Protein proteolysis and the multi-dimensional electrochromatographic separation of histidine-containing peptide fragments on a chip |
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Authors: | Slentz Benjamin E Penner Natalia A Regnier Fred E |
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Institution: | Department of Chemistry, Purdue University, 1393 Brown Building, West Lafayette, IN 47907-1393, USA. |
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Abstract: | This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 microm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example. |
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