Photoimmobilization of proteins for affinity capture combined with MALDI TOF MS analysis

Affinity capture surfaces can be prepared in a number of ways. A method of obtaining such surfaces through UV-activated immobilization of binding proteins using a benzophenone derivative is reported. Photoimmobilized protein G was used to selectively capture and preconcentrate bovine IgG from a mixture with BSA, and the affinity of photoattached concanavalin A toward ovalbumin was compared with that of commercially available concanavalin A on agarose beads. The results of the capture after tryptic digestion were analyzed by MALDI TOF MS. Immobilized trypsin was also prepared through photoimmobilization and later used to digest hemoglobin. Immobilized enzyme digestion resulted in more partial cleavages than solution-phase digestion. More methionine and tryptophan oxidation was also observed. Photoimmobilization was shown to be a quick and easy way of immobilizing ligands on surfaces.