  Orientation and three-dimensional structure of immobilized proteins on
bio-devices are very important to assure their high performance. Time-of-flight
secondary ion mass spectrometry (TOF-SIMS) is able to analyze upper surface of
one layer of molecules. Orientation of immobilized proteins can be evaluated
based on determination of a partial structure, representing ensemble of amino
acids, on the surface part. In this study, a monolayer of cytochrome b5 was
reconstituted onto gold substrate and investigated by surface plasmon resonance
(SPR). After freeze-drying, the resulted protein self-assembly was evaluated
using TOF-SIMS with the bismuth cluster ion source, and then TOF-SIMS spectra
were analyzed to select peaks specific to cytochrome b5 and identify their
chemical formula and ensembles of amino acids. The results from TOF-SIMS
spectra analysis were compared to the amino acid sequence of the modified
cytochrome b5 and three-dimensional structure of cytochrome b5 registered in
the protein data bank. Finally, fragment-ion-generating parts of the
immobilized-cytochrome b5 are determined based on the suggested residues and
three-dimensional structure. These results suggest the actual structure and
confirm the expected orientation of immobilized protein.
