Imaging of Protein Distribution

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TOF-SIMS is the most useful technique for evaluating proteins on surfaces because it provides chemical mapping at a high spatial resolution. The chemical mapping of oligopeptides immobilized on an array of gold mi-croelectrodes was obtained as secondary ion images with the peptide-dried ion C4H8N+ (Mathieu 2001). In this case, the peptide image was obtained by conventional spectrum analysis, because components of the substrate were different from the peptide, and the fragment ions from the substrate and the peptide were therefore readily distinguishable.

One specific kind of protein found on substrates, which are composed of substances different from proteins, such as metallic substrates and polymers free of nitrogen, can be easily imaged by conventional TOF-SIMS techniques (Aubagnac et al. 1999; Belu et al. 2001). However, it is difficult to obtain one specific protein mapping from out of a collection of other proteins, especially on complicated materials containing nitrogen, because TOF-SIMS is not suitable for the ionization of large, intact molecules. In order to obtain chemical images of proteins, the specific peaks related to each protein must be selected from out of the TOF-SIMS spectra. Therefore, appropriate data analysis techniques are required for the characterization of TOF-SIMS spectra with fragment ions from large molecules. Moreover, since every protein consists of the same 20 amino acids, it is extremely difficult to discriminate between proteins based on a simple comparison of the fragment ions. Multivariate analysis techniques such as PCA and LDA have been employed to interpret TOF-SIMS spectra using fragment ions related to proteins.

Although LDA is considered suitable for the selection of specific peaks, the selection process requires either a great deal of data, or pretreatment of the data so as to prevent spurious discrimination. Fragment ions from bio-devices are often unpredicted because they contain complicated chemical additives in order to make them highly biocompatible and have high performance. In addition, since some of fragment ions from substrate materials generate complicated protein-like fragment ions, it is difficult to select appropriate peaks of fragment ions for data analysis of TOF-SIMS

spectra. Therefore an analysis technique which can be applied all peaks in spectra is required. Information theory has been employed to help analyze TOF-SIMS data on biomaterials containing proteins to enable a selection of several important peaks from all peaks (Aoyagi et al. 2003,2004a,b).

Mutual information, as defined by information theory, describes the specificity of every peak in the TOF-SIMS spectra of a sample in comparison with another sample, such as a reference sample. With mutual information, important and specific peaks can be selected out of numerous candidate peaks, even in numbers ranging from several hundreds to 1,000 or more. Then selected peaks, however, should be checked based on their peak properties, such as chemical structures, noises, and distributions, because sometimes inappropriate peaks are selected by means of automatic analysis. Finally, the selected specific peaks provide the distribution of a particular protein on bio-devices. For example, Fig. 7 shows the distribution of immobilized protein on a glass surface with a chromium pattern. Protein molecules are immobilized on the glass surface through a covalent bond using the hydroxyl group. The images of amino acid fragment ions related to the protein mach the image of the glass surface shown by the Si+ image.

The protein adsorption that takes place on hollow-fiber dialysis membranes with nanosized pores, which are used for the treatment of patients

Fig. 7. TOF-SIMS images of amino acid fragment ions related to proteins on a chromium-patterned glass surface

C4H10N+: Val C4H6NO+: Glu, Gin

Fig. 7. TOF-SIMS images of amino acid fragment ions related to proteins on a chromium-patterned glass surface

on a hollow-fiber membrane a hollow-fiber membrane

(TOF-SIMS image) Fig. 8. TOF-SIMS image of protein adsorbed onto hollow-fiber membranes with renal failure, has been evaluated by TOF-SIMS imaging in an effort to develop higher-performance dialysis techniques. The adsorption of a model protein, BSA, onto three commercially available, hollow-fiber dialysis membranes of different components and different structures, was measured with TOF-SIMS, and then the SIMS spectra were analyzed in the light of mutual information to select specific peaks for chemical imaging (Aoyagi et al. 2003, 2004a).

Figure 8 shows the secondary ion images of BSA adsorbed onto hollow-fiber membranes composed of polyacrylonitrile, according to the secondary positive ions related to the adsorbed BSA obtained from TOF-SIMS data analyzed with mutual information. Since TOF-SIMS secondary ion images are influenced by sample topography and the useful ion yield is dependent on certain chemical features, chemical images are needed for comparison with the total ion images.

The distribution and immobilization processes (Aoyagi et al. 2005; Chat-terjee 2004; Hagenhoff 1995; Loyd and O'Keefe 2004) of proteins on biosen-sorsurfaceshavebeenmeasuredusingTOF-SIMSwith mutual information or multivariate analysis. In order to allow a detailed control and specific tailoring of interfaces, a surface analytical tool is required that is able to localize, identify, and quantify the molecular structures of interest (Souda 2004). The distribution patterns of two proteins on a glass plate have been observed by TOF-SIMS imaging (Aoyagi et al. 2005). Both protein A, immobilized on the glass plate, and immunoglobulin G (IgG), bound to the immobilized protein A, were measured with TOF-SIMS and then their spectra were analyzed using PCA and mutual information. The PC score plots indicate that the fragment ions from protein A and those from the IgG could be distinguished with the TOF-SIMS spectra, and chemical images were obtainable based on mutual information. The TOF-SIMS images of each protein reveal the distributions of protein A and IgG on the glass plate, and indicate that protein A is immobilized on the plate homogeneously, and that the reaction between the immobilized protein A and the IgG is not specifically localized.

This technique will be of special value for the evaluation of bio-devices containing biomolecules on their surfaces in order to confirm both the immobilization processes and the orientation of the immobilized molecules.

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