This article describes the evaluation and observation of proteins immobilized on substrates by means of time-of-flight mass spectrometry (TOF-SIMS), and discusses briefly the evaluation of reactions between immobilized protein and free protein using TOF-SIMS imaging at the submicron level.

The monitoring of proteins on material surfaces is useful for the development of high-performance bio-devices such as artificial organs and biosensors. TOF-SIMS is one of the most useful techniques for the eval-

Satoka Aoyagi: Faculty of Life and Environmental Science, Department of Regional Development, Shimane University 1060 Nishikawatsu-cho, Matsue-shi, Shimane, 690-8504, Japan, Shimane University, Japan, E-mail: [email protected]

Masahiro Kudo: Department of Materials and Life Science, Seikei University, Japan

Principles and Practice Proteins at Solid-Liquid Interfaces Philippe Dejardin (Ed.) © Springer-Verlag Berlin Heidelberg 2006

uation of bio-device surfaces, because it provides a submicron-scale look at the distribution of proteins on materials. In addition, TOF-SIMS requires no pretreatment of samples, such as labeling with a fluorescent probe or coating with metallic thin films, to prepare the samples, although radioisotope labeling is sometimes employed to enhance detection sensitivity. Furthermore, both the identification of proteins and investigation of the orientation of immobilized proteins may be carried out using this technique. However, at present TOF-SIMS is not suitable for the ionization of large molecules such as proteins. Therefore, special data analysis techniques have often been employed for the characterization of TOF-SIMS spectra, utilizing fragment ions from large molecules.

Secondary ion mass spectrometry (SIMS) is a method for detecting ions, including those molecular ions produced by collisions between primary ions and atoms and molecules on a sample surface. SIMS provides qualitative and quantitative information on the sample surfaces via mass spectra, depth profile, chemical structure, and chemical mapping, in the form of secondary ion images. TOF-SIMS is one of the SIMS techniques carried out with a TOF mass spectrometer, which is the most sensitive analyzer, and which has the widest mass spectrum range.

Certain analytic techniques have often been employed for the characterization of TOF-SIMS spectra with fragment ions from large molecules such as proteins and polymers, because TOF-SIMS is not suitable for the ionization of intact macromolecules. In particular, there is a problem in the case of the measurement of proteins with TOF-SIMS: since every protein consists of the same 20 amino acids, it is difficult to discriminate between proteins based on a simple comparison of the fragment ions. Certain types of polymers also have this same problem. In order to overcome this problem, appropriate spectrum analysis techniques are needed.

Multivariate analysis techniques, such as principal component analysis (PCA) and linear discriminant analysis (LDA), have been employed to interpret TOF-SIMS spectra using fragment ions related to proteins (Belu et al. 2003; Lhoest et al. 1998,2001; Mantus et al. 1993; Wagner and Castner 2001; Wagner et al. 2002, 2004). PCA is especially useful for characterizing the TOF-SIMS spectra of protein samples. However, it is sometimes difficult to select specific peaks for the chemical imaging of proteins with PCA. Accordingly, information theory (Shannon and Weaver 1947) has been employed to analyze TOF-SIMS data on protein-containing biomaterials (Aoyagi et al. 2003). This innovation is now sufficiently developed so as to be able to obtain chemical images of protein samples. Mutual information (Shannon and Weaver 1947; Eckschlager et al. 1990), a technical term defined by information theory, characterizes the specificity of every peak in the TOF-SIMS spectra of a sample compared with another sample, such as a reference sample. With mutual information specific, the desired peaks can be selected out of a great number of peaks that appear in TOF-SIMS spectra. In this article, the application of TOF-SIMS to protein measurement is described, and the latest research in this field is reviewed.

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