Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II) |
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Authors: | Ren Diya Penner Natalia A Slentz Benjamin E Inerowicz Halina D Rybalko Marina Regnier Fred E |
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Institution: | Department of Chemistry, Purdue University, 1393 Brown Building, West Lafayette, IN 47907, USA. |
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Abstract: | 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. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and beta-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity. |
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