The pH dependence of predictive models relating electrophoretic mobility to peptide chemico‐physical properties in capillary zone electrophoresis

Abstract

We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C-terminus and carboxylic group of the side-chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591-593), who suggested a proportionality between the polymer Stokes radius and M(2/3). The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C-terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25-2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V(-1)s(-1) is given by mu = 85.4(Z/M(0.68))10(-8).

Evaluation of Electrokinetic Flow Mobility Using Isotacho-Electrophoresis Techniques

“…Therefore, pK a could be slightly dependent within the range of the present study. At constant pK a , the electrophoretic mobility abruptly decreases as the pH value increases at the range between 2.0 and 4.0 [13]. However, there was a slight decrease in electrophoretic mobility above pH 5.0.…”

Identification and Characterization of Peptides and Proteins Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

“…The chromatographic retention or electrophoretic migration time contains information on the physiochemical properties of the peptides, such as hydrophobicity in RPC and size and charge in standard capillary zone electrophoresis (CZE). A model of chromatographic retention [124][125][126][127][128] or electrophoretic migration [129][130][131][132][133][134][135][136][137][138] can be fitted to experimental data from known proteins. These models are then used to predict the retention time for candidate peptides in a database.…”

“…In addition to the accurate mass measurement and distribution of peptides in the protein sequence, containing information on the non-random behaviour of trypsin, protein structure, post-translational modifications and database sequence errors, there is also information on the physiochemical properties of the individual peptides. This is primarily hydrophobicity in reversed-phase chromatography [124][125][126][127][128] and size and charge in capillary zone electrophoresis [129][130][131][132][133][134][135][136][137][138]. A predictor of retention time according to Eq.…”

“…In this separation, the pH of the mobile phases was ~3. The pH has been shown to be very important in reversed-phase chromatographic retention and electrophoretic migration of peptides [138,167]. Table 4.…”

Capillary electrophoresis‐mass spectrometry of peptides from enzymatic protein hydrolysis: Simulation and optimization

“…A solution that can help to overcome these limitations is the development of theoretical models [28][29][30][31][32][33][34][35] able to predict the CE migration time and peak shape of the peptide in different pH and buffers. Following this idea, a system for the prediction of peptide migration (SPPM) for CE-MS was tested in a previous paper [36].…”