Protein analysis using enzymes immobilized to paramagnetic beads

A new method combining protein chemistry with enzymes immobilized to parama gnetic beads is presented. The immobilized enzymes can substitute for regul ar enzymes in a number of protein chemistry protocols, resulting in faster reaction times, less sample contamination, and improved interfacing to mode rn procedures, like mass spectrometry. Trypsin was used as a model enzyme t o test the amount of protein coupled to glass beads and the degree of autod igestion when analyzed by MALDI-MS and HPLC. Immobilization of trypsin resu lted in digestions comparable with standard solution digestions using fetui n as a model substrate. Furthermore, fetuin was used to test the stability of the enzyme-coated beads. No apparent loss of enzyme activity was observe d after 10 times reuse of trypsin-coated beads. Immobilization of exo- and endoglycosidases to paramagnetic beads resulted in high sensitivity, faster sequential glycosidase digestion of glycopeptides, and reduced sample cont amination. All digestions could be performed in less than 24 h, when a tryp tic glycopeptide from human lung proteinosis surfactant protein A was used as model compound. (C) 1999 Academic Press.