Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II).

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC.

Histidine-rich peptide selection and quantification in targeted proteomics.

Agarose based immobilized copper (II) affinity chromatography (Cu(II)-IMAC) in tandem with reversed-phase chromatography was applied to a yeast protein extract. Histidine-rich peptides were selected and, in the process, samples were substantially simplified prior to mass spectral analysis. Samples of proteins from the yeast extract at fermentation time periods of 2.

Capillary electrochromatography of peptides on microfabricated poly(dimethylsiloxane) chips modified by cerium(IV)-catalyzed polymerization.

Vinylsulfonic acid, acrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (AMPS), 4-styrenesulfonic acid, and stearyl methacrylate were used for successful modification of the surface of poly(dimethylsiloxane) (PDMS) by cerium(IV) catalyzed polymerization on microfabricated collocated monolith support structures microchips. Reproducible and stable coatings were obtained allowing highly efficient separations of a peptide mixture with RSD for retention times below 2.6%.

Evaluating immobilized metal affinity chromatography for the selection of histidine-containing peptides in comparative proteomics.

Agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containing peptides in comparative proteomics. Recovery, binding specificity, and reproducibility were investigated with model proteins. Cu(II)-IMAC was found to be highly selective for histidine containing peptides; moreover, a low degree of nonspecific selection was observed.

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.

Sampling BIAS at channel junctions in gated flow injection on chips.

The commonly used gated injection scheme was examined and found to suffer from multiple levels of electrokinetic sampling bias, including a new type based on transradial electrokinetic selection (TREKS). TREKS occurs as analytes of differing electrophoretic mobilities migrate around the corner at a channel junction in a microchip. The overall sample bias in gated injection was shown to be time-dependent and resulted in a larger sample bias against components of negative electrophoretic mobility.