Capillary zone electrophoresis in stationary, isoelectric buffers is a novel method for generating peptide maps of protein digests. The buffer system developed is composed of iminodiacetic acid (IDA), whose physico-chemical parameters were found - by theoretically modeling and experimental verification - to be: pI 2.23 (at 100 mM concentration), pK(1) = 1.73 and pK(2) = 2.73 (no attempts were made at measuring the pK of the primary amino group, since such a low pI value would be compatible with any pK value of the basic group, down to as low as pK 5.5). IDA is compatible with most hydro-organic solvents, including trifluoroethanol (TFE), up to at least 40% v/v, typically used for modulating peptide mobility. In naked capillaries, a buffer comprising 50 mM IDA, 10% TFE and 0.5% hydroxyethylcellulose (HEC) allows generation of peptide maps with high resolution, reduced transit times and no interaction of even large peptides with the wall. However, the best background electrolyte was found to be a solution of 50 mM IDA in 0.5% HEC and 6-8 M urea, one of the best solubilizers of proteins and peptides known. In this last electrolyte system, peptide maps of beta-casein digests (known to contain also very large peptides, up to 6000 Da) could be generated with excellent resolution and half the transit times as compared with the standard buffer adopted in peptide analysis (80 mM phosphate buffer, pH 2.0). IDA thus appears to be another valid isoelectric buffer system, operating in a different pH window (pH 2.33 in 50 mM IDA) as compared to the other amphotere previously adopted (50 mM Asp, pH 2.77) for the same kind of analysis.
Generation of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid.
BOSSI, Alessandra Maria;
1997-01-01
Abstract
Capillary zone electrophoresis in stationary, isoelectric buffers is a novel method for generating peptide maps of protein digests. The buffer system developed is composed of iminodiacetic acid (IDA), whose physico-chemical parameters were found - by theoretically modeling and experimental verification - to be: pI 2.23 (at 100 mM concentration), pK(1) = 1.73 and pK(2) = 2.73 (no attempts were made at measuring the pK of the primary amino group, since such a low pI value would be compatible with any pK value of the basic group, down to as low as pK 5.5). IDA is compatible with most hydro-organic solvents, including trifluoroethanol (TFE), up to at least 40% v/v, typically used for modulating peptide mobility. In naked capillaries, a buffer comprising 50 mM IDA, 10% TFE and 0.5% hydroxyethylcellulose (HEC) allows generation of peptide maps with high resolution, reduced transit times and no interaction of even large peptides with the wall. However, the best background electrolyte was found to be a solution of 50 mM IDA in 0.5% HEC and 6-8 M urea, one of the best solubilizers of proteins and peptides known. In this last electrolyte system, peptide maps of beta-casein digests (known to contain also very large peptides, up to 6000 Da) could be generated with excellent resolution and half the transit times as compared with the standard buffer adopted in peptide analysis (80 mM phosphate buffer, pH 2.0). IDA thus appears to be another valid isoelectric buffer system, operating in a different pH window (pH 2.33 in 50 mM IDA) as compared to the other amphotere previously adopted (50 mM Asp, pH 2.77) for the same kind of analysis.
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