Protein Analysis Using Enzymes Immobilized to Paramagnetic Beads

✍ Scribed by Thomas N. Krogh; Torben Berg; Peter Højrup

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 140 KB
• Volume: 274
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4254

✦ Synopsis

A new method combining protein chemistry with enzymes immobilized to paramagnetic beads is presented. The immobilized enzymes can substitute for regular enzymes in a number of protein chemistry protocols, resulting in faster reaction times, less sample contamination, and improved interfacing to modern procedures, like mass spectrometry. Trypsin was used as a model enzyme to test the amount of protein coupled to glass beads and the degree of autodigestion when analyzed by MALDI-MS and HPLC. Immobilization of trypsin resulted in digestions comparable with standard solution digestions using fetuin as a model substrate. Furthermore, fetuin was used to test the stability of the enzyme-coated beads. No apparent loss of enzyme activity was observed 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 contamination. All digestions could be performed in less than 24 h, when a tryptic glycopeptide from human lung proteinosis surfactant protein A was used as model compound.