Cu(II)-loaded iminodiacetic acid-silica particles for protein profiling of human serum samples using surface-enhanced affinity capture: support porosity effects.

Silica particles of different porosity were functionalised with iminodiacetic acid (IDA) and loaded with Cu(II) ions to yield Cu(II)-IDA-silica. These immobilised metal affinity chromatography (IMAC) materials were subjected to a comprehensive characterisation study. The Cu(II) content--determined via UV/Vis spectroscopy and atomic absorption spectroscopy (AAS)--was found to be linearly dependent on the specific surface area of the silica particles. The evaluation of protein adsorption isotherms provided information on binding properties towards biomolecules. The data fitted Langmuir's adsorption theory. Binding capacity of hen egg white lysozyme (HEWL) was highest for Cu(II)-IDA-silica with mean pore diameter of 120 A, reaching nearly 350 mg/g. All derivatised materials were applied to the fractionation of human serum samples and subsequent mass spectrometric analysis (m/z: 2000-10,000) according to a surface-enhanced affinity capture (SEAC) protocol. Pore size of the support material affected the appearance of the mass spectra to a great extent, showing that surface morphology is another parameter that has to be considered in addition to surface chemistry. Signal intensity as well as the number of detected masses were strongly dependent on the pore diameter, indicating that the carrier material has to be carefully chosen to assure best results.