Selective extraction of small proteins from biological samples using a novel restricted access column with cation exchange properties

The determination of proteins utilising a polymer-based restricted access support material with ion exchange properties (IERAM) is outlined. Solid phase extraction coupled on-line with a microbore reversed phase HPLC system for the quantitation of small marker proteins is demonstrated. The cation-exchange restricted access packings were characterised with respect to their adsorption and desorption kinetics. The IERAM material was also investigated by capacity, selectivity, and biocompatibility determinations when applied to the quantification of small molecular weight proteins such as cytochrome C, Lysozyme, Ribonuclease A, Myoglobin, Insulin, human serum albumin, and a Tryptic inhibitor. The integrated system was coupled to mass identity of selected proteins by MALDI-TOF mass spectrometry. The chromatographic outlet was interfaced to an "Interplate" fractionation collecter that sampled 1 μL volumes directly onto the MALDI target plate. The fully automated coupled column system was run unattended overnight and applied to protein quantitations in human plasma samples. Recovery data for a selected number of proteins varied between 90-96% (n = 10) with a limit of quantification around 2 μM with an injection volume of 100 μL. The RSD data were typically less than 8% at a 50 μM protein level (n = 7).