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Generation of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid
Author(s) -
Bossi Alessandra,
Righetti Pier Giorgio
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181122
Subject(s) - iminodiacetic acid , capillary electrophoresis , chemistry , peptide , chromatography , isoelectric point , isoelectric focusing , electrophoresis , electrolyte , resolution (logic) , analytical chemistry (journal) , biochemistry , chelation , inorganic chemistry , electrode , artificial intelligence , computer science , enzyme
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: p I 2.23 (at 100 m M concentration), p K 1 = 1.73 and p K 2 = 2.73 (no attempts were made at measuring the p K of the primary amino group, since such a low p I value would be compatible with any p K value of the basic group, down to as low as p K 5.5). IDA is compatiable with most hydro‐organic solvents, including trifluoreoethanol (TFE), up to at least 40% v/v, typically used for modulating peptide mobility. In naked capillaries, a buffer comprising 50 m M 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 m M 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 β‐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 m M 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 m M IDA) as compared to the other amphotere previously adopted (50 m M Asp, pH 2.77) for the same kind of analysis.