TOF-SIMS structural characterization of self-assembly monolayer of cytochrome b5 onto gold substrate

摘要

Orientation and three-dimensional structure of immobilized proteins onbio-devices are very important to assure their high performance. Time-of-flightsecondary ion mass spectrometry (TOF-SIMS) is able to analyze upper surface ofone layer of molecules. Orientation of immobilized proteins can be evaluatedbased on determination of a partial structure, representing ensemble of aminoacids, on the surface part. In this study, a monolayer of cytochrome b5 wasreconstituted onto gold substrate and investigated by surface plasmon resonance(SPR). After freeze-drying, the resulted protein self-assembly was evaluatedusing TOF-SIMS with the bismuth cluster ion source, and then TOF-SIMS spectrawere analyzed to select peaks specific to cytochrome b5 and identify theirchemical formula and ensembles of amino acids. The results from TOF-SIMSspectra analysis were compared to the amino acid sequence of the modifiedcytochrome b5 and three-dimensional structure of cytochrome b5 registered inthe protein data bank. Finally, fragment-ion-generating parts of theimmobilized-cytochrome b5 are determined based on the suggested residues andthree-dimensional structure. These results suggest the actual structure andconfirm the expected orientation of immobilized protein.