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Evaluation of immobilized-lysozyme by means of TOF-SIMS
Applied Surface Science 255 (2008) 1104–1106
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Evaluation of immobilized-lysozyme by means of TOF-SIMS Keigo Okada a, Satoka Aoyagi a,*, Makoto Dohi a, Nobuhiko Kato b, Masahiro Kudo b, Miyako Tozu c, Takuya Miyayama c, Noriaki Sanada c a
Faculty of Life and Environmental Science, Shimane University, 1060 Matsue-shi, Shimane 690-8504, Japan Department of Applied Physics, Faculty of Engineering, Seikei University 3-3-1, Kitamachi, Kichijioji, Musashino, Tokyo 180-8633, Japan c ULVAC-PHI Inc., 370 Enzo, Chigasaki, Kanagawa 253-8522, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history:
Evaluation of immobilized-proteins on bio-devices is important for the development of sophisticated devices. Lysozyme molecules immobilized on substrates were evaluated by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS). Two types of the lysozyme-immobilized samples were prepared by controlling the binding parts, i.e., the amino groups or carboxyl groups, of the protein. The TOF-SIMS spectra of each sample were analyzed with mutual information to select fragment ions specific to each sample. According to the results, differences between the samples being immobilized in the different ways are suggested, and the surface structure of the lysozyme molecule immobilized at amino groups is determined based on three-dimensional structure of lysozyme in the Protein Data Bank. ß 2008 Elsevier B.V. All rights reserved.
Available online 8 May 2008 Keywords: Lysozyme Orientation TOF-SIMS Immobilization Three-dimensional structure
1. Introduction The evaluation of immobilized-proteins is crucial to the further development sophisticated bio-devices. Surface analysis techniques such as scanning electron microscopy (SEM) [1] and atomic force microscopy (AFM) [2] have been applied to the evaluation of immobilization or substrate surface conditions based on the shape of the sample surface. Secondary ion mass spectrometry (SIMS) and X-ray photoelectron spectroscopy (XPS) have been applied to the analysis of impurities and the composition [3,4] of metal or semiconductor materials such as microelectrodes, and recently these techniques are also applied to the evaluation of bio-devices. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has a special feature of providing detailed chemical information of the upper surface part of a macromolecule. Moreover, TOF-SIMS is able to determine a partial structure of a particular protein immobilized on a substrate, based on an analysis of fragment ions in TOF-SIMS spectra [5]. In this study, a protein of 129 amino acid residues, egg white lysozyme, was employed as a model protein [6,7]. Two types of the lysozyme-immobilized samples were prepared by controlling the binding parts, i.e., amino groups or carboxyl groups, of the protein. Their orientations were evaluated by determining their surface structures. First, lysozyme molecules were immobilized on an
* Corresponding author. E-mail address: [email protected] (S. Aoyagi). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.05.050
aminosilanized indium tin oxide (ITO) coated glass slide at their amino groups by covalent bonding (N-lysozyme samples). Second, lysozyme molecules were immobilized at their carboxyl groups by ionic bonding (C-lysozyme samples). Both samples were measured with TOF-SIMS using a gold cluster ion source. Specific fragment ions of each sample were selected based on values of mutual information [8]. Then, differences between the samples immobilized in the different ways were evaluated in terms of orientation. 2. Materials and methods 2.1. Sample preparation An indium tin oxide (ITO) coated glass slide (Sigma–Aldrich Co., St. Louis, MO, USA) of 8 mm 8 mm was aminosilanized with aminopropyltrimethoxysilan (Tokyo Kasei, Tokyo, Japan). The aminosilanized ITO glass plates were activated by glutaraldehyde and then soaked in a 0.1 M phosphate buffered saline (PBS) solution at pH 7.4 containing lysozyme (from chicken egg white, Sigma) and allowed to react in the dark for 30 h at 277 K, and then lysozyme molecules were immobilized on the ITO plates at amino groups by covalent bonding [9,10]. This protein immobilization was confirmed with a fluorescence sensor in a previous study [9]. After the ITO glass plates were washed in the PBS solution, with some of the sample plates washed in pure water and the others in PBS solution, respectively, sonic waves were applied for 10 s to remove adsorbed proteins. Next, the aminosilanized ITO plates were soaked in the PBS solution with lysozyme directly, allowed to
K. Okada et al. / Applied Surface Science 255 (2008) 1104–1106
react in the dark for 30 h at 277 K, and then lysozyme molecules were immobilized on the ITO plates at carboxyl groups by ion bonding. These samples were dried with a freeze dryer (VD-250F, Taitech, Saitama, Japan) before TOF-SIMS measurement. The N- and C-lysozyme samples were not rinsed with pure water to avoid protein structural change depending on pH. The affect of the salts was canceled when N-lysozyme data were compared with C-lysozyme data.
1105
Table 1 Chemical formulas and ensembles of amino acids (N-lysozyme) m/z
Chemical formulas possible amino acid
Amino acids identified
133.01
C4H7NO2S C7H3NO2, C6HN2O2
[C] Less specificity
281.05
C10H11N5O3S C15H11N3OS C10H9N4O6
[RC], [CH], [CRG] [MW] Less specificity
2.2. TOF-SIMS measurement Positive ion spectra obtained with TOF-SIMS, TRIFT-IV (Physical Electronics, Eden Prairie, MN) using a 30 kV Au3+ primary ion source, were acquired at up to 1000 m/z while maintaining the primary ion dose at less than 1012 ions/cm2 to ensure static conditions [11]. All the spectra, composed of positive ion TOF-SIMS spectra, were calibrated to the CH3+, C2H5+ and C3H5+ peaks before data analysis. 2.3. Spectrum analysis The calculation steps and the basic concept of classification by means of mutual information were described in previous papers [8,12–14]. The values of the mutual information were calculated comparing the N-lysozyme with the N-lysozyme rinsed with water or C-lysozyme. The peaks of secondary ions at m/z = 40–400 were used for the calculation of mutual information.
Table 1 shows the chemical formulas of peaks at m/z = 133 or 281 and the possible combinations of amino acid residues generating these fragment ions. The chemical formula candidates having too many possibilities were omitted because it is highly unlikely they would be specific only to N-lysozyme. ‘‘Less specificity’’ in Table 1 indicates the following: since the C10H9N4O6 fragment can be derived from many amino acid residues or their pairs, which are included in many parts of the amino acid sequence of the protein, every sample spectrum must show the peak. Therefore, the C10H9N4O6 peak cannot be specific only to N-lysozyme. The chemical formula of m/z 133 was determined to be C4H7NO2S and it is a fragment ion from cysteine. Lysozyme has eight cysteines in one molecule and the following cysteines (C) are bound by a disulfide bond: C6–C127, C30–C115, C64–C80, and C76–C94. The numbers after each residue is the order in the sequence. On the other hand, the chemical formula of m/z 281 is C10H11N5O3S or C15H11N3OS. The structure of C10H11N5O3S, an RC fragment, is considered to be the following:
2.4. Peak identification and matching The peaks of fragment ions from combinations of amino acids are identified by searching through every combination of amino acids based on the following hypothesis: (1) double bonds are not cut, (2) the numbers of carbon, oxygen, nitrogen and sodium are considered, (3) SS bonding is taken into consideration when there are more than two sodium atoms. Then the origin of the identified fragment ion, formed from a combination of amino acids, is found through the primary sequence of lysozyme. 3. Results and discussion 3.1. Lysozyme immobilized at amino groups (N-lysozyme) Data for N-lysozyme were compared with N-lysozyme rinsed with water or C-lysozyme to find the peaks of fragment ions specific to N-lysozyme. Peaks related to proteins, immobilized on the ITO glass or native glass substrates, are detectable with TOFSIMS, as shown in previous studies comparing protein-immobilized substrates and substrates without proteins [8,15,16]. As a result, the peaks of secondary ions at m/z = 75, 133, 281, 325, and 327 were obtained as fragment ions specific to N-lysozyme by comparing the TOF-SIMS spectra of each sample based on the mutual information. The peak at m/z = 75 was omitted because it is so small that it is difficult to clarify the original parts. In addition, peaks at m/z = 325, 327 were also ignored because they have too many possible chemical formulas to determine. In this case, the reference samples were N-lysozyme rinsed with pure water and C-lysozyme. The specific peaks are considered to come from the protein because the contaminations were cancelled when compared with the reference samples prepared in the same condition. Moreover, not only single amino acids but also every possible combination of amino acids should be considered to determine the chemical formulas of fragment ions because fragmentation does not always occur at a single amino acid, and some fragment ions could be related to multiple amino acids.
C15H11N3OS is omitted because a sequential combination of methionine (M) and tryptophan (W) is not included in the amino acid sequence of chicken egg white lysozyme [7]. Therefore, the possible combination of residues are ‘‘cysteines’’ and ‘‘glycine (G)4– arginine (R)5–C6’’, ‘‘R114–C115’’, and ‘‘R125–G126–C127–R128’’. According to these results, the parts generating the fragment ions specific to N-lysozyme in the amino acid sequence of egg white lysozyme were determined, and then the three-dimensional configurations were also determined based on three-dimensional structure of chicken egg white lysozyme (1DPW.PDB) registered in the Protein Data Bank. Finally, the side containing ‘‘G4–R5–C6’’, ‘‘R114–C115’’, ‘‘R12–G126–C127–R128’’, and four cysteines, C6– C127, C30–C115, was found as shown in Fig. 1. This side is considered to be the surface of the N-lysozyme. Since glutaraldehyde mainly reacts with lysine, which has an epsilon-amino group [10], the underside of the immobilized protein should have lysine residues. The egg white lysozyme has six lysine residues. The backside of the side indicated as the surface side shown in Fig. 1 includes three lysine residues, although most of the other sides do not include them. Therefore, it is also suggested that the side shown in Fig. 1 can be the surface side when the protein is immobilized with glutaraldehyde due to the protein structure.
1106
K. Okada et al. / Applied Surface Science 255 (2008) 1104–1106
not been confirmed yet if the protein of C-lysozyme is immobilized with an ion bond between the carboxyl groups of lysozyme and the amino groups of the aminosilanized ITO glass plates, it can be said that the protein molecules of C-lysozyme must be immobilized in a different way than N-lysozyme, and therefore the C-lysozyme sample is useful as a reference sample to help select specific peaks. 4. Conclusions
Fig. 1. 3D structure of chicken egg white lysozyme (1DPW). The numbers show the sequence numbers.
3.2. Lysozyme immobilized at carboxyl groups (C-lysozyme) Peaks of the secondary ions at m/z = 94.93, 103.94, 160.92, 163.95, 164.93, 174.90, 196.87, 211.84, 212.85, 218.86, 234.83, 278.82 and 282.85 were obtained as specific fragment ions of lysozyme immobilized at carboxyl groups (C-lysozyme) by comparing the TOF-SIMS spectra of each sample based on mutual information. Since they are smaller than their nearest integral values, they are thought to be related to metals in the substrate or PBS. Some of these peaks are observed also in the TOF-SIMS spectra of N-lysozyme, though they are mainly specific to C-lysozyme. The TOF-SIMS spectra of N-lysozyme and those of N-lysozyme washed with pure water after protein immobilization to eliminate PBS were compared to estimate the potential influence of metals in the PBS. As a result, some of them were detected in the spectra of Nlysozyme, but not detected in those of N-lysozyme washed with pure water. Therefore, they are related to PBS, and thus they should be omitted. The remaining peaks are m/z = 174.90 (C2HNOSn), 234.83 (C3H3N2OSSn), 278.82, which is omitted because an appropriate formula was not found, and 282.85 (C6H2NO3SIn), which is related to metal ingredients of the substrate, such as In+ and Sn+. Since the protein fragment parts are too small to determine the particular parts in the protein molecule, the surface portions of C-lysozyme were not determinable in this condition. Therefore, it is indicated that the lysozyme molecules are arranged in a non-oriented manner in the case of C-lysozyme. Though it has
The orientation of lysozyme molecules immobilized at amino groups is here evaluated by comparing the upper surface structure indicated by TOF-SIMS analysis with the three-dimensional structure of lysozyme in the Protein Data Bank. It is suggested that the lysozyme molecules immobilized at carboxyl groups are not immobilized in orientation in this preparation condition. Thus, TOF-SIMS analysis is very useful to evaluate the orientation and surface structure of immobilized macromolecules, such as proteins. Further study will provide a promising technique for the evaluation of bio-devices, thus contributing to the continued development of more sophisticated devices. Acknowledgment This research was partly supported by the Kao foundation for arts and sciences. References [1] K. Abe, A. Ishii, M. Hirano, J.F. Rusling, Electroanalysis 17 (24) (2005) 2266. [2] D.V. Nicolau, T. Taguchi, H. Taniguchi, S. Yoshikawa, Langmuir 14 (7) (1998) 1927. [3] D.C. Gerlach, J.B. Cliff, D.E. Hurley, B.D. Reid, W.W. Little, G.H. Meriwether, A.J. Wickham, T.A. Simmons, Appl. Surf. Sci. 252 (19) (2006) 7041. [4] T. Kubo, S. Fujiwara, H. Nanao, I. Minami, S. Mori, Tribol. Lett. 23 (2) (2006) 171. [5] S. Aoyagi, M. Dohi, N. Kato, M. Kudo, S. Iida, M. Tozu, N. Sanada, e-J. Surf. Sci. Nanotech. 4 (2006) 1. [6] C. Brinkmann, M.S. Weiss, E. Weckert, Acta Cryst. D 62 (4) (2006) 349. [7] R.E. Canfield, J. Biol. Chem. 238 (8) (1963) 2698. [8] S. Aoyagi, Y. Kawashima, M. Kudo, Nucl. Instr. Methods Phy. Res. B 232 (2005) 146. [9] S. Aoyagi, R. Imai, K. Sakai, M. Kudo, Biosens. Bioelectron. 18 (2003) 791. [10] D. Hopwood, Histochem. J. 4 (1972) 267. [11] G. Marletta, S.M. Catalano, S. Pignataro, Surf. Interf. Anal. 16 (1990) 407. [12] S. Aoyagi, M. Hayama, U. Hasegawa, K. Sakai, M. Tozu, T. Hoshi, M. Kudo, e-J. Surf. Sci. Nanotech. 1 (2003) 67. [13] C.E. Shannon, W. Weaver, The Mathematical Theory of Information, University of Illinois Press, Urbana, IL, 1947. [14] T. Fujikura, K. Sakamoto, J.T. Shimozawa, Anal. Chim. Acta 351 (1–3) (1997) 387. [15] S. Aoyagi, Y. Oiwa, M. Kudo, Appl. Surf. Sci. 231–232 (2004) 432. [16] S. Aoyagi, M. Kudo, Biosens. Bioelectron. 20 (8) (2005) 1626.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Evaluation of immobilized-lysozyme by means of TOF-SIMS Keigo Okada a, Satoka Aoyagi a,*, Makoto Dohi a, Nobuhiko Kato b, Masahiro Kudo b, Miyako Tozu c, Takuya Miyayama c, Noriaki Sanada c a
Faculty of Life and Environmental Science, Shimane University, 1060 Matsue-shi, Shimane 690-8504, Japan Department of Applied Physics, Faculty of Engineering, Seikei University 3-3-1, Kitamachi, Kichijioji, Musashino, Tokyo 180-8633, Japan c ULVAC-PHI Inc., 370 Enzo, Chigasaki, Kanagawa 253-8522, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history:
Evaluation of immobilized-proteins on bio-devices is important for the development of sophisticated devices. Lysozyme molecules immobilized on substrates were evaluated by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS). Two types of the lysozyme-immobilized samples were prepared by controlling the binding parts, i.e., the amino groups or carboxyl groups, of the protein. The TOF-SIMS spectra of each sample were analyzed with mutual information to select fragment ions specific to each sample. According to the results, differences between the samples being immobilized in the different ways are suggested, and the surface structure of the lysozyme molecule immobilized at amino groups is determined based on three-dimensional structure of lysozyme in the Protein Data Bank. ß 2008 Elsevier B.V. All rights reserved.
Available online 8 May 2008 Keywords: Lysozyme Orientation TOF-SIMS Immobilization Three-dimensional structure
1. Introduction The evaluation of immobilized-proteins is crucial to the further development sophisticated bio-devices. Surface analysis techniques such as scanning electron microscopy (SEM) [1] and atomic force microscopy (AFM) [2] have been applied to the evaluation of immobilization or substrate surface conditions based on the shape of the sample surface. Secondary ion mass spectrometry (SIMS) and X-ray photoelectron spectroscopy (XPS) have been applied to the analysis of impurities and the composition [3,4] of metal or semiconductor materials such as microelectrodes, and recently these techniques are also applied to the evaluation of bio-devices. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has a special feature of providing detailed chemical information of the upper surface part of a macromolecule. Moreover, TOF-SIMS is able to determine a partial structure of a particular protein immobilized on a substrate, based on an analysis of fragment ions in TOF-SIMS spectra [5]. In this study, a protein of 129 amino acid residues, egg white lysozyme, was employed as a model protein [6,7]. Two types of the lysozyme-immobilized samples were prepared by controlling the binding parts, i.e., amino groups or carboxyl groups, of the protein. Their orientations were evaluated by determining their surface structures. First, lysozyme molecules were immobilized on an
* Corresponding author. E-mail address: [email protected] (S. Aoyagi). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.05.050
aminosilanized indium tin oxide (ITO) coated glass slide at their amino groups by covalent bonding (N-lysozyme samples). Second, lysozyme molecules were immobilized at their carboxyl groups by ionic bonding (C-lysozyme samples). Both samples were measured with TOF-SIMS using a gold cluster ion source. Specific fragment ions of each sample were selected based on values of mutual information [8]. Then, differences between the samples immobilized in the different ways were evaluated in terms of orientation. 2. Materials and methods 2.1. Sample preparation An indium tin oxide (ITO) coated glass slide (Sigma–Aldrich Co., St. Louis, MO, USA) of 8 mm 8 mm was aminosilanized with aminopropyltrimethoxysilan (Tokyo Kasei, Tokyo, Japan). The aminosilanized ITO glass plates were activated by glutaraldehyde and then soaked in a 0.1 M phosphate buffered saline (PBS) solution at pH 7.4 containing lysozyme (from chicken egg white, Sigma) and allowed to react in the dark for 30 h at 277 K, and then lysozyme molecules were immobilized on the ITO plates at amino groups by covalent bonding [9,10]. This protein immobilization was confirmed with a fluorescence sensor in a previous study [9]. After the ITO glass plates were washed in the PBS solution, with some of the sample plates washed in pure water and the others in PBS solution, respectively, sonic waves were applied for 10 s to remove adsorbed proteins. Next, the aminosilanized ITO plates were soaked in the PBS solution with lysozyme directly, allowed to
K. Okada et al. / Applied Surface Science 255 (2008) 1104–1106
react in the dark for 30 h at 277 K, and then lysozyme molecules were immobilized on the ITO plates at carboxyl groups by ion bonding. These samples were dried with a freeze dryer (VD-250F, Taitech, Saitama, Japan) before TOF-SIMS measurement. The N- and C-lysozyme samples were not rinsed with pure water to avoid protein structural change depending on pH. The affect of the salts was canceled when N-lysozyme data were compared with C-lysozyme data.
1105
Table 1 Chemical formulas and ensembles of amino acids (N-lysozyme) m/z
Chemical formulas possible amino acid
Amino acids identified
133.01
C4H7NO2S C7H3NO2, C6HN2O2
[C] Less specificity
281.05
C10H11N5O3S C15H11N3OS C10H9N4O6
[RC], [CH], [CRG] [MW] Less specificity
2.2. TOF-SIMS measurement Positive ion spectra obtained with TOF-SIMS, TRIFT-IV (Physical Electronics, Eden Prairie, MN) using a 30 kV Au3+ primary ion source, were acquired at up to 1000 m/z while maintaining the primary ion dose at less than 1012 ions/cm2 to ensure static conditions [11]. All the spectra, composed of positive ion TOF-SIMS spectra, were calibrated to the CH3+, C2H5+ and C3H5+ peaks before data analysis. 2.3. Spectrum analysis The calculation steps and the basic concept of classification by means of mutual information were described in previous papers [8,12–14]. The values of the mutual information were calculated comparing the N-lysozyme with the N-lysozyme rinsed with water or C-lysozyme. The peaks of secondary ions at m/z = 40–400 were used for the calculation of mutual information.
Table 1 shows the chemical formulas of peaks at m/z = 133 or 281 and the possible combinations of amino acid residues generating these fragment ions. The chemical formula candidates having too many possibilities were omitted because it is highly unlikely they would be specific only to N-lysozyme. ‘‘Less specificity’’ in Table 1 indicates the following: since the C10H9N4O6 fragment can be derived from many amino acid residues or their pairs, which are included in many parts of the amino acid sequence of the protein, every sample spectrum must show the peak. Therefore, the C10H9N4O6 peak cannot be specific only to N-lysozyme. The chemical formula of m/z 133 was determined to be C4H7NO2S and it is a fragment ion from cysteine. Lysozyme has eight cysteines in one molecule and the following cysteines (C) are bound by a disulfide bond: C6–C127, C30–C115, C64–C80, and C76–C94. The numbers after each residue is the order in the sequence. On the other hand, the chemical formula of m/z 281 is C10H11N5O3S or C15H11N3OS. The structure of C10H11N5O3S, an RC fragment, is considered to be the following:
2.4. Peak identification and matching The peaks of fragment ions from combinations of amino acids are identified by searching through every combination of amino acids based on the following hypothesis: (1) double bonds are not cut, (2) the numbers of carbon, oxygen, nitrogen and sodium are considered, (3) SS bonding is taken into consideration when there are more than two sodium atoms. Then the origin of the identified fragment ion, formed from a combination of amino acids, is found through the primary sequence of lysozyme. 3. Results and discussion 3.1. Lysozyme immobilized at amino groups (N-lysozyme) Data for N-lysozyme were compared with N-lysozyme rinsed with water or C-lysozyme to find the peaks of fragment ions specific to N-lysozyme. Peaks related to proteins, immobilized on the ITO glass or native glass substrates, are detectable with TOFSIMS, as shown in previous studies comparing protein-immobilized substrates and substrates without proteins [8,15,16]. As a result, the peaks of secondary ions at m/z = 75, 133, 281, 325, and 327 were obtained as fragment ions specific to N-lysozyme by comparing the TOF-SIMS spectra of each sample based on the mutual information. The peak at m/z = 75 was omitted because it is so small that it is difficult to clarify the original parts. In addition, peaks at m/z = 325, 327 were also ignored because they have too many possible chemical formulas to determine. In this case, the reference samples were N-lysozyme rinsed with pure water and C-lysozyme. The specific peaks are considered to come from the protein because the contaminations were cancelled when compared with the reference samples prepared in the same condition. Moreover, not only single amino acids but also every possible combination of amino acids should be considered to determine the chemical formulas of fragment ions because fragmentation does not always occur at a single amino acid, and some fragment ions could be related to multiple amino acids.
C15H11N3OS is omitted because a sequential combination of methionine (M) and tryptophan (W) is not included in the amino acid sequence of chicken egg white lysozyme [7]. Therefore, the possible combination of residues are ‘‘cysteines’’ and ‘‘glycine (G)4– arginine (R)5–C6’’, ‘‘R114–C115’’, and ‘‘R125–G126–C127–R128’’. According to these results, the parts generating the fragment ions specific to N-lysozyme in the amino acid sequence of egg white lysozyme were determined, and then the three-dimensional configurations were also determined based on three-dimensional structure of chicken egg white lysozyme (1DPW.PDB) registered in the Protein Data Bank. Finally, the side containing ‘‘G4–R5–C6’’, ‘‘R114–C115’’, ‘‘R12–G126–C127–R128’’, and four cysteines, C6– C127, C30–C115, was found as shown in Fig. 1. This side is considered to be the surface of the N-lysozyme. Since glutaraldehyde mainly reacts with lysine, which has an epsilon-amino group [10], the underside of the immobilized protein should have lysine residues. The egg white lysozyme has six lysine residues. The backside of the side indicated as the surface side shown in Fig. 1 includes three lysine residues, although most of the other sides do not include them. Therefore, it is also suggested that the side shown in Fig. 1 can be the surface side when the protein is immobilized with glutaraldehyde due to the protein structure.
1106
K. Okada et al. / Applied Surface Science 255 (2008) 1104–1106
not been confirmed yet if the protein of C-lysozyme is immobilized with an ion bond between the carboxyl groups of lysozyme and the amino groups of the aminosilanized ITO glass plates, it can be said that the protein molecules of C-lysozyme must be immobilized in a different way than N-lysozyme, and therefore the C-lysozyme sample is useful as a reference sample to help select specific peaks. 4. Conclusions
Fig. 1. 3D structure of chicken egg white lysozyme (1DPW). The numbers show the sequence numbers.
3.2. Lysozyme immobilized at carboxyl groups (C-lysozyme) Peaks of the secondary ions at m/z = 94.93, 103.94, 160.92, 163.95, 164.93, 174.90, 196.87, 211.84, 212.85, 218.86, 234.83, 278.82 and 282.85 were obtained as specific fragment ions of lysozyme immobilized at carboxyl groups (C-lysozyme) by comparing the TOF-SIMS spectra of each sample based on mutual information. Since they are smaller than their nearest integral values, they are thought to be related to metals in the substrate or PBS. Some of these peaks are observed also in the TOF-SIMS spectra of N-lysozyme, though they are mainly specific to C-lysozyme. The TOF-SIMS spectra of N-lysozyme and those of N-lysozyme washed with pure water after protein immobilization to eliminate PBS were compared to estimate the potential influence of metals in the PBS. As a result, some of them were detected in the spectra of Nlysozyme, but not detected in those of N-lysozyme washed with pure water. Therefore, they are related to PBS, and thus they should be omitted. The remaining peaks are m/z = 174.90 (C2HNOSn), 234.83 (C3H3N2OSSn), 278.82, which is omitted because an appropriate formula was not found, and 282.85 (C6H2NO3SIn), which is related to metal ingredients of the substrate, such as In+ and Sn+. Since the protein fragment parts are too small to determine the particular parts in the protein molecule, the surface portions of C-lysozyme were not determinable in this condition. Therefore, it is indicated that the lysozyme molecules are arranged in a non-oriented manner in the case of C-lysozyme. Though it has
The orientation of lysozyme molecules immobilized at amino groups is here evaluated by comparing the upper surface structure indicated by TOF-SIMS analysis with the three-dimensional structure of lysozyme in the Protein Data Bank. It is suggested that the lysozyme molecules immobilized at carboxyl groups are not immobilized in orientation in this preparation condition. Thus, TOF-SIMS analysis is very useful to evaluate the orientation and surface structure of immobilized macromolecules, such as proteins. Further study will provide a promising technique for the evaluation of bio-devices, thus contributing to the continued development of more sophisticated devices. Acknowledgment This research was partly supported by the Kao foundation for arts and sciences. References [1] K. Abe, A. Ishii, M. Hirano, J.F. Rusling, Electroanalysis 17 (24) (2005) 2266. [2] D.V. Nicolau, T. Taguchi, H. Taniguchi, S. Yoshikawa, Langmuir 14 (7) (1998) 1927. [3] D.C. Gerlach, J.B. Cliff, D.E. Hurley, B.D. Reid, W.W. Little, G.H. Meriwether, A.J. Wickham, T.A. Simmons, Appl. Surf. Sci. 252 (19) (2006) 7041. [4] T. Kubo, S. Fujiwara, H. Nanao, I. Minami, S. Mori, Tribol. Lett. 23 (2) (2006) 171. [5] S. Aoyagi, M. Dohi, N. Kato, M. Kudo, S. Iida, M. Tozu, N. Sanada, e-J. Surf. Sci. Nanotech. 4 (2006) 1. [6] C. Brinkmann, M.S. Weiss, E. Weckert, Acta Cryst. D 62 (4) (2006) 349. [7] R.E. Canfield, J. Biol. Chem. 238 (8) (1963) 2698. [8] S. Aoyagi, Y. Kawashima, M. Kudo, Nucl. Instr. Methods Phy. Res. B 232 (2005) 146. [9] S. Aoyagi, R. Imai, K. Sakai, M. Kudo, Biosens. Bioelectron. 18 (2003) 791. [10] D. Hopwood, Histochem. J. 4 (1972) 267. [11] G. Marletta, S.M. Catalano, S. Pignataro, Surf. Interf. Anal. 16 (1990) 407. [12] S. Aoyagi, M. Hayama, U. Hasegawa, K. Sakai, M. Tozu, T. Hoshi, M. Kudo, e-J. Surf. Sci. Nanotech. 1 (2003) 67. [13] C.E. Shannon, W. Weaver, The Mathematical Theory of Information, University of Illinois Press, Urbana, IL, 1947. [14] T. Fujikura, K. Sakamoto, J.T. Shimozawa, Anal. Chim. Acta 351 (1–3) (1997) 387. [15] S. Aoyagi, Y. Oiwa, M. Kudo, Appl. Surf. Sci. 231–232 (2004) 432. [16] S. Aoyagi, M. Kudo, Biosens. Bioelectron. 20 (8) (2005) 1626.