Protein analysis using enzymes immobilized to paramagnetic beads.
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.
Krogh
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TN
Department of Molecular Biology, Odense University, Campusvej 55, Odense M, DK-5230, Denmark.
Berg
T
T
Højrup
P
P
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D006801:Humans; D008280:Magnetics; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D004800:Enzymes, Immobilized; D011506:Proteins