Protein proteolysis and the multi-dimensional electrochromatographic separation of histidine-containing peptide fragments on a chip.
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.
Slentz
Benjamin E
BE
Department of Chemistry, Purdue University, 1393 Brown Building, West Lafayette, IN 47907-1393, USA.
Penner
Natalia A
NA
Regnier
Fred E
FE
D006639:Histidine / Q000737:chemistry*; D006868:Hydrolysis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D011506:Proteins; D006639:Histidine