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

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 threedimensional structure. These results suggest the actual structure and confirm the expected orientation of immobilized protein.

Preparation of flat gold terraces for protein chip developments

“…Concerning the size, we managed to prepare terraces from 22 500 nm 2 Nevertheless, there is a maximum heating duration beyond which the gold surface is completely disorganized like "melt" on/with mica (sample V and Fig3A). AFM images do not present gold terraces anymore and the surface topography is a sort of intermediary between classical rough gold film and highly flat mica.…”

“…a unique sulfhydryl residue at the periphery of the 3D structure, will naturally self-assemble on gold substrate. Bearing this sulfhydryl moiety, cytochrome b5 is able to perform direct selfassembly onto gold surface by chemisorption process leading to a packed and oriented self assembled monolayer (SAM) 2 .…”

Image Processing Quality Analysis for Particle Based Peptide Array Production on a Microchip

“…The TOF-SIMS method is thus selected as method of choice, further encouraged by literature, demonstrating that single molecule detection, especially amino acid detection is possible with this method, for example in Aoyagi et al (2008) or Hagenhoff (2000. Furthermore, it is possible to chemically characterise self-assembled monolayers (SAMs) by means of TOF-SIMS Graham & Ratner (1994), which can become important for peptide synthesis on CMOS chips as well, since SAMs are currently being established as chemically active surface coating.…”

Simulation of Fragmentation of Polyethylene Glycol by Quantum Molecular Dynamics for TOF-SIMS Spectral Analysis

“…Therefore, we need detailed analysis of such organic molecules in TOF-SIMS spectra. Recently, Aoyagi and coworkers selected specific peaks which can distinguish different protein samples by using spectral analytical method owing to information theory [1] and applied the analytical method to clarify the structure for biosensor materials [2]. On the other hand, in order to determine the structure of complicated fragment ions from organic molecules such as proteins, we think it is useful to simulate fragmentation process of such organic molecules from theoretical standing-points.…”