ionisable behaviour of the different fragments as a function of pH to determine their pKs. The accuracy of the pKs was demonstrated predicting the electrophoretic behaviour of the studied fragments using the classical semiempirical relationships between m and peptide charge-to-mass ratio (m vs. q/M, classical polymer model, q=charge and M=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 pKs of the fragments were used to estimate the pKs of the Aβ peptides 1-40 and 1-42 (C and D 3.1, E 4.6 and Y 10.8 for acidic amino acids and N-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 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.

Evaluation of on-line solid-phase extraction capillary electrophoresis–mass spectrometry with a nanoliter valve for the analysis of peptide biomarkers

“…The LOD for the analysis of the opioid peptides by CE-MS was 100 µg•L -1 (Table 1), similar to the value obtained in previous studies [24,31]. The same strategy was followed to select pH 3.0 for the separation of a mixture of the four Aβ peptide fragments (Aβ 1-15, 10-20, 20-29, and 25-35) [29]. Figure S-1A shows the EIEs for the analysis of standards at 10 mg•L -1 by CE-MS using 50 mM HAc:50 mM HFor, pH 3.0, as BGE and the four Aβ peptide fragments were separated.…”

“…In previous works, the acid-base properties of opioid peptides [28] and Aβ peptide fragments [29] were characterized by CE-UV to establish electrophoretic migration models and systematically optimize their separation. Accurate pK a values were determined, which can be used to easily obtain charge-to-mass ratios (q/M r ) of the peptides and to calculate the multicriterium optimization function T ' for a simple, experiment-free and reliable selection of optimized separation conditions [30].…”

Determination of acidity constants, ionic mobilities, and hydrodynamic radii of carborane‐based inhibitors of carbonic anhydrases by capillary electrophoresis

“…CE is a powerful separation method suitable for the physicochemical characterization of ionogenic compounds [20] including determination of their acidity constant, p K a [21, 22]. CE is widely used for the determination of the p K a s of acidic, basic, and amphoteric compounds in aqueous [23–33], hydro‐organic [34], and organic solvents [35, 36]. Recently, a new computer program AnglerFish was introduced for direct determination of the thermodynamic p K a s and limiting ionic mobilities from the raw CE experimental data [37].…”

Improving separation optimization in capillary electrophoresis by using a general quality criterion

“…In contrast to liquid chromatography (LC) [4,5], resolution (Rs) is a difficult parameter to predict in CE, because many phenomena affect peak widths and shapes [1,2,6]. Therefore, quality criteria based on migration velocities or electrophoretic mobilities (me), which solely consider ratios or differences on the separation between pairs of compounds, have been traditionally preferred [2,[7][8][9][10]. Nowadays, selectivity between a pair of compounds (αij) in CE is widely accepted to be defined as the ratio of their me (Equation 1).…”

“…amyloid beta (Aβ) peptide fragments [10], apothioneins [20], peptides and glycopeptides from tryptic digests of glycoproteins [21] and quinolones [22] when good pKa values are available in the literature for charge calculations. For instance, the migration behavior of Aβ peptide fragments was accurately modelled using the classical polymer model (me vs. q/Mr 1/2 ) [10].…”