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

TL;DR

This study uses TOF-SIMS to analyze the surface structure and orientation of cytochrome b5 monolayers immobilized on gold, providing insights into their three-dimensional arrangement and confirming their expected orientation.

Contribution

It demonstrates a novel application of TOF-SIMS for detailed structural characterization and orientation analysis of immobilized proteins on bio-devices.

Findings

Identified specific amino acid residues on the surface of immobilized cytochrome b5.

Correlated TOF-SIMS spectra with known protein structures.

Confirmed the orientation of cytochrome b5 on gold surfaces.

Abstract

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.