Evaluation of oriented lysozyme immobilized with monoclonal antibody

The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.     

An orientation analysis method for protein immobilized on quantum dot particles

The evaluation of orientation of biomolecules immobilized on nanodevices is crucial for the development of high performance devices. Such analysis requires ultra high sensitivity so as to be able to detect less than one molecular layer on a device. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has sufficient sensitivity to evaluate the uppermost surface structure of a single molecular layer. The objective of this study is to develop an orientation analysis method for proteins immobilized on nanomaterials such as quantum dot particles, and to evaluate the orientation of streptavidin immobilized on quantum dot particles by means of TOF-SIMS. In order to detect fragment ions specific to the protein surface, a monoatomic primary ion source (Ga⁺) and a cluster ion source (Au₃⁺) were employed. Streptavidin-immobilized quantum dot particles were immobilized on aminosilanized ITO glass plates at amino groups by covalent bonding. The reference samples streptavidin directly immobilized on ITO plates were also prepared. All samples were dried with a freeze dryer before TOF-SIMS measurement. The positive secondary ion spectra of each sample were obtained using TOF-SIMS with Ga⁺ and Au₃⁺, respectively, and then they were compared so as to characterize each sample and detect the surface structure of the streptavidin immobilized with the biotin-immobilized quantum dots. The chemical structures of the upper surface of the streptavidin molecules immobilized on the quantum dot particles were evaluated with TOF-SIMS spectra analysis. The indicated surface side of the streptavidin molecules immobilized on the quantum dots includes the biotin binding site.     

Evaluation of immobilized-lysozyme by means of TOF-SIMS

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.     

Mutual information theory for biomedical applications: Estimation of three protein-adsorbed dialysis membranes

Protein-adsorbed dialysis membranes are evaluated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical imaging technique. Protein adsorption causing permeability change is one of big issues in the development of dialysis membranes. Bovine serum albumin adsorption onto three kinds of dialysis membranes has been evaluated with TOF-SIMS. In the present study three kinds of proteins, bovine serum albumin, α-chymotripsinogen A, and cytochrome C adsorbed onto hollow-fiber dialysis membranes, were measured by means of TOF-SIMS and then TOF-SIMS spectra were analyzed using mutual information. Then specific peaks of fragment ions related to α-chymotripsinogen A and bovine serum albumin were found, respectively. In this condition, however, specific peaks to cytochrome C were not able to find compared with other samples. Finally, chemical images of α-chymotripsinogen A and bovine serum albumin, respectively, adsorbed onto the membranes with co-existing proteins were obtained. The results of TOF-SIMS images of the proteins on the membranes show different tendency of adsorption depending on co-existing proteins. Further study is needed to study more detailed protein adsorption onto the membranes with co-existing proteins.     

Embedded structure of silicon monoxide in SiO2 films

The structure of SiO_x (x = 1.94) films has been investigated using both X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The SiO_x films were deposited by vacuum evaporation. XPS spectra show that SiO_1.94 films are composed of silicon suboxides and the SiO₂ matrix. Silicon clusters appeared only negligibly in the films in the XPS spectra. Si₃O⁺ ion species were found in the TOF-SIMS spectra with strong intensity. These results reveal the structure of the films to be silicon monoxide embedded in SiO₂, and this structure most likely exists as a predominant form of Si₃O₄. The existence of Si-Si structures in the SiO₂ matrix will give rise to dense parts in loose glass networks.     

Optical manipulation of proteins in aqueous solution

Optical trapping of lysozyme, cytochrome c, or myoglobin based on photon pressure generated by focusing 1064 nm laser beam in an aqueous solution was explored. For all the proteins, microparticle formation was observed at the focal point under an optical microscope. Furthermore, the microparticles were identified to the molecular assemblies of the corresponding protein by means of confocal Raman microspectroscopy. For lysozyme, molecular clusters in solution were optically trapped to form the microparticle and it took more than 1 h to produce the microparticle. By contrast, molecular assembling proceeded within 1 min for cytochrome c and myoglobin. Since heme in cytochrome c or myoglobin would have a high polarizability, that would contribute to rapid assembling of the protein. Thus we demonstrated that a focused laser beam was a powerful tool to manipulate protein molecules in solution.     

Protein fragment imaging using ink jet printing digestion technique

We have developed a capable tool for visualizing protein distribution at the sub-cell level with TOF-SIMS. This new method of visualizing a protein includes: (1) printing a digestive enzyme with ink jet (IJ) printing technology onto a tissue section, (2) measuring the two-dimensional distributions of decomposed peptide ions derived from digested protein on the tissue section by TOF-SIMS with a bismuth cluster ion (Bi₃⁺) source and using the aqueous ion enhancement method, and (3) analyzing the two-dimensional distributions of proteins using the results of proteome analysis. We have not yet tried this method onto a real tissue section. To show the possibility of this method, TOF-SIMS results of visualizing dot-patterned digested fragment peptide ions using IJ printing digestion technique onto a protein film are presented.     

Investigations of the interactions of CO2, O3 and UV light with silver surfaces by in situ IRRAS/QCM and ex situ TOF-SIMS

The characterization of the reactions occurring during the atmospheric corrosion of silver surfaces at the metal-atmosphere interface is of great importance as only the exact knowledge of the degradation mechanisms enables the development of suitable corrosion inhibiting steps. For in situ surface analysis three complementary experimental techniques have been combined in order to study the surface chemistry of silver samples exposed to humidified air, CO₂, ozone (O₃) and ultraviolet (UV) light. In situ infrared reflection absorption spectroscopy (IRRAS) combined with a quartz crystal microbalance (QCM) as well as ex situ time of flight secondary ion mass spectrometry (TOF-SIMS) provide an overall picture of the processes occurring at the metal-atmosphere interface. Investigations were carried out by exposing polycrystalline silver samples to synthetic air containing 50-90% relative humidity (RH) and 250 ppm CO₂. Additionally, the samples were exposed to UV light or O₃ for certain time periods. The formation of the main corrosion products on the sample surfaces, depending on the relative humidity, O₃ content and UV light exposure could be observed in a time resolved way. These investigations reveal the different reaction mechanisms and reaction rates occurring on a silver surface.     

A group of glycosphingolipids found in an invertebrate: Their structures and biological significance

A novel group of glycosphingolipids was identified in the nervous tissue and skin of the mollusc, Aplysia kurodai, which lacks gangliosides. More than 30 glycolipids were detected on HPTLC plates and the structures of 9 major glycolipids were determined. They were pentaosylglycosphingolipids and their common core structure was GalNAcα1→3Galβ1→4Glcβ1→1ceramide, except for one glycolipid in which Galβ of the core structure was replaced by Galα. 3-O-MeGalβ or 4-O-MeGlcNAcα or 3,4-O-carboxyethylideneGalβ was at their non-reducing ends. Galα or Fucα binds to Gal of the core structure at 2C as a side chain sugar. One to three 2-aminoethylphosphonic acids and/or phosphoethanolamine link to the glycolipids. Immunohistochemically, glycolipids having carboxyethylideneGal at their non-reducing ends were localized exclusively in nerve bundles. Glycolipids activated cAMP-dependent protein kinase in the rat brain and may directly activate cAMP-dependent protein kinase in a manner similar, but not identical, to that of cAMP. The biological functions of glycolipids may share neurobiological functions proposed for gangliosides in vertebrates.     

Analysis of surface and bulk polymerization of a thin film using a chemical derivatization technique and TOF-SIMS

A chemical derivatization technique in TOF-SIMS along with ultra-low angle sample cutting technique were used to perform a quantitative study of the surface and in-depth double bond profile of the photo-initiated polymerized thin film. We found out that the characteristic peaks at m/z 185 and 199 were obtained from the thin film composed of acrylate monomer and methacrylate monomer, respectively, after reaction with bromine gas. The detection sensitivity of certain chemical indicators is affected by changing the primary ion species. The Bi₃⁺ primary ion results in the best chemical sensitivity. The surface double-bond density obtained by TOF-SIMS and the Br 3d signal intensity of XPS showed a good linear relationship in the limited region due to the effect of matrix hardness.The thin film was cut with microtome about 1° angle and was left to react with bromine and was measured using TOF-SIMS. It was clearly observed from this technique that double bond of acrylate and methacrylate monomer remained much more at the surface of the photo-initiated polymerized thin film, due to the inhibition of polymerization by oxygen. From the surface to 1 μm depth, both monomers show the same behavior, but the rate of polymerization of methacrylate monomer was lower than that of acrylate in deeper layers.     

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive C Labeling and Two-Dimensional Solid-State NMR

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive ¹³C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective ¹³C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-¹³C]glucose preferentially labels the ends of the side chains, while [2-¹³C]glycerol labels the C^α of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic–anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively ¹³C labeled protein were performed using ¹⁵N–¹³C 2D correlation spectroscopy. From the time dependence of the ¹⁵N–¹³C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective ¹³C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.     

TOF-SIMS analysis of magnetic materials in chum salmon head

A piece of tissue extracted from a chum salmon Oncorhynchus keta head was measured with time-of-flight secondary ion mass spectrometry (TOF-SIMS) in order to evaluate the distribution and composition of magnetic materials in the tissue, which may concern with geomagnetic navigation of long-distance migrating salmon. Several depositions of iron compounds were detected in the tissue by TOF-SIMS analysis. Comparing with total ion images providing a topological tissue structure, specific distribution of iron ion in the tissue was clearly shown. Higher magnification TOF-SIMS analysis revealed the existence of the aggregations of iron particles. Iron oxide clusters comprising many submicron particles were also detected in the tissue using scanning electron microscopy and X-ray analysis, suggesting the common existence of submicron-scale iron oxides in salmon heads. These results suggest that TOF-SIMS analysis is a valid method to clarify detailed structures and chemical properties of candidate magnetoreceptors in fish heads.     

Application of multivariate analysis of TOF-SIMS spectra for studying the effect of high glucose intake on aortic lipid profile

Aim

To study the effect of long-term glucose feeding on aortic lipid composition by using the time of flight-secondary ion mass spectrometry (TOF-SIMS).

Method

Rats were divided into two groups, drinking water with or without 10% glucose from birth to 6 months of age. The aortic wall was dissected out, high-pressure frozen, freeze-fractured, freeze-dried and analyzed by TOF-SIMS using a Bismuth cluster ion source. Surface spectra were taken from standardized regions of the vessel wall.

Results

Different peaks, such as cholesterol, fatty acids (FAs) and diacylglycerols (DAGs), were identified by the principal component analysis as carries of variance between two groups. These peaks were then compared by conventional t-test. Our data showed that the intensity of cholesterol, but not FAs and DAGs, was significantly decreased in the glucose-drinking rat. Moreover, the long-term glucose intake changed ratios between different FAs in the aorta.

Conclusion

The long-term glucose intake led to decreased cholesterol intensity in the aortic wall and this effect was revealed through a global analytical approach with objective selection of significant variables.     

Microwave structural studies of organoselenium compounds 1. Microwave spectra, molecular structure, and methyl barrier to internal rotation of dimethyl diselenide

The rotational spectra of nine isotopomers of dimethyl diselenide, CH₃SeSeCH₃, have been measured with a molecular-beam Fourier transform microwave spectrometer. The spectra were complex due to the presence of many isotopomers in natural abundance and the splitting caused by the interactions with two methyl internal rotors. The spectra were assigned and fit to experimental precision to an effective rotational Hamiltonian for molecules with two periodic internal motions. The spectra of the symmetric isotopomers are consistent with a C₂ equilibrium structure. The rotational constants were used to determine the r_s structure of the C-Se-Se-C frame with the results r(SeSe)=2.306(3) Å, r(SeC)=1.954(6) Å, ?(CSeSe)=99.8(2)°, ?(CSeSeC)=85.2(1)°. A barrier to internal rotation of the methyl groups of 395 ± 2 cm⁻¹ was derived from the internal rotation splittings.     

Quantum molecular dynamics simulation for fragmentation of arginine molecule induced by ion impact

15 keV Ga⁺ ion impact and the thermal decomposition of arginine molecules has been simulated by quantum molecular dynamics (QMD). We obtained the calculated mass spectra which express the distribution for fragment molecule generated by ion impact or thermal decomposition. From the comparison between the experimental TOF-SIMS spectra and the calculated spectra, we discussed the fragmentation mechanism of arginine molecule by Ga⁺ ion impact.     

Oxygen distribution in nickel silicide films analyzed by time-of-flight secondary ion mass spectrometry

The oxygen distribution in Ni₂Si and NiSi films formed during a two-step silicidation process was analyzed by time-of-flight secondary ion mass spectrometry (TOF-SIMS). TOF-SIMS mass spectra revealed that both silicon and nickel reacted with oxygen at the Ni₂Si surface. In addition, silicon nitride was formed at the surface by the reaction of silicon with nitrogen in the TiN capping layer during the first silicidation annealing. The amount of nitrogen at the NiSi surface varied with silicidation annealing temperature and with the formation conditions of the TiN capping layer. We also showed that a small amount of oxygen was penetrated into the NiSi film and strongly affected the level of junction leakage current in n⁺–p junctions in n-channel MOSFETs. The oxygen concentration in the NiSi film decreased with an increase in the amount of nitrogen at the NiSi surface.     

TOF-SIMS analysis of the interface between bone and titanium implants—Effect of porosity and magnesium coating

Implant healing was studied with regard to the mineralization of the implant-tissue interface. Titanium discs were surface-modified and implanted in rat tibia for 4 weeks. After implantation, the bone was embedded in resin and cross sections of bone and implant were made using a low speed saw equipped with a diamond wafering blade. The sections were analyzed with imaging TOF-SIMS using a Bi₃⁺ cluster ion source. This ion source has recently been shown to enable identification of hydroxyapatite (HA) fragments in bone samples. The area within 40 μm from the implant surface was selected for analysis, corresponding to bone-implant interface, from which positive spectra were recorded. In conclusion, differences were observed between the implants tested regarding signal intensity of fragments specific for HA. Coating of the implants with magnesium and porosity were shown to influence the mineral content of the bone-implant interface. This technique might be useful for biocompatibility assessment and for studying the mineralization process at implant surfaces.     

Molecular characterization of O-methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis japonica

O-Methyltransferases, which catalyze the production of small molecules in plants, play a crucial role in determining biosynthetic pathways in secondary metabolism because of their strict substrate specificity. Using three O-methyltransferase (OMT) cDNAs that are involved in berberine biosynthesis, we investigated the structure that was essential for this substrate specificity and the possibility of creating a chimeric enzyme with novel substrate specificity. Since each OMT has a relatively well-conserved C-terminal putative S-adenosyl-L-methionine-binding domain, we first exchanged the N-terminal halves of different OMTs. Among the 6 combinations that we tested for creating chimeric OMTs, 5 constructs produced detectable amounts of recombinant proteins, and only one of these with an N-terminal half of 6-OMT and a C-terminal half of 4′-OMT (64′-OMT) showed methylation activity with isoquinoline alkaloids as a substrate. Further enzymological analysis of 64′-OMT reaction product indicated that 64′-OMT retained the regio-specificity of 6-OMT. Further examination of the N-terminal region of 64′-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity. The creation of OMTs with novel reactivity is discussed.     

Temperature dependent analysis of three classes of fluorescence spectra from p-6P nanofiber films

Discontinuous nanofiber films of para-hexaphenylene molecules can be routinely fabricated via vacuum deposition on muscovite mica. The fibers emit upon UV illumination blue fluorescence with excitonic spectral peaks. Their intense fluorescence makes them very attractive for the use in photonic devices, given that the spectra are reproducible for varying surface temperatures. A detailed investigation as a function of surface temperature variation from 300 to 30 K reveals three classes of spectra: (a) spectra with well resolved excitonic peaks, which shift 35 meV to the blue with decreasing temperature, (b) similar spectra with an additional intermediate broadening around 150 K, and (c) excitonic spectra similar to (b), but with a green defect emission band. Quantitative fitting of type (a) spectra results in an exciton-phonon coupling factor of and an average phonon temperature of . The Huang-Rhys factor decreases linearly from 1.2 at 300 K to 1.0 at 30 K. Fitting of type (b) spectra reveals that the apparent intermediate temperature broadening is due to additional fluorescence peaks, the relative importance of which increasing with decreasing temperature.     

The effect of angle of incidence to low damage sputtering of organic polymers using a C60 ion beam

The effect of angle of incidence of C₆₀ ion beam for low damage polymer depth profiling on TOF-SIMS and XPS has been investigated. In this study, TOF-SIMS and XPS depth profiles were taken at several angles of incidence of C₆₀ ion beam and the results were compared with each other. By using a higher angle of incidence, in XPS analysis, the changes of atomic concentration for polyethyleneterephthalate (PET) were suppressed. In TOF-SIMS analysis, the degradations of fragment ion intensity for PET and polystyrene (PS) were also suppressed at a higher angle of incidence. Although the information depth of TOF-SIMS is different from that of XPS, both results suggested that a higher angle of incidence is a better condition for low damage polymer depth profiling.