Review

Received: 3 October 2008 Accepted: 6 October 2008 Published online in Wiley Interscience: 28 November 2008

(www.interscience.wiley.com) DOI 10.1002/sia.2989

Review of TOF-SIMS bioanalysis using mutual information

Satoka Aoyagi
The evaluation of proteins on biodevices, such as the distribution or orientation of immobilized proteins, is one of the most important issues for the development of sophisticated biodevices. In this review, a description is provided of the application of one of the most sensitive surface analysis methods, time-of-ight secondary ion mass spectrometry (TOF-SIMS), to protein evaluation and TOF-SIMS spectra analysis using mutual information. TOF-SIMS is useful for the evaluation of biodevice surfaces, because TOF-SIMS provides submicron-scale chemical mapping and information on the chemical structures of the upper surface part of a protein. In addition, TOF-SIMS requires no pretreatment of samples, such as labeling with a uorescent probe or coating with metallic thin lms. Data analysis methods are, however, required to interpret the protein sample TOF-SIMS data, because the fragment ions from proteins are so complicated that it is difcult to predict them. In order to identify hidden important peaks related to protein samples out of the hundreds of peaks in TOF-SIMS spectra, mutual information has been used. Protein distribution on biodevices and the orientation of an immobilized protein obtained with this method are described in this review. Copyright c 2008 John Wiley & Sons, Ltd. Keywords: TOF-SIMS; mutual information; protein; distribution; imaging; orientation

Introduction
This article reviews the application of information theory to biodevice analysis by means of time-of-ight secondary ion mass spectrometry (TOF-SIMS). The evaluation of proteins on biodevice surfaces is crucial for the development of high-performance biodevices such as articial organs and biosensors. TOF-SIMS is one of the most useful techniques for such evaluation of biodevice surfaces, because TOF-SIMS provides chemical information on the most upper surface of a protein monolayer and the distribution of proteins on materials at a submicron scale. Although TOF-SIMS is one of the most powerful techniques for analyzing biodevices, it has not come into widespread use in the eld of biology because of its complicated spectrum interpretation. Many analytic techniques have been used for the characterization of TOF-SIMS spectra[1 5] with fragment ions from organic molecules or biomolecules, such as polymers and proteins, because TOF-SIMS is not suitable for the ionization of intact macromolecules. Moreover, it is difcult to discriminate among proteins based on a simple comparison of the fragment ions, because every protein consists of amino acids from the same basic set of 20 amino acids. Certain types of polymers also have this same problem. Therefore, TOF-SIMS spectra of protein samples contain a number of unpredictable and complicated peaks. Multivariate data such as TOF-SIMS spectra can be solved in a variety of ways. In this article, one of the new approaches to analyzing the TOF-SIMS spectra of protein samples is described. Mutual information,[6 8] dened by information theory, has been applied to analyzing TOF-SIMS spectra to characterize the specicity of every peak in the TOF-SIMS spectra of a sample by comparing it with another sample, such as a reference sample. Mutual information has been applied to chromatography for selecting signicant peaks.[8] Because TOF-SIMS spectra, especially of protein samples, contain complicated peaks, they require a critical peak selection method to obtain images of particular proteins and also to evaluate their orientation. Secondary ion

images of a particular protein[9 12] can be obtained and the orientation of immobilized proteins[13 16] can be evaluated using the specic peaks selected with the mutual information. In this article, the concept of mutual information and the spectra analysis method using mutual information[8,9] are explained, and certain results using this method are presented.

TOF-SIMS Spectra Analysis

TOF-SIMS spectra analysis with mutual information Mutual information is obtained by subtracting a posteriori entropy (uncertainty) from a priori entropy (uncertainty). In this formulation, a posteriori entropy is dened as the information entropy that occurs after an event. The calculation steps are the following: Assume the number of TOF-SIMS spectra is N and they are classied into two categories, the sample and the reference sample. The number of spectra belonging to the sample is n(a1 ) and that belonging to the reference sample is n(a2 ). In terms of sample categories, information entropy S(A) is dened by the following Eqn (1): S(A) = p(ai ) log2 p(ai ) (1)

where the probability p(ai ) = n(ai )/N (i = 1, 2), S(A) is the amount of information needed to determine the a priori category of a spectrum. With a certain peak threshold V, the set of spectra are

Correspondence to: Satoka Aoyagi, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue-shi, Shimane 6908504, Japan. E-mail: aoyagi@life.shimane-u.ac.jp Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue-shi, Shimane 690-8504, Japan

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TOF-SIMS application to bioanalysis using mutual information split into two subsets, B1 and B2 . The peak intensity greater than V is classied as B1 and the number of the spectra containing these peaks as n(b1 ), while that less than V is classied as B2 and the number of the spectra containing these peaks as n(b2 ). Therefore, the information entropy of splitting induced by V, S(B) is dened by the Eqn (2): (2) S(B) = p(bj ) log2 p(bj ) where the probability p(bj ) = n(bj )/N (j = 1, 2) Mutual information I(A;B) is dened by Eqn (3): I(A; B) = S(A) S(A|B) S(A|B) = p(bj )p(ai |bj ) log2 p(ai |bj ) (3) (4) sample. Therefore, only peaks having high values of mutual information should be checked, based on their peak properties such as chemical structures, noise and distribution, to see if they are appropriate and useful for further analysis. An example would be mutual information as calculated for sample 1. Assume there are three spectra of sample 1 and ve spectra of sample 2. Table 1 shows the intensities of several peaks in the TOF-SIMS spectra. The mutual information of the peak at m/z133 is calculated. First, S(A) is calculated with these sample numbers as follows: The number of sample 1 data: n(a1 ) = 3 The number of sample 2 data: n(a2 ) = 5 The number of all data: N = 8 Therefore, p(a1 ) = n(a1 )/N = 3/8 p(a2 ) = n(a2 )/N = 5/8 Therefore, S(A) = p(ai )log2 p(ai ) = {p(a1 )log2 p(a1 ) (6) (5)

Table 1. Intensities of peaks m/z Sample 1 Sample 1 Sample 1 Sample 2 Sample 2 Sample 2 Sample 2 Sample 2 46 24 747 14 056 21 176 12 349 25 371 18 076 20 468 22 006 129 3621 3309 3431 2516 3039 2944 2837 2444 130 4073 5616 2488 5998 6283 7338 6715 5467 131 4752 5417 4664 3963 4151 4259 3962 3449 132 133 134 135

2329 10 053 2424 3940 2512 11 961 2825 4018 2038 10 761 2456 3693 2028 5826 1917 2421 2125 5688 2124 2814 2144 4874 2014 2243 2015 4488 1935 2300 1630 3921 1602 2264

where the probability p(ai |bj ) = n(ai |bj )/n(bj ), S(A) means the a priori uncertainty and S(A|B) is the a posteriori uncertainty. The term n(ai |bj ) is the number of spectra belonging to sample category i out of the spectra containing peaks greater than V. The best value of V is chosen to provide the largest I(A; B).[7,8] When I(A; B) = S(A), the peak intensity of each spectra is classiable to the correct category. For example, there are TOF-SIMS spectra of two samples; 1 and 2. We can compare the intensities of a certain peak, as shown in Fig. 1. When the peaks are completely classied by the threshold V, for example, the intensities of the peaks in the all of TOF-SIMS spectra of the sample 1 are higher than those of sample 2, the a posteriori entropy is zero and the mutual information is 1. In other words, when mutual information equals the a priori entropy, this peak is the most important peak for classifying the samples. On the other hand, when intensities of the peaks cannot be classied with an appropriate threshold V at all, for example, the intensities of the peaks in most of TOF-SIMS spectra of sample 1 are almost as high as those of sample 2, the value of the a priori entropy equals the a posteriori entropy. This a posteriori entropy is information entropy after the estimation of the peak intensity with threshold V. In this case, the mutual information is zero, and nothing is claried by the evaluation of the peak intensity with V. When a peak of a secondary ion is specic to sample 1, its intensity in the sample 1 is higher than that in sample 2. Considering the intensity uctuation in the measurements and background, the intensity in sample 1 may not always be higher than sample 2, even though it is indeed often higher. Therefore, identication is not easy by way of a simple comparison. Furthermore, it is not easy to check every peak in all spectra. The mutual information values indicate the relative importance of a peak. Peaks having low mutual information values can be omitted because they are considered not to be specic to a particular

+ p(a2 )log2 p(a2 )} = {(3/8)log2 (3/8) + (5/8)log2 (5/8)} = 0.95

Next, the most appropriate threshold V was determined by trial and error to obtain the best mutual information. For example, the best value of the mutual information, 0.95, is obtained when V is 6000. S(A|B) is calculated: The number of data with a peak intensity at m/z133 higher than 6000: n(b1 ) = 3 The number of data with a peak intensity at m/z133 lower than 6000: n(b2 ) = 5

Sample 1

Sample 2

Sample 1

Sample 2

Mutual information 1
Figure 1. Classication concept based on mutual information.

Mutual information 0

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S. Aoyagi The number of sample 1 data with a peak intensity at m/z133 higher than 6000: n(a1 |b1 ) = 3 The number of sample 1 data with a peak intensity at m/z133 lower than 6000: n(a1 |b2 ) = 0 The number of sample 2 data with a peak intensity at m/z133 higher than 6000: n(a2 |b1 ) = 0 The number of sample 2 data with a peak intensity at m/z133 lower than 6000: n(a2 |b2 ) = 5 Therefore, p(a1 |b1 ) = n(a1 |b1 )/n(b1 ) = 3/3 = 1 p(a1 |b2 ) = n(a1 |b2 )/n(b2 ) = 0/5 = 0 p(a2 |b1 ) = n(a2 |b1 )/n(b1 ) = 0/3 = 1 p(a2 |b2 ) = n(a2 |b2 )/n(b2 ) = 5/5 = 0 Therefore, S(A|B) = p(bj )p(ai |bj )log2 p(ai |bj ) (7) principal chain or principal chains. If it is assumed that a fragment ion is generated from a part containing two neighbor residues, its formula can be one of the combinations of two of the residues in Table 2 and fragments of the two principle chains in Table 3. In addition, when the fragment ion generation part contains a proline residue, the possible fragments in Table 4 should be considered.

Evaluation of Proteins on Biodevices

Imaging of protein distribution TOF-SIMS is the most useful technique to evaluate the distribution and immobilization processes of proteins on biodevice surfaces[12,17 19] because it provides chemical mapping at high spatial resolution. However, it is difcult to obtain one specic protein mapping from out of a collection of other proteins, especially on complicated materials containing nitrogen, because fragment ions from protein samples are very complicated and not predictable at present. In order to obtain chemical images of proteins, the specic peaks related to each protein must be selected out of all the peaks in the TOF-SIMS spectra. Because there are so many possible combinations of amino acid residues and so many possible structures of fragment ions from amino acid combinations, it is very difcult to nd out a specic secondary ion from a particular protein. In addition, some of the fragment ions from substrate materials have the appearance of complicated, protein-like fragment ions, which makes it more difcult to select appropriate peaks of the fragment ions related to the protein for further analysis of the TOF-SIMS data. When TOF-SIMS spectra analysis using mutual information is used to study protein samples, this analysis method indicates several important peaks that can be used to obtain secondary ion images of the particular protein. For example, in order to evaluate the protein adsorption that takes place on hollow ber

= {p(b1 )p(a1 |b1 )log2 p(a1 |b1 ) + p(b2 )p(a1 |b2 )log2 p(a1 |b2 ) + p(b1 )p(a2 |b1 )log2 p(a2 |b1 ) + p(b2 )p(a2 |b2 )log2 p(a2 |b2 )} = 0 Finally, mutual information I(A; B) is I(A; B) = S(A) S(A|B) = 0.95 (9) (8)

S(A) is 1 when the spectra numbers of both samples are equal. When mutual information on the peaks at m/z46, 134 is calculated in the same way focusing on sample 1, the best values of the mutual information are approximately 0.5 and 0.95, respectively. Although a low value of mutual information means a particular peak is not specic to a particular sample, a high value of mutual information does not always ensure the specicity of a particular peak to a particular sample. For example, the peak at m/z134 cannot be selected as a specic peak in sample 1 because the difference in the intensities of sample 1 and sample 2 is not large enough to allow classication, even though the intensities of sample 1 data are higher than those of the sample 2 data. Therefore, every peak selected as a specic peak using mutual information should be checked to determine whether it is really appropriate. On the other hand, peaks having low mutual information values can be omitted from further consideration. This method reduces the target peaks on TOF-SIMS spectra. Usually several to several dozen peaks are retained. Peak identication and protein matching Fragment ion peaks from ensembles of amino acids are identied by searching every combination of amino acids based on the following hypothesis: (i) recombination is not considered, (ii) double bonds are not cut completely, (iii) hydrogen addition and hydrogen desorption are considered exibly (i.e. the numbers of carbon, oxygen, nitrogen and sulfur are considered) and (iv) sulfursulfur bonding is considered present when there are more than two sulfur atoms. Possible fragments from every part of a protein molecule were considered with structures of the 20 amino acid residues composing a protein. Table 2 shows fragments from the side chains of the residues, and Table 3 shows fragments from the

Table 2. Possible fragments generated from amino acid residue side chains Amino acid Gly Ala Val Leu Ile Met Pro Phe Trp Ser Thr Asn Gln Tyr Cys Lys Arg His Asp Glu G A V L I M P F W S T N Q Y C K R H D E Formula of side chain H CH3 C3 H7 C4 H9 C4 H9 C3 H7 S C3 H6 C7 H8 C9 H7 N CH3 O C2 H5 O C2 H4 NO C3 H6 NO C7 H8 O CH3 S C4 H11 N C4 H11 N3 C4 H5 N2 C2 H2 O2 C3 H4 O2 Possible fragment (except for hydrogen) C C3 C4 C4 C3 S C3 C7 C9 N CO C2 O C2 NO C3 NO C7 O CS C4 N C4 N3 C4 N2 C2 O2 C3 O2 C2 C3 C3 C2 S C2 C C C C2 C2 O C3 O C7 C C4 C4 N2 C C2 O C3 O C C2 C2 C2 C CO C C2 C C3 C3 N C C2 C C C C C C2 C3 C C C2 C

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TOF-SIMS application to bioanalysis using mutual information

Table 3. Possible fragments generated from principle chains of amino acid residues Number of residues 1 2 3 4 5 Formula of principle chain C2 H2 NO C4 H4 N2 O2 C6 H6 N3 O3 C8 H8 N4 O4 C10 H10 N5 O5 C3N2O2 C5N3O3 C7N4O4 C9N5O5 C11N6O6 Possible fragment from principle chain (except for hydrogen) C3NO2 C5N2O3 C7N3O4 C9N4O5 C11N5O6 C2N2O C4N3O2 C6N4O3 C8N5O4 C10N6O5 C2NO C4N2O2 C6N3O3 C8N4O4 C10N5O5 C2O C4NO2 C6N2O3 C8N3O4 C10N4O5 CN C3N2O C5N3O2 C7N4O3 C9N5O4 C C3NO C5N2O2 C7N3O3 C9N4O4

Table 4. Possible fragments generated from the principle chains containing proline Number of residues 1 2 3 4 5 Possible fragment from principle chain (except for hydrogen) N C2N2O C4N3O2 C6N4O3 C8N5O4 C2NO C4N2O2 C6N3O3 C8N4O4 C5N3O3 C7N4O4 C9N5O5

dialysis membranes with nanosize pores used for the treatment of patients with renal failure, secondary ion images related to the protein are useful. The protein adsorption onto the dialysis membranes has been evaluated by TOF-SIMS imaging[9,10] in an effort to develop higher performance dialysis techniques. Solutions of a model protein, bovine serum albumin (BSA), were fed into the hollow of three commercially available dialysis membranes made of polysulfone (APS), polyether polymer alloy (FLX) and poly(acrylonitrile) (PAN), respectively, for 7 h, and then rinsed with distilled water and dried in a desicator before TOFSIMS measurement. In addition, dried native membranes were prepared as reference samples. The values of mutual information of SIMS spectra of each sample were calculated to select peaks specic to BSA-adsorbed samples. The peaks with higher values of

mutual information were checked to determine whether they were appropriate for chemical mapping or not, for example, whether their intensities were sufcient for secondary ion imaging. Because the dialysis membranes contain complicated additives such as polyvinylpyrrolidone (PVP) to obtain high biocompatibility, they produce complicated, protein-like fragment ions. In order to select peaks appropriate for protein imaging, peaks only specic to the target protein must be found from among all the peaks in the spectra. The spectra analysis with the mutual information is helpful for such a purpose. For example, according to the high mutual information values, the peaks of CHS, C2 H6 NO, C4 H10 N and C3 H6 NO2 at m/z = 45, 60, 72 and 88, respectively, were selected as peaks specic to BSA adsorbed on the APS membrane, and the C4 H2 , C5 H8 N, C5 H10 O and C6 H10 NO peaks at m/z = 50, 82, 86 and 112, respectively, were selected as peaks specic to the APS membrane.[9,10] Protein images on the membranes were obtained by integrating these specic secondary ion images. Figure 2 shows typical secondary ion images of BSA adsorption on membranes. Because these samples are inuenced by topography, which may alter the images of BSA distribution, all images were divided by their total images to reduce the topographical effect. The peaks related to BSA were selected by comparing the TOF-SIMS spectra of BSA-adsorbed membranes and those of native membranes using mutual information. According to the native membrane gures (upper gures) and BSA-treated membrane gures (lower gures), BSA is adsorbed on the inside and outside surfaces of the APS membrane, the inside and outside

CCD image of the hollow-fiber membrane

APS

FLX

PAN

100 mm

Native membranes

APS (BSA)

FLX(BSA)
BSA-treated membranes

PAN(BSA)

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Figure 2. TOF-SIMS image of the protein adsorbed onto hollow ber membranes.

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S. Aoyagi

Mass Spectrum

Specific fragment ion Amino acid residues

m/z

Amino acid sequence

Secondary ion generation Surface part 2 nm

Substrate: biosensor, particle, electrode etc

Figure 3. Basic concept of orientation evaluation with TOF-SIMS.

surfaces as well as on cross-section of the FLX membrane, and the inside surface of the PAN membrane. Distributions of two or three proteins were also obtained using TOF-SIMS with the mutual information analysis.[11 13] For example, the distribution patterns of two proteins on an optic biosensor were observed by TOF-SIMS imaging.[11,12] Protein A was immobilized on the sensor surface to detect the sample protein, immunoglobulin G (IgG). In order to conrm immobilization of protein A and also to evaluate the reaction between the immobilized protein A and IgG in a sample solution, both protein A immobilized on the glass plate and IgG bound to the immobilized protein A were measured with TOF-SIMS, and then their spectra were analyzed by means of principal component analysis (PCA) and mutual information. Principal component (PC) score plots indicated that the fragment ions from protein A and those from IgG could be distinguished with the TOF-SIMS spectra.[12] According to the PC scores and loadings, though peaks related to the protein A were not found searching through loadings of the PCs, peaks related to the IgG sample were found to be at m/z = 43, 71, 82, 84, 102, 136, 159 and 170. In addition, some of these peaks, at m/z = 84, 102, 159 and 170, have high values of mutual information. Unfortunately, they were not appropriate for imaging because their intensities in the IgG sample and the protein A sample were not so different. Peaks thought to be specic to each protein were selected using the mutual information. Candidate peaks specic to the IgG sample were at m/z = 59, 60, 70, 72, 74, 86, 87, 100, 110, 112, 130, 136, 159, 166, 170 and 184, and those specic to the protein A sample were at m/z = 40, 44, 45, 46, 47, 48, 127 and 182. The mutual information values of these peaks are more than 0.8. After checking to determine whether they are really specic to the proteins by comparing their intensities in the protein A sample to those in the IgG sample and by evaluating the secondary ion images related to each protein, peaks specic to protein A and

IgG were determined to be at m/z = 182 and 184, respectively.[12] Finally, both protein distributions on the biosensor chip were obtained with the peaks selected with this analysis method of the TOF-SIMS spectra using mutual information to reduce the number of unimportant peaks. In protein TOF-SIMS spectra, the relationship among peaks is usually hidden, because several peaks of fragment ions generated from different parts of a protein often have very similar mass values, or the same formula, which makes attribution of a particular peak uncertain. Because a peak in TOF-SIMS spectra from protein samples can be generated by several different parts of a protein because of complicated fragmentation, this may affect the results obtained with PCA or other commonly used multivariate techniques. Those peaks selected using PCA may not always include all the important peaks specic to a particular sample. Therefore, sophisticated analysis methods are sometimes not successful for interpreting the TOF-SIMS spectra from proteins. On the other hand, information theory, which has been applied to reduce the inuence of noise in information technology, can help simplify the TOF-SIMS spectra from protein samples to select the unimportant peaks. Peaks acting almost the same in different samples can be found based on the mutual information. In other words, peaks acting differently in the different samples can be identied using mutual information. When TOF-SIMS devices having much higher mass resolution or MS/MS TOF-SIMS are released in the near future, TOF-SIMS spectra from proteins will be analyzed with sophisticated statistical techniques. Currently, comparatively simple comparison methods, such as the mutual information method, are useful to identify the important hidden peaks. Moreover, the mutual information analysis method is able to contribute as the rst step of TOF-SIMS data analysis to reduce the amount of less important information. Then the data treated with the mutual information analysis method can be applied to other multivariate analysis techniques.

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TOF-SIMS application to bioanalysis using mutual information Investigation of conformation and orientation of proteins on substrates The orientation and three-dimensional structure of immobilized proteins on biodevices are very important to assure their high performance. TOF-SIMS has been already used in the steric analysis of proteins such as an evaluation of orientation and protein conformation.[14 16,20 25] Because the sampling depth of TOF-SIMS in the static mode is less than 2 nm,[26] a thickness much less than for most proteins, TOF-SIMS is able to provide the chemical structure of the surface side of an immobilized protein molecule, which indicates the orientation of the immobilized protein. In other words, the orientation of immobilized proteins can be evaluated based on the determination of a partial structure, representing ensembles of amino acids, on the surface side. When peaks generated from these amino acid ensembles are identied, the surface side can be determined. The important peaks indicating the surface side are specic to a specic target sample when reference samples are appropriately prepared. Using the mutual information analysis, the important peaks specic to the target protein can be selected by a comparison with TOF-SIMS spectra of the reference samples. Lysozyme[16,20] , cytochrome b5[15] and BSA[14] have been investigated with regard to their orientation by means of TOFSIMS with mutual information analysis. Specic peaks related to an oriented immobilized protein were obtained by a comparison with reference samples such as nonoriented immobilized proteins, differently oriented proteins and different protein immobilized samples. Because proteins in general produce similar fragment ions, specic fragment ions are not always found in strong peaks of the spectra. Every peak in the spectra should be checked in comparison with the reference samples to nd the relevant specic peaks. For example, in case of egg white lysozyme,[16] the following peaks were selected using mutual information analysis: main peaks specic to N-lysozyme, immobilized at epsilon amine groups, are at m/z = 133 and 281, and a peak specic to C-lysozyme, immobilized at carboxyl groups, is at m/z = 175, 234 and 282.[16] When the data of the lysozyme samples and reference samples, enkephalin-immobilized samples and BSA-immobilized samples, were performed with PCA using a peak set of reported fragment ions related to the amino acids composing proteins,[27 29] it is suggested that candidate peaks related to C-lysozyme are at m/z = 70, 81, 82, 83, 84, 86, 88, 110 and 159, and those related to N-lysozyme are at m/z = 43, 44, 60, 68, 69, 72, 73 and 74. Some of the peaks, peaks at m/z = 70, 84, 86, 88, 110 and 159 for C-lysozyme and at m/z = 43, 73 and 74 for N-lysozyme, have high mutual information values though they were omitted in the checking process after the calculation of mutual information. The peaks selected with PCA are not helpful to determine surface or near surface sides of the immobilized protein because they are mainly generated from one residue existing in almost every side of the protein. Moreover, the intensities of the peaks are not so different in both lysozyme sample spectra. After nding the specic peaks, their possible chemical formulas are searched by means of considering every possible fragment from amino acid residues composing a protein, or an ensemble of the residues. Then the amino acid residues, being able to generate fragment ions having certain indicated chemical formulas in the amino acid sequence of the protein are searched. Because the side containing most of these residues is considered to be the surface side of the protein, every side of the three-dimensional protein structure is checked. Finally, the surface side containing most of these residues is found, which indicates the orientation of the protein. Figure 3 shows the basic concept of these steps. In order to evaluate the results obtained with the mutual information method, the orientational states of the protein samples were controlled, and the TOF-SIMS spectra of the samples were analyzed. For example, egg white lysozyme was immobilized on an indium tin oxide (ITO)-coated glass slide activated by glutaraldehyde at an epsilon amine group of lysine.[16] Because glutaraldehyde mainly reacts with lysine, which has an epsilon amine group,[30] the underside of the immobilized protein should have many lysine residues. The egg white lysozyme contains

Figure 4. The indicated surface side of the egg white lysozyme immobilized at lysine residues.

Figure 5. The backside of the indicated surface side of the egg white lysozyme.

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S. Aoyagi six lysine residues in one molecule. As a result, Fig. 4 shows the indicated surface side of this lysozyme[16] and Fig. 5 shows the backside of the indicated surface side. Because the backside includes four lysine residues (13, 96, 97 and 116) out of six lysine residues, this lysozyme is mainly immobilized at this backside. Therefore, the orientation indicated with the TOF-SIMS analysis results is rational and in line with the expected orientation based on the 3D structure obtained with X-ray analysis. The actual 3D structures of protein samples may be different from their reported structures obtained with X-ray analysis or nuclear magnetic resonance (NMR), because the structures will change depending on the conditions. However, their main structures are preserved, and these structural changes are minor and partial changes, which can be detected with this TOF-SIMS analysis method. In the near future, this TOF-SIMS analysis method is expected to be able to indicate not only the orientation but also both real protein structures and structural changes depending on sample conditions.

References
[1] M. S. Wagner, D. G. Castner, Langmuir 2001, 17, 4649. [2] E. S. Berman, K. S. Kulp, M. G. Knize, L. Wu, E. J. Nelson, D. O. Nelson, K. J. Wu, Anal. Chem. 2006, 78, 6497. [3] J. L. S. Lee, I. S. Gilmore, M. P. Seah, Surf. Interface Anal. 2007, 40, 1. [4] F. M. Green, E. J. Dell, I. S. Gilmore, M. P. Seah, Int. J. Mass Spectrom. 2008, 272, 38. [5] S. Aoyagi, M. Kudo, in Proteins at Solid-liquid Interfaces (Ed.: P. D jardin), Springer-Verlag: Berlin, Hidelberg, 2006, pp. 151. e [6] C. E. Shannon, W. Weaver, The Mathematical Theory of Information, University of Illinois Press: Urbana, 1947. [7] K. Eckschlager, V. Stepanek, K. Danzer, J. Chemometr. 1990, 4, 195. [8] T. Fujikura, K. Sakamoto, J. T. Shimozawa, Anal. Chim. Acta 1997, 351, 387. [9] S. Aoyagi, M. Hayama, U. Hasegawa, K. Sakai, M. Tozu, T. Hoshi, M. Kudo, e-J. Surf. Sci. Nanotechnol. 2003, 1, 67. [10] S. Aoyagi, M. Hayama, U. Hasegawa, K. Sakai, M. Tozu, T. Hoshi, M. Kudo, Appl. Surf. Sci. 2004, 411, 231. [11] S. Aoyagi, Y. Kawashima, M. Kudo, Nucl. Instrum. Methods Phys. Res., Sect. B 2005, 232, 146. [12] S. Aoyagi, M. Kudo, Biosens. Bioelectron. 2005, 20, 8 1626. [13] S. Aoyagi, A. Takesawa, A. C. Yamashita, M. Kudo, Appl. Surf. Sci. 2006, 252, 6697. [14] S. Aoyagi, M. Dohi, N. Kato, M. Kudo, S. Iida, M. Tozu, N. Sanada, e-J. Surf. Sci. Nanotechnol. 2006, 4, 614. [15] S. Aoyagi, A. Rouleau, W. Boireau, Appl. Surf. Sci. (in press). [16] K. Okada, S. Aoyagi, M. Dohi, N. Kato, M. Kudo, M. Tozu, T. Miyayama, N. Sanada, Appl. Surf. Sci. (in press). [17] B. Hagenhoff, Biosens. Bioelectron. 1995, 10, 885. [18] R. Chatterjee, Appl. Surf. Sci. 2004, 437, 231. [19] K. G. Lloyd, D. P. O. Keefe, Appl. Surf. Sci. 2004, 207, 231. [20] S. Aoyagi, K. Okada, A. Shigyo, N. Man, A. Karen, Appl. Surf. Sci. (in press). [21] P. Bertrand, Appl. Surf. Sci. 2006, 252, 6986. [22] R. Michel, S. Pasche, M. Textor, D. G. Castner, Langmuir 2005, 21, 12327. [23] K. Leufgen, M. Mutter, H. Vogel, W. Szymczak, J. Am. Chem. Soc. 2003, 125, 8911. [24] N. Xia, C. J. May, S. L. McArthur, D. G. Castner, Langmuir 2002, 18, 4090. [25] N. Xia, D. G. Castner, J. Biomed. Mater. Res. 2003, 67A, 179. [26] R. Michel, D. G. Castner, Surf. Interface Anal. 2006, 38, 1386. [27] D. S. Mantus, B. D. Ratner, B. A. Carlson, J. F. Moulder, Anal. Chem. 1993, 65, 1431. [28] J.-B. Lhoest, E. Detrait, P. van den B. de Aguilar, P. Bertrand, J.Biomed. Mater. Res. 1998, 41, 95. [29] J.-B. Lhoest, M. S. Wagner, C. D. Tidwell, D. G. Castner, J. Biomed. Mater. Res. 2001, 57, 432. [30] D. Hopwood, Histochem. J. 1972, 4, 267.

Summary
SIMS spectra analysis techniques are necessary to interpret complicated and nonpredictable fragment ions from macromolecules, even though SIMS, with the cluster of primary ion sources, enhances the intensity of high-mass secondary ions. Spectra analysis with mutual information is one of the most useful techniques for identifying important, sometimes hidden, fragment ion peaks related to a particular protein sample to obtain either secondary ion images of the protein or chemical structures on the surface side of the protein. At this point, further improvement of the production of larger and greater numbers of fragment ions and higher mass resolution or MS/MS application to SIMS to identify high-mass fragment ions is required. Both data analysis and ion analysis techniques, including the enhancement of ion production, are expected to be considerably improved in the near term, and hence will enable a more precise measurement of various proteins on biodevices. In the near future, conformation change in proteins after reactions or environmental changes such as pH and orientation of the binding sites of immobilized protein will be measurable with TOF-SIMS.

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