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. 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.
Ren
Diya
D
Department of Chemistry, Purdue University, 1393 Brown Building, West Lafayette, IN 47907, USA.
Penner
Natalia A
NA
Slentz
Benjamin E
BE
Inerowicz
Halina D
HD
Rybalko
Marina
M
Regnier
Fred E
FE
D000042:Absorption; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry*; D006868:Hydrolysis; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008670:Metals / Q000737:chemistry*; D010316:Particle Size; D010455:Peptides / Q000737:chemistry; D011492:Protein Hydrolysates / Q000737:chemistry; D012995:Solubility; D014168:Transferrin / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry
D007202:Indicators and Reagents; D008670:Metals; D010455:Peptides; D011492:Protein Hydrolysates; D014168:Transferrin; D003300:Copper; D001616:beta-Galactosidase; D014357:Trypsin