JP2011027477A - Sample capturing alloy, mass spectrometer, mass spectrometry, method of capturing sample, sample capturing alloy, and method of manufacturing sample capturing alloy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample capturing alloy or the like for efficiently capturing samples. <P>SOLUTION: One aspect is the sample capturing alloy which has a modified surface, contains Fe, Pt and Cu, and captures samples. With this configuration, the sample capturing alloy for efficiently capturing samples is obtained. The orther aspect is the sample capturing alloy defined in claim 1 which is imparted with charge by modification of the surface. With this configuration, the sample capturing alloy which more efficiently captures samples by charge is obtained. Further the other aspect is the sample capturing alloy defined in claim 1 of which surface is modified by a thiol group. With this configuration, the sample capturing alloy for further easily introducing other substituents is obtained since the surface is modified by a thiol group. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a sample capture alloy, a mass spectrometer, a mass spectrometry method, a sample capture method, a sample capture alloy, and a sample capture alloy manufacturing method.

The mass spectrometry is a technique used when obtaining a mass-to-charge ratio (a value obtained by dividing a mass by the number of charges) of a sample. In mass spectrometry, an object to be measured is ionized, and ions are separated and detected based on the difference in mass-to-charge ratio (m / z).

When a substance is irradiated with laser light, the substance absorbs light, and photoelectron transfer proceeds and is ionized. This ionization method by direct laser irradiation is called laser desorption / ionization (LDI). However, LDI has a drawback in that efficient electron transfer is not performed depending on the type of substance, and the sample is damaged by the laser.

Therefore, by mixing an ionization aid (matrix agent) with the measurement sample and applying a laser beam (for example, wavelength: 337 nm), the matrix agent absorbs the light and a rapid temperature rise occurs at the irradiated site, resulting in sample molecules. So far, methods for ionization (soft ionization) without decomposing have been studied widely. This technique is a technique called Matrix Assisted Laser Desorption / Ionization (MALDI) -MS.

[MALDI-MS]

The matrix-assisted laser desorption / ionization method developed by Tanaka et al. And Hillenkamp et al. (See Non-Patent Documents 1 and 2) enables soft ionization of thermally unstable substances and high molecular weight substances. It has been used to measure the molecular weight of substances such as polymers and metal complexes (see Non-Patent Documents 3, 4, 5, and 6). In MALDI measurement, it is necessary to create a mixed crystal of a low molecular weight organic molecule that absorbs UV laser light called a matrix and a target sample on a plate. In MALDI, it is important to select an appropriate matrix for the target sample. Those that absorb the laser light used with high efficiency, those that act as proton donors for basic compounds, and those that act as proton acceptors for acidic compounds are selected. In the case of a low molecular weight sample, the matrix-derived peak and the sample-derived peak should not overlap. It is necessary to select a matrix in consideration of the above.

In MALDI, matrix molecular ions and their cluster ions are observed with high intensity in the low molecular weight region of the spectrum, and these ions interfere with the measurement of low molecular weight samples, making analysis difficult. There is. In addition, when the target compound is a mixture, interference with samples other than the target compound and suppression of ionization may occur, making detection difficult (ion suppression). Against this background, in recent years, LDI-MS not using an organic matrix has attracted attention.

[SALDI-MS]

Laser desorption ionization method using stable inorganic nanoparticles is
It was first reported by Tanaka et al. (See Non-Patent Document 7). We succeeded in mass spectrometry of protein lysozyme by mixing 30 nm cobalt nanoparticles and glycerol solution which is a liquid matrix. Subsequently, Sunner et al. (See Non-Patent Document 8) proposed a laser desorption ionization method using 2-150 μm graphite particles mixed with glycerol as a matrix, and used it as a surface-assisted laser desorption ionization method (surface- assisted laser desorption / ionization: SALDI). Kinumi et al. Have reported SALDI-MS using various inorganic fine particles (see Non-Patent Document 9). The mechanism of laser desorption / ionization using inorganic fine particles is not clear enough, but as one model, "reaching high temperature under rapid heating conditions" is important for raising the sample without decomposing it, It has been proposed (see Non-Patent Documents 7, 10, and 11).

In recent years, SALDI using substrates carrying nanoparticles such as carbon nanotubes (see Non-Patent Documents 12 and 13), silicon nanoparticles (see Non-Patent Document 14), and gold nanoparticles (see Non-Patent Document 15-22) Is attracting attention. These are characterized by not using glycerol, which is a liquid matrix, and are also called Dry-SALDI-MS.

McLean et al. Ionize without destroying peptide (substance P) samples using gold nanoparticles of about 2-10 nm (see Non-Patent Document 15). This is because gold nanoparticles are irradiated with a pulse laser (N ₂ laser 337 nm), the temperature rapidly rises locally, and sample molecules such as peptides are softly ionized without being decomposed. The ionization efficiency of the sample depends on the particle size of the gold nanoparticles, and particles of about 10-20 nm are considered good. There are studies on selective ionization of target molecules using specific interactions with gold nanoparticles (see Non-Patent Documents 16 and 17) and quantitative analysis of glucose in urine (see Non-Patent Document 18). Further, since the organic substance adsorbed on the gold nanoparticle surface is preferentially ionized, SALDI-MS is used for analyzing the adsorbed substance on the nanoparticle surface (see Non-Patent Documents 19 and 20). Thus, SALDI-MS (Au-SALDI-MS) using gold nanoparticles has advantages such as simplicity, high ionization efficiency per particle, and selective ionization of adsorbed particles on the particle surface. However, some problems remain in Au-SALDI. For example, since the melting point of gold nanoparticles decreases due to the nanosize effect, gold cluster ions generated by laser irradiation appear in the low molecular weight region as interference ion peaks. In Au-SALDI, a substrate on which colloidal gold colloid is dried is generally used as an ionization substrate. During the drying process, gold nanoparticles aggregate, and the aggregation state affects the ionization efficiency and reproducibility of the sample. May give. From such a viewpoint, the influence of the concentration of gold colloid applied by Au-SALDI and the application method on the ionization efficiency has been studied (see Non-Patent Document 21).

SALDI-MS using platinum (Pt) nanoparticles is also being studied. Compared to Au metal, Pt metal has a large melting point of 2045 K and low thermal conductivity, so the temperature is locally highest by laser irradiation. Therefore, desorption ionization can be expected to be promoted. Yonezawa et al. Synthesized new platinum nanoparticles (called nanoflowers) with petal-like protrusions on top of the unique features of platinum. And we are developing platinum nanoflowers to SALDI-MS (see Non-Patent Document 23). Four typical peptide samples using platinum nanoflowers are ionized, and in all cases, detection is performed with high sensitivity. We have also succeeded in soft ionization of cytochrome C with a molecular weight of 10,000 or more using platinum nanoflowers. SALDI using platinum nanoflowers is much more sensitive than ordinary spherical platinum nanoparticles and vapor deposited platinum substrates, and can detect even high molecular weight peptides. This shows that the petal-like protrusion shape of platinum nanoflowers acts on high sensitivity and high molecular weight detection of peptides. This indicates that in the application of nanoparticles to SALDI, particle shape control is also effective in increasing the ionization efficiency of the sample.

[LDI-MS using nanoparticle affinity]

Magnetic separation using magnetic particles is used for the purpose of separating, extracting, and removing a specific sample from a biomolecule mixed solution. Chen et al. Have developed magnetic separation of magnetic nanoparticles in SALDI-MS (see Non-Patent Documents 24 and 25). Fe ₃ O ₄ / TiO ₂ nanoparticles coated with titania thin film on the surface of iron oxide nanoparticles were synthesized by sol-gel reaction, and the effectiveness for SALD-MS was demonstrated. In the field of bioanalytical chemistry, selective enrichment of phosphorylated peptides obtained by enzymatic degradation of proteins has become an important research subject. Peptide mixture sample solution from β-casein digest and this nanoparticle are mixed in a test tube, a magnet is in contact with the outer wall of the test tube, and the phosphorylated peptide adsorbed on the nanoparticle surface is magnetically separated. Yes. By using the magnetic nanoparticles as they are for SALD-MS, the phosphorylated peptide in the peptide mixture is detected with high sensitivity. In this method, the chemoinitiity between the titania thin film and the phosphorylated peptide is used to concentrate the sample on the nanoparticle surface and detect it with high sensitivity by “direct” SALDI-MS. Thereby, there is an advantage that the substance adsorbed on the particle surface can be directly detected without performing a complicated separation operation such as normal solvent extraction. On the other hand, in surface modification with a coupling agent such as sol-gel reaction, uniform modification of a single molecular layer is difficult and there are few types of functional groups that can be introduced compared to thiol surface modification.

A biological substance contained in urine or serum that serves as an index for quantitatively grasping biological changes in the living body is called a biomarker. Since the biomarker changes quantitatively in correlation with a specific disease or body condition, it is possible to diagnose the disease or establish an effective treatment method by measuring the amount. However, due to the complexity of human ecology and the multifactorial nature of disease, it is difficult to predict disease based on a single biomarker alone. Thus, there is an urgent need to develop multiple immunoassays that can simultaneously screen multiple biomarkers. Wang et al. Used MALDI-TOF to enrich biomarkers in serum using magnetic nanoparticles conjugated with antibodies.
Multiple immunoassay for identification and quantification by MS is performed (see Non-Patent Document 26).

P. Pelagatti, M. Carcelli, F. Calbiani, C. Cassi, L. Elviri, C. Pelizzi, U. Rizzotti, D. Rogolino. Transfer Hydrogenation of Acetophenone Catalyzed by Half-Sandwich Ruthenium (II) Complexes ContainingAmino Amide Ligands. of the Catalytic Intermediates by ElectrosprayIonization Mass Spectrometry. Organometallics., 2005; 24: 5836-5844. LA Paim, DV Augusti, D. Ilza, TMA Alves.R. Augusti, HGL Siebald.Electrospray ionization and tandemmass spectrometry characterization of novel heterotrimetallic Ru (η5-C5H5) (dppf) SnX3 complexes andtheir heterobimetallic Ru (η5-C5H5) (dppf ) X precursors.Polyhedron., 2005; 24: 1153-1159. G. Bhaskar, MA Chary, MK Kumar, K. Syamasundar, M. Vairamani, S. Prabhakar. Electrosprayionization studies of transition-metal complexes of 2-acetylbenzimidazolethiosemicarbazone using collision-induced dissociation and ion-molecule reactions.Rapid Commun. Mass Spectrometry., 2005 ; 19: 1536-1544. G. Maerkl, K. Gschwendner, I. Roetzer, P. Kreitmeier. Tetrakis (diethyl phosphonate), tetrakis (ethyl phenylphosphinate)-, tetrakis (diphenylphosphine oxide)-substituted phthalocyanines. HelveticaChimica Acta., 2004; 87: 825-844. H. Takeda, A. Kawasaki, M. Takahashi, A. Yamada, T. Koike.Matrix-assisted laser desorption / ionization time-of-flight massspectrometry of phosphorylated compounds using a novel phosphate capturemolecule.Rapid Commun.Mass Spectrometry., 2003 ; 17: 2075-2081. J. E. Ham, B. Durham, J. R. Scott. Comparison of Laser Desorption and Matrix-Assisted Laser Desorption / Ionization for Rutheniumand Osmium Trisbipyridine Complexes Using Fourier Transform Mass Spectrometry. J. Am. Soc. Mass Spectrom., 2003; 14: 393-400. K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida: Rapid Commun. Mass Spectrom., 2, 151 (1988). D.S.Peterson: Mass Spectrom. Rev., 26, 19 (2007). T. Kinumi, T. Saisu, M. Takayama, H. Niwa: J. Mass Spectrom., 35,417 (2000). M. Schu1renberg, K. Dreisewerd, F. Hillenkamp: Anal.Chem., 71, 221 (1999). Shimadzu Corporation Koichi Tanaka Memorial Institute for Mass Spectrometry, “MADI-MS Technical Reports”, Rev .: 29 / Jun / 2007. S.Y.Xu, Y.F.Zou, J. S. Qiu, Z. Guo, B. C. Guo: Anal.Chem., 75,6191 (2003). M.V.Ugarov, T. Egan, D.V.Khabashesku, J. A. Schultz, H. Peng, V. N. Khabashesku, H. Furutani, K. S. Prather, H. W. J. Wang, S. N. Jackson, A. S. Woods: Anal. Chem., 76, 6734 (2004). X. Wen, S. Dagan, V. H. Wysocki: Anal. Chem., 79, 434 (2007). J. A. McLean, A. A. Stumpo, D. H. Russell: J. Am. Chem. Soc., 127, 5304 (2005). Y. F. Huang, H. T. Chang: Anal. Chem., 78, 1485 (2006). Y. F. Huang, H. T. Chang: Anal. Chem., 79, 4852 (2007). C. L. Su, W. L. Tseng: Anal. Chem., 79, 1626 (2007). H. Kawasaki, T. Yonezawa, K. Nishimura, R. Arakawa: Chem. Lett., 36, 1038 (2007). H. Kawasaki, M. Uota, T. Yoshimura, D. Fujikawa, G. Sakai, R. Arakawa, T. Kijima: Langmuir, 23, 11540 (2007). H-P. Wu, C-L. Su, H-C. Chang, W-L.Tseng: Anal. Chem., 79, 6215 (2007). A. Tarui, H. Kawasaki, T. Taiko, T. Watanabe, T. Yonezawa, R. Arakawa: Journal of Nanoscience and Nanotechnology, inpress (2008). H. Kawasaki, T. Yonezawa, T. Watanabe, R. Arakawa: J. Phys. Chem. C, 11, 16278 (2007). C-T. Chen, Y-C. Chen: Anal. Chem., 77, 5912 (2005). W-Y. Chen, Y-C. Chen: Anal. Bioanal. Chem., 386, 699 (2006). Kai-Yi Wang, Szu-An Chuang, Po-Chiao Lin, Li-ShingHuang, Shu-Hua Chen, Saib Ouarda, Wen-Harn Pan, X Ping-Ying Lee, O Chun-ChengLin, Yu-Ju Chen: Anal. Chem., 80, 6159-6167 (2008)

The present invention has been made in view of the above-described background art, and an object thereof is to provide a sample-trapping alloy that can capture a sample with high efficiency.

According to this invention, in order to achieve the above-mentioned object, the configuration as described in the claims is adopted. Hereinafter, the present invention will be described in detail.

The first aspect of the present invention is:
The surface is modified,
Containing Fe, Pt and Cu,
A sample-trapping alloy characterized by capturing a sample.

According to this configuration, a sample capturing alloy capable of capturing a sample with high efficiency can be obtained.

Examples of surface modification include surface modification with sulfonic acid, and methods using amines, carboxylic acids, aromatics, hydrocarbons, and fluorocarbons. In addition to the case of surface modification via a thiol group, a method via an amino group, a phosphine group, a carboxyl group or the like can be mentioned as an example. These points are the same not only for the following FePtCu alloys but also for FePt alloys.

The second aspect of the present invention is
2. The sample-trapping alloy according to claim 1, wherein a charge is applied by modifying the surface.

According to this configuration, a sample capturing alloy capable of capturing a sample with higher efficiency by electric charge can be obtained.

The third aspect of the present invention is
2. The sample-trapping alloy according to claim 1, wherein a thiol group is bonded, and a surface is modified through the thiol group.

According to this configuration, since the surface is modified with a thiol group, a sample-trapping alloy in which other substituents can be easily introduced can be obtained.

The fourth aspect of the present invention is
2. The sample-trapping alloy according to claim 1, wherein the surface is modified with sulfonic acid.

According to this configuration, a sample capturing alloy having high selectivity can be obtained.
The fifth aspect of the present invention provides
2. The sample-trapping alloy according to claim 1, wherein the sample-trapping alloy supports ionization of the sample captured by laser irradiation.

According to this configuration, the sample can be efficiently captured and the efficiency up to ionization can be increased.

The sixth aspect of the present invention provides
6. The sample-trapping alloy according to claim 5, wherein the sample-trapping alloy supports ionization of the sample captured by the surface-assisted laser desorption ionization method.

According to this configuration, a sample capturing alloy suitable for ionization of a low molecular weight sample can be obtained.

The seventh aspect of the present invention provides
A mass spectrometer equipped with the sample-trapping alloy according to any one of claims 1 to 6.

The eighth aspect of the present invention is
A mass spectrometric method is characterized in that mass spectrometric analysis of a sample supplemented with the sample trapping alloy according to any one of claims 1 to 6 is performed.

The ninth aspect of the present invention provides
A sample capturing method is characterized in that a sample is captured by an alloy containing a modified surface and containing Fe, Pt and Cu.

According to this configuration, a method capable of capturing a sample with high efficiency is obtained.

According to a tenth aspect of the present invention, there is provided a sample capturing alloy containing Fe, Pt and Cu, wherein a SO ₃ ^- group is bonded to the surface via a thiol group, and the sample is captured.

According to this configuration, a sample capturing alloy capable of capturing a sample with high efficiency is obtained.

An eleventh aspect of the present invention resides in a method for producing a sample-trapping alloy, characterized in that surface modification is performed on an alloy containing Fe, Pt, and Cu via a thiol group.

According to this configuration, a sample capturing alloy capable of capturing a sample with high efficiency can be obtained by a simple method.

According to a twelfth aspect of the present invention, there is provided a sample-trapping alloy which is substantially composed of Fe and Pt and captures a sample.

According to this configuration, a sample capturing alloy having an excellent capturing ability can be obtained.

Here, “substantially” means that other elements such as Cu are not included so as to greatly affect the physical properties of the alloy.

The thirteenth aspect of the present invention is
13. The sample-trapping alloy according to claim 12, wherein the surface is modified.

According to this configuration, a sample capturing alloy capable of capturing a sample with higher efficiency can be obtained.

The fourteenth aspect of the present invention provides
Consisting essentially of Fe and Pt,
The SO ₃ ^- group is attached to the surface via a thiol group,
A sample-trapping alloy characterized by capturing a sample.

According to this configuration, a sample capturing alloy capable of capturing a sample with high efficiency is obtained.

In the present specification, ionization support refers to supporting both desorption (vaporization) and ionization of a sample to be captured by laser irradiation. Any size such as diameter is acceptable. Examples of the alloy include fine powder, fine particles, and nanoparticles described later. The size (diameter) of the primary particles of the fine powder is, for example, preferably from 1 nm to 1 μm, preferably from 5 nm to 600 nm, and more preferably from 5 nm to 300 nm. Also included are secondary aggregates. Furthermore, the structure which has the protrusion part of the above-mentioned magnitude | size among structures larger than a fine powder may be sufficient. For example, a structure in which the protrusion has a size of 3 nm to 300 nm, preferably 5 nm to 200 nm is conceivable.

According to this configuration, a sample capturing alloy or the like that can capture a sample with high efficiency can be obtained.

Other objects, features, or advantages of the present invention will become apparent from the detailed description based on the embodiments of the present invention described later and the accompanying drawings.

It is a figure which shows the chemical structure, molecular weight, and acid dissociation constant of a drug sample. It is a figure which shows the chemical structure, molecular weight, isoelectric point of an oligopeptide sample. It is a figure which shows the chemical structure and molecular weight of Gly-Gly-His and nonafluorobutanesulfonic acid. FePtCu-SO ₃ ^- is a diagram showing an outline of capture and extraction steps of the target molecule using nanoparticles. It is a figure which shows the FE-SEM photograph (25000 times) of the FePtCu nanoparticle synthesize | combined in presence of the protective agent SDS. It is a figure which shows the structural element (EDX spectrum in the expansion range (25,000 times) of (X)) of the FePtCu nanoparticle synthesize | combined in presence of the protective agent SDS. Element distribution of FePtCu nanoparticles synthesized in the presence of the protective agent SDS (element distribution of (a) Fe, (b) Pt, (c) Cu in (X) expanded range (25,000 times)) FIG. FE-SEM photograph of FePtCu nanoparticles synthesized in the absence of protective agent ((a) 25,000 times, (b) near the left large particle of (a), 50000 times, (c) in the center of (a) It is a figure which shows a small particle vicinity, 50000 times). It is a figure which shows the TEM photograph (100,000 times) of the FePtCu nanoparticle synthesize | combined in the absence of a protective agent. It is a figure which shows the structural element ((a) EDX spectrum and (b) quantitative analysis in the expansion range (25000 times) of (X)) of the FePtCu nanoparticle synthesize | combined in the absence of a protective agent. Element distribution of FePtCu nanoparticles synthesized in the absence of protective agent ((a) Fe, (b) Pt, (c) Cu element distribution in (X) expanded range (25000 times) (d) Fe, (e) Pt, (f) Cu element mass distribution). FT-IR (ATR) spectra ((a) FePtCu nanoparticles before surface modification, (b) FePtCu nanoparticles after HSCH ₂ CH ₂ SO ₃ Na surface modification, (c) HSCH ₂ CH ₂ SO ₃ Na powder) FIG. It is a figure which shows the SALDI mass spectrum of angiotensin I (10 fmol). It is a figure which shows the SALDI mass spectrum of insulin human (5 pmol). It is a figure which shows the SALDI mass spectrum of cytochrome C (5 pmol). It is a figure which shows the mass spectrum of caffeine (100 pmol). It is a figure which shows the SALDI mass spectrum of GGH (100 pmol). It is a figure which shows the mass spectrum of amitriptyline hydrochloride (1 pmol). FIG. 3 is a diagram showing a mass spectrum of dextromethorphan hydrobromide (1 pmol). Oligopeptide GGYR (1 μM) FePtCu-SO 3 was recovered after the capture and extraction operations ^- shows the SALDI mass spectrum of the nanoparticles. Any FePtCu- SO in pH ₃ ^- illustrates the charge state of the nanoparticles and oligopeptides GGYR, and capture and extraction results oligopeptide GGYR. It is a figure which shows the SALDI mass spectrum of the supernatant collect | recovered after the capture and extraction operation of oligopeptide GGYR (1 micromol). Three mixed oligopeptides (10 μM) FePtCu-SO ₃ was recovered after the capture and extraction operations ^- shows the SALDI mass spectrum of the nanoparticles. FePtCu- SO ₃ at any pH ^- shows charge states of the nanoparticles and oligopeptides, and oligopeptides three capture and extraction results. It is a figure which shows the MALDI mass spectrum of the egg white lysozyme (100 micromol) added in serum. It is a figure which shows the MALDI mass spectrum of the egg white lysozyme (10 micromol) added in the serum. It is a figure which shows the SALDI mass spectrum of Ang I using FePt nanoparticles and the SALDI mass spectrum of Ang I using FePtCu nanoparticles. It is a figure which shows the SALDI mass spectrum of Ang I using FePt nanoparticles and the SALDI mass spectrum of Ang I using FePtCu nanoparticles.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[Overview]

The present inventors synthesized FePtCu nanoparticles (alloy nanoparticles composed of iron, platinum, and copper) as an inorganic matrix with an affinity function, and developed and studied SALDI-MS using nanoparticle affinity separation. Went. These FePtCu nanoparticles have high ionization efficiency compared to single metal nanoparticles by alloying, and introduce various functional groups that interact with target molecules by surface modification with thiol groups. be able to. It becomes possible to capture target molecules from the mixture onto the particle surface, and to separate and extract using magnetic properties such as FePtCu nanoparticles or simple centrifugation. Furthermore, the target molecule captured on the particle surface can be identified by SALDI-MS. To take advantage of these features, surface-modified with a thiol acid is a strong electrolyte such as FePtCu nanoparticles, it was applied a negative surface charge that is independent of pH (e.g. FePtCu- SO ₃ ^- nanoparticles). We also demonstrated that low-molecular-weight oligopeptides and high-molecular-weight proteins with positive charge can be electrostatically captured and extracted and detected by SALDI / MALDI-MS.

[Background to the present invention, background]

The present inventors have aimed to achieve higher ionization efficiency compared to single metal nanoparticles by using alloyed FePtCu nanoparticles (see the following non-patent document).

56th Mass Spectrometry Discussion Meeting (2008) Abstracts 246-247

In order to realize further progress, the present inventors have surface-modified FePtCu nanoparticles and the like with thiols and attempted to capture and extract target molecules that interact with thiol ends. As a previous study, Benjamin et al. Captured a positively charged peptide on the particle surface using gold nanoparticles that were negatively charged by surface modification of gold nanoparticles with an alkanethiol terminated with a carboxyl group. The extracted peptide is detected by MALDI-MS (see the following non-patent document).

Benjamin N. Y. Vanderpuije, Gang Han, Vincent M. Rotello, Richard W. Vachet: Anal. Chem. 78 (2006) 5491-5496

However, since the carboxyl group hardly dissociates under the condition of pH 6 or less, the negative charge is lost, and there is a restriction on usable pH that makes it difficult to use electrostatic affinity. In addition, since the MALDI method is used, it is difficult to measure low molecular weight compounds such as pharmaceuticals due to the influence of the matrix.

In this study, FePtCu-SO ₃ ^- was developed and research LDI-MS using affinity of the nanoparticles. Such synthesized new FePtCu nanoparticles, was surface-modified with a thiol acid is a strong electrolyte, it was applied a negative surface charge that is independent of pH (e.g. FePtCu- SO ₃ ^- nanoparticles). The FePtCu- SO ₃ ^- The use of nanoparticles as the inorganic matrix SALDI, the low molecular weight oligopeptides from the mixed sample has a positive charge electrostatically captured and extracted, detects them as samples SALDI-MS It was a goal. Also, many of contaminants to the basic protein FePtCu- SO ₃ from serum containing ^- captured with nanoparticles, it was examined to detect it in MALDI-MS.

Next, experiments actually performed will be described.

[reagent]

Iron (II) chloride (FeCl ₂・ 4H ₂ O), platinum (IV) chloride (H2PtCl ₆・ H ₂ O), copper sulfate pentahydrate (II) (CuSO ₄・ 5H ₂ O), hydrazine monohydrate Japanese (N ₂ H ₄・ H ₂ O), 0.1 M hydrochloric acid (for volumetric analysis), 25% aqueous ammonia, citric acid (anhydrous), nonafluorobutanesulfonic acid
(NFA), trifluoroacetic acid (TFA), sodium dodecyl sulfate (CH ₃ (CH ₂ ) ₁₁ OSO ₃ Na, SDS), angiotensin I, insulin human (recombinant), caffeine (anhydrous), dextromethorphan bromide Hydronate monohydrate, lysozyme hydrochloride (derived from chicken egg white), ethanol
(EtOH, 99.5%, special grade), methanol (MeOH, for HPLC), acetonitrile (MeCN, special grade), acetone (special grade), tetrahydrofuran (THF, for organic synthesis, containing stabilizer) were purchased from Wako Pure Chemical Industries. Distilled water (for LC-MS) was purchased from Kanto Chemical. Distilled water is distilled water production equipment (RFD250NB,
ADVANTEC). Cytochrome C (from horse heart)
Was purchased from Calbiochem. Gly-Gly-Tyr-Arg
(GGYR) and Gly-Gly-His (GGH) were purchased from Peptide Institute. Diammonium citrate was purchased from Fluka. Sodium 2-mercaptoethanesulfonate (HSCH ₂ CH ₂ SO ₃ Na) and Gly-Gly-Gly-Gly (GGGG) were purchased from Tokyo Chemical Industry. α-Cyano-4-hydroxycinnamic acid (CHCA), Asp-Asp-Asp-Asp (DDDD), amitriptyline hydrochloride were purchased from Sigma Aldrich. Human serum is CHEMICON
Purchased from International.

FIG. 1 is a diagram showing the chemical structure, molecular weight, and acid dissociation constant of a drug sample. The chemical structure, molecular weight, and acid dissociation constant of caffeine, amitriptyline hydrochloride, and dextromethorphan hydrobromide are shown.
FIG. 2 is a diagram showing the chemical structure, molecular weight, and isoelectric point of an oligopeptide sample. Asp-Asp-Asp-Asp
The chemical structure, molecular weight, and isoelectric point (pI) of (DDDD), Gly-Gly-Gly-Gly (GGGG), and Gly-Gly-Tyr-Arg (GGYR) are shown. The pI value was obtained using the calculation program of http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=pepstats. FIG. 3 shows the chemical structure and molecular weight of Gly-Gly-His (GGH) and nonafluorobutanesulfonic acid.

[Synthesis of FePtCu alloy nanoparticles]
<Synthesis of FePtCu alloy nanoparticles in the presence of the protective agent SDS>

0.8 mmol FeCl ₂・ 4H ₂ O, 0.8 mmol H ₂ PtCl ₆・ H ₂ O, 0.192 mmol CuSO ₄・ 5H ₂ O, and 1.6 mmol SDS in distilled water (40
mL, 70 ° C) to give concentrations of 20 mM, 20 mM, 4.8 mM, and 40 mM, respectively (see the following non-patent document). The complex of Fe and SDS produced here was dissolved by adding 40 mM N ₂ H ₄ (10 mL, 0.4 mmol N ₂ H ₄ · H 2 O dissolved in distilled water) little by little. To this, quickly add 390 mM N ₂ H ₄ (40 mL, 70 ° C, 15.6 mmol N ₂ H ₄ · H ₂ O dissolved in water) with vigorous stirring (530-rpm, using a magnetic stirring bar). Reduced. The reaction proceeded in several seconds, and then refluxing and stirring were continued at 70 ° C. for 3 hours. After natural cooling, the mixture was centrifuged (4000 rpm, 15 min, KUBOTA3200), and the supernatant was discarded. The reducing agent may be sodium borohydride or lithium borohydride.

P. Gibot, E. Tronc, C. Chaneac, J.P. Jolivet, D. Fiorani, A.M.Testa: J. Magn. Magn. Mater. 290-291 (2005) 555-558

MeOH (˜8 mL) was added to the remaining precipitate of FePtCu nanoparticles and dispersed by irradiating with ultrasonic waves (˜3 min). Agitation (~ 3 min) and centrifugation (4000
After the rpm, 15 min, KUBOTA3200), the supernatant was discarded. Subsequently, distilled water (~ 8 mL) is added to the remaining precipitate of FePtCu nanoparticles and irradiated with ultrasound.
(~ 3 min) to disperse. After stirring (~ 3 min) and centrifugation (4000 rpm, 15 min, KUBOTA3200), the supernatant was discarded. The above washing was performed 5 times. Finally, FePtCu nanoparticles were dried under reduced pressure to form a powder and stored at room temperature.

<Synthesis of FePtCu alloy nanoparticles in the absence of protective agent>
Dissolve 2 mmol FeCl ₂・ 4H ₂ O, 2 mmol H ₂ PtCl ₆・ H2O, and 0.48 mmol CuSO4 ・ 5H ₂ O in distilled water (100 mL, 70 ° C), 20 mM, 20 mM, 4.8 mM, respectively. Concentration. 400 mM N ₂ H ₄ (100 mL, 70 ° C, 40 mmol N ₂ H ₄ · H ₂ O dissolved in water) is quickly added to this with vigorous stirring (530-rpm, using a magnetic stirring bar). Reduced. The reaction proceeded in several seconds, and then refluxing and stirring were continued at 70 ° C. for 3 hours. After natural cooling, the supernatant was discarded.

EtOH (˜8 mL) was added to the remaining FePtCu nanoparticle precipitates and dispersed by irradiation with ultrasonic waves (˜3 min). Agitation (~ 3 min) and centrifugation (4000
After rpm, 5 min, KUBOTA3200), the supernatant was discarded. Subsequently, distilled water (~ 8 mL) is added to the remaining precipitate of FePtCu nanoparticles and irradiated with ultrasound.
(~ 3 min) to disperse. After stirring (~ 3 min) and centrifugation (4000 rpm, 5 min, KUBOTA3200), the supernatant was discarded. The above washing was performed 3 times. Finally, FePtCu nanoparticles were dried under reduced pressure to form a powder and stored at room temperature.

[Structural evaluation of FePtCu alloy nanoparticles]

FePtCu nanoparticles are field emission scanning electron microscope (Field Emission Scanning).
Electron Microscope, FE-SEM) and Transmission Electron Microscope (Transmission
The size and shape were confirmed by Electron Microscope, TEM, and JEOL, and the constituent elements were confirmed by Energy Dispersive X-ray Spectroscopy (EDX). (FE-SEM and EDX are Prof. Toru Yonezawa at the University of Tokyo) To request measurement).

[Surface modification of FePtCu alloy nanoparticles with thiolsulfonic acid]

FePtCu nanoparticles (100 mg) synthesized in the absence of protective agent were added to EtOH (~ 8
mL) was added and dispersed by irradiating with ultrasound (~ 3 min). Agitation (~ 3 min) and centrifugation (4000
After rpm, 5 min, KUBOTA3200), the supernatant was discarded. Subsequently, distilled water (~ 8 mL) is added to the remaining precipitate of FePtCu nanoparticles and irradiated with ultrasound.
(~ 3 min) to disperse. After stirring (~ 3 min) and centrifugation (4000 rpm, 5 min, KUBOTA3200), the supernatant was discarded. The above washing was performed 5 times, and it was confirmed that the electrical conductivity of the supernatant discarded last was 25 mS / m or less (CM-30G, DKK / TOA).

EtOH (50 mL) is added to the washed FePtCu nanoparticles and irradiated with ultrasound (5 min, US-3R, AS
ONE) to disperse. To this, 1M HS (CH ₂ ) ₂ SO ₃ Na (5.6 mL, 5.6 mmol, dissolved in water) was quickly added with stirring (500-770 rpm, LT400, yamato scientific). Thereafter, stirring was continued at 25 ° C. for 24 hours (see the following non-patent document). In the middle, precipitation of HS (CH ₂ ) ₂ SO ₃ Na was observed, and distilled water (˜10 mL) was added to dissolve it. After the reaction, the supernatant was discarded.

Simard, J. M., Briggs, C., Boal, A. K., Rotello, V. M .: Chem. Commun. 19 (2000) 1943-1944 McIntosh, C. M., Esposito, E. A., Boal, A. K., Simard, J. M., Martin, C. T., & Rotello, V. M.J. Am. Chem. Soc. 123 (2001) 7626-7629

Distilled water (˜1.5 mL) was added to the remaining FePtCu nanoparticle precipitate. After stirring (~ 3 min) and centrifugation (14000 rpm, 5 min, centrifuge 5417R, eppendorf), the supernatant was discarded. The above washing was performed 5 times, and it was confirmed that the electrical conductivity of the supernatant discarded last was 1 mS / m or less (CM-30G, DKK / TOA). Subsequently, acetone (˜8 mL) was added to the remaining FePtCu nanoparticle precipitate. Stirring (~ 3
min) and centrifugation (4000 rpm, 10 min, KUBOTA3200), the supernatant was discarded. The above washing was performed twice. Finally, FePtCu nanoparticles were dried under reduced pressure to form a powder and stored at −30 ° C.

FePtCu nanoparticles that have undergone the above surface modification treatment will be referred to as FePtCu-
SO ₃ ^- represents the nanoparticles.

[FePtCu- SO ₃ ^- surface modification Evaluation of Nanoparticles

FePtCu- SO ₃ ^- nanoparticles, Fourier transform infrared spectroscopy (Fourier Transform Infrared Spectroscopy,
Surface modification was confirmed by Attenuated Total Reflection (ATR) of FT-IR, FT / IR-4200, JASCO.

[Sample preparation, solution preparation]
<Preparation of aqueous dispersion of FePtCu nanoparticles (before surface modification)>
Powder (~ 1 mg) of FePtCu nanoparticles (before surface modification) was placed in a microtube, and distilled water (~ 200 μL) was added. Stirring (~ 1 min) and ultrasonic irradiation (15 min, US-3R, AS ONE) were performed 3 or 4 times to disperse the nanoparticles. The concentration was 1.5 mg / mL.

<FePtCu- SO ₃ ^- Preparation of an aqueous dispersion of nanoparticles>
FePtCu- SO ₃ ^- nanoparticle powder (to 6 mg) is taken up in a glass centrifuge tube, to which THF of (to 8 mL) was added and dispersed by irradiation (to 3 min) ultrasound. Agitation (~ 3 min) and centrifugation (4000
After rpm, 5 min, KUBOTA3200), the supernatant was discarded. The above washing was performed twice. Next, the remaining FePtCu-
SO ₃ ^- distilled water precipitation nanoparticles (to 8 mL) was added and dispersed by irradiation (to 3 min) ultrasound. Agitation (~ 3 min) and centrifugation (4000
After rpm, 5 min, KUBOTA3200), the supernatant was discarded. The above washing was performed twice.

Finally, the washed FePtCu- SO ₃ ^- distilled water (4 mL) was added to the nanoparticles, ultrasonic waves
(~ 3 min) to disperse. Concentration is 1.5
mg / mL.

<Preparation of cationizing agent>
As the cationizing agent, citrate buffer, TFA, or NFA was selected. When using a citrate buffer with a sample with a molecular weight of 10,000 or less, the composition of 10 mM aq citrate / 5 mM diammonium citrate aq = 1/3 (v / v) is used with a sample with a molecular weight of 10,000 or more. In this case, the composition was 23 mMaq citrate / 20 mMaq diammonium citrate = 1.1 / 5 (v / v). TFA and NFA were diluted with distilled water and adjusted to a concentration of 0.1 v% each.

<Preparation of matrix>
CHCA was selected as the matrix agent used in MALDI-MS. Distilled water / MeCN = 1/1 (v / v) was used as a solvent to prepare a concentration of 10 mg / mL. Here, CHCA was used as the organic matrix, but sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid), ferulic acid (trans-4-) was also used depending on the molecular weight of the sample. Hydroxy-3-methoxycinnamic acid), gentisic acid, DHBA (2,5-dihydroxybenzoic acid), HPA (3-hydroxypicolinic acid), disranol (1,8-dihydroxy-9,10-dihydroanthracene-9- ON) may be used.

[FePtCu alloy nanoparticles / FePtCu- SO ₃ ^- SALDI-MS using nanoparticles]
<SALDI-MS using FePtCu nanoparticles (before surface modification)>
The SALDI characteristics of FePtCu nanoparticles before surface modification were studied.

Angiotensin I, insulin human, and cytochrome C were selected as samples. Distilled water was used as a solvent to adjust the concentration to 20 nM, 10 μM, and 10 μM, respectively.

Apply an aqueous dispersion of FePtCu nanoparticles (before surface modification) to a 384-well MALDI stainless steel plate (0.5
μL), air dried. Subsequently, the sample solution and the cationizing agent were applied thereon (0.5 μL each) and dried. A citrate buffer was selected as the cationizing agent.

<FePtCu- SO ₃ ^- SALDI-MS using nanoparticles>
The low molecular pharmaceutical compounds as a sample, FePtCu- SO ₃ ^- and SALDI mass spectrum using nanoparticles, was compared with MALDI mass spectra.

Caffeine, amitriptyline hydrochloride, and dextromethorphan hydrobromide were selected as samples. Distilled water was used as a solvent to prepare concentrations of 100 μM, 1 μM, and 1 μM, respectively. When caffeine was used as a sample, 0.1 v% NFA was selected as a cationizing agent. No cationizing agent was used when amitriptyline hydrochloride and dextromethorphan hydrobromide were used as samples.

384-well MALDI stainless plate FePtCu- SO ₃ ^- an aqueous dispersion of nanoparticles was applied (1 [mu] L), and air-dried. Subsequently, the sample solution (and cationizing agent) was applied (1 μL) thereon and dried.

{MALDI-MS}
Apply CHCA, sample solution (and cationizing agent) in sequence to a 384-well MALDI stainless steel plate.
(1 μL each) and dried.

<FePtCu- SO ₃ ^- capture experiment LDI-MS of the target molecule using affinity of nanoparticles>
4, FePtCu- SO ₃ ^- is a diagram showing an outline of capture and extraction steps of the target molecule using nanoparticles. Here, FePtCu- SO ₃ ^- work between the negative charge of the nanoparticle surface, a peptide-protein with a positive charge

We tried to capture and extract peptides and proteins using electrostatic affinity and detect them with SALDI-MS / MALDI-MS.

<Single Oligopeptide Capture Experiment and SALDI-MS>
GGYR was selected for a single oligopeptide. The concentration was adjusted to 50 μM using distilled water as a solvent (20 μL). Dilute it to 1 μM with 1 × 10 ⁻⁴ M hydrochloric acid, distilled water, 3 × 10 ⁻⁴ % ammonia water, 1 × 10 ⁻² % ammonia water (980 μL) (1 mL) The pH was adjusted to 4, 6, 8, and 10, respectively.

These FePtCu- SO ₃ ^- aqueous dispersion of nanoparticles (100 [mu] L) was added, stirred
(10 min,
Thermomixer comfort, eppendorf) ・ Stand overnight to capture GGYR. Thereafter, simple centrifugation (6200 rpm, 5 min, MICRO SIX) was performed, and the supernatant (950 μL) was recovered.

Remaining FePtCu- SO ₃ ^- the precipitation of the nanoparticles, 1 × 10 ^-4 M hydrochloric acid to keep the same pH as when capturing, distilled water, 3 × 10 ^-4% ammonia water, 1 × 10 ^-2% ammonia Any of the water (1 mL) was added and stirred (˜1 min). Simple centrifugation (6200
After rpm, 5 min, MICRO SIX), the supernatant (1 mL) was discarded. Each of the above washings was performed twice.

After washing, leave 100 μL of supernatant and irradiate with ultrasound.
(~2 min,), FePtCu- SO 3 - redispersing the nanoparticles. These were applied to a 384-well MALDI stainless steel plate (1.0 μL) and air dried. Subsequently, a cationizing agent was applied thereon (1 μL) and dried. A citrate buffer was selected as the cationizing agent.

{SALDI-MS of supernatant collected immediately after capture}
FePtCu- SO ₃ ^- applying an aqueous dispersion of nanoparticles 384-well MALDI stainless plates (1.0 [mu] L), and air-dried. Subsequently, the collected supernatant and cationizing agent were applied (1 μL each) and dried. A citrate buffer was selected as the cationizing agent.

<Capture experiment of mixed oligopeptide and SALDI-MS>
Three types of oligopeptides, DDDD, GGGG, and GGYR, were selected. Distilled water was used as a solvent to adjust the concentration to 300 μM. These were mixed in equal amounts to prepare a concentration of 100 μM (100 μL). Dilute this solution to a concentration of 10 μM with 1 × 10 ^−2.5 M hydrochloric acid, 3 × 10 ⁻⁴ % aqueous ammonia, or 1 × 10 ⁻² % aqueous ammonia (900 μL) (1 mL). , 8, 10 adjusted.

These FePtCu- SO ₃ ^- aqueous dispersion of nanoparticles (100 [mu] L) was added, stirred
(10 min, Thermomixer comfort, eppendorf) ・ Stand overnight to capture the oligopeptide. Thereafter, simple centrifugation (6200 rpm, 5 min, MICRO SIX) was performed, and the supernatant (950 μL) was discarded.

Remaining FePtCu- SO ₃ ^- the precipitation of the nanoparticles, 1 × 10 ^-2.5 M hydrochloric acid to keep the same pH as when capturing, any 3 × 10 ^-4% ammonia water, 1 × 10 ^-2% aqueous ammonia (1 mL) was added and stirred (~ 1 min). Simple centrifugation (6200
After rpm, 5 min, MICRO SIX), the supernatant (1 mL) was discarded. Each of the above washings was performed twice.

After washing, leaving supernatant to 100 [mu] L was irradiated with ultrasonic waves _{(~2 min), FePtCu- SO 3} - redispersing the nanoparticles. These were applied to a 384-well MALDI stainless steel plate (1.0 μL) and air dried. Subsequently, a cationizing agent was applied thereon (1 μL) and dried. A citrate buffer was selected as the cationizing agent.

<Capture experiment of protein in serum and MALDI-MS>
Proteins added to the serum to a target molecule, FePtCu- SO ₃ ^- was compared MALDI mass spectra with and without the capture and extraction using nanoparticles. Egg white lysozyme was selected as the protein (MW 14306, pI = 11.1). Lysozyme is an enzyme protein whose serum concentration increases in monocytic leukemia, kidney disease and meningitis (according to the following non-patent document).

Levinson, S. S .; Elin, R. J .; Yam, L. Clin. Chem. 2002, 48, 1131-1132. Harrison, J. F .; Lunt, G. S .; Scott, P .; Blainey, J. D. Lancet 1968, 1,371-375. Klockars, M .; Reitamo, S .; Weber, T .; Kerttula, Y. Acta Med. Scand. 1978, 203, 71-74.

Lysozyme was prepared at a concentration of 5 mM or 500 μM using distilled water as a solvent (20 μL). This was diluted with human serum (980 μL, pH 8) to a concentration of 100 μM and 10 μM (1 mL).

Serum samples above FePtCu- SO ₃ ^- aqueous dispersion of nanoparticles (200 [mu] L) was added, stirring to (1 h, Thermomixer comfort, eppendorf ) was trapped in the lysozyme. Thereafter, simple centrifugation (6200 rpm, 5 min, MICRO SIX) was performed, and the supernatant (1.19 mL) was discarded.

Remaining FePtCu- SO ₃ ^- the precipitation of the nanoparticles, distilled water (1.3 mL) was added, and stirred (to 1 min). Simple centrifugation
(6200 rpm, 5 min, MICRO
After SIX), the supernatant (1.29 mL) was discarded. The above washing was performed 3 times.

After washing, distilled water was added to 100 [mu] L was irradiated with ultrasonic waves _{(~2 min), FePtCu- SO 3} - redispersing the nanoparticles. This was applied to a 384-well MALDI stainless steel plate (1.0 μL) and air dried. Subsequently, CHCA and a cationizing agent were applied thereon (1 μL each) and dried. A citrate buffer was selected as the cationizing agent.

{MALDI-MS without preprocessing}
The above serum sample, CHCA, and cationizing agent were sequentially applied to a 384-well MALDI stainless steel plate (1 μL, 0.6 μL, and 0.6 μL, respectively) and dried. A citrate buffer was selected as the cationizing agent.

[SALDI / MALDI-MS measurement conditions]

The 384-well MALDI stainless steel plate coated with the sample as described above is introduced into a time-of-flight mass spectrometer (AXIMA-CFR or AXIMA-CFR plus, nitrogen laser light wavelength 337 nm, Shimadzu-Kratos). SALDI / MALDI-TOF-MS measurement was performed in positive ion mode. For mass calibration, CHCA [M + H] ⁺ ion, Angiotensin I [M + H] ⁺ ion, Insulin human [M + H] ⁺ ion, Lysozyme [M + H] ⁺ ion (m / z 190.05, 1296.69, 5806.65, 14303.83, respectively) , Most
abundunt).

[Structural evaluation of FePtCu alloy nanoparticles]

The FE-SEM image, EDX spectrum, and element distribution of FePtCu nanoparticles synthesized in the presence of the protective agent SDS will be described. FIG. 5 is a diagram showing an FE-SEM photograph (25,000 times) of FePtCu nanoparticles synthesized in the presence of the protective agent SDS. FIG. 6 is a diagram showing constituent elements (EDX spectrum in the expanded range (25,000 times) of (X) 2) of FePtCu nanoparticles synthesized in the presence of the protective agent SDS. Fig. 7 shows the element distribution of FePtCu nanoparticles synthesized in the presence of the protective agent SDS (elements of (a) Fe, (b) Pt, (c) Cu in the (X) expanded range (25,000 times)) It is a figure which shows distribution.

From the FE-SEM images, it was found that the FePtCu nanoparticles synthesized in the presence of the protective agent SDS had a spherical shape with a diameter of about 10 nm (Fig. 5). In addition, EDX analysis confirmed that it was an alloy of iron, platinum, and copper (Fig. 6), and there was no difference in element distribution.
(Figure 7).

Next, FE-SEM images, TEM images, EDX spectra, and element distributions of FePtCu nanoparticles synthesized in the absence of a protective agent will be described. Figure 8 shows FE-SEM images of FePtCu nanoparticles synthesized in the absence of a protective agent ((a) 25000 times, (b)
(a) In the vicinity of the large particle on the left side, 50000 times (c) (a) In the vicinity of the small particle in the center of (a), 50000 times). FIG. 9 is a diagram showing a TEM photograph (100,000 times) of FePtCu nanoparticles synthesized in the absence of a protective agent. FIG. 10 is a diagram showing constituent elements of FePtCu nanoparticles synthesized in the absence of a protective agent ((a) EDX spectrum and (b) quantitative analysis) in the expanded range (25000 times) of (X) 2. Figure 11 shows the element distribution of FePtCu nanoparticles synthesized in the absence of a protective agent (element distribution of (a) Fe, (b) Pt, (c) Cu in (X) expanded range (25000 times))
(d) Elemental distribution of Fe, (e) Pt, (f) Cu). Figure 12 shows FT-IR (ATR) spectra ((a) FePtCu nanoparticles before surface modification, (b) FePtCu nanoparticles after surface modification of HSCH ₂ CH ₂ SO ₃ Na, (c) HSCH ₂ CH ₂ SO ₃ It is a figure which shows Na powder.

From the FE-SEM image (Fig. 8) and the TEM image (Fig. 9), FePtCu nanoparticles synthesized in the absence of a protective agent are about 10-20 nm nanoparticles and 100-200 nm particles. It was found to be a mixture. Also, EDX analysis confirmed that all were alloys of iron, platinum and copper, but the former had a high platinum content (Fig. 11a, b) and the latter had a high iron content (Fig. 11d, e). These biases are considered to have occurred because the reduction reaction proceeded non-uniformly compared to the case where the protective agent SDS was present in the synthesis system.

FePtCu nanoparticles modified with thiolsulfonic acid (FePtCu-
SO ₃ ^- is shown in FIG. 12 FT-IR spectra of the nanoparticles). Thiol acid is in the vicinity of a wavelength of 1200 nm ^- SO ₃ - 2 peaks strong derived from is seen in feature (Figure 12c). Focusing on this peak, comparing before surface modification (Fig. 12a) and after surface modification (Fig. 12b), it is a weak peak intensity, but after surface modification (Fig.
around nm ^- SO ₃ - 2 peaks considered to be derived from are newly appeared. FePtCu-
SO ₃ ^- nanoparticles, compared to FePtCu nanoparticles prior to the surface modification was readily dispersed in water, since the settling speed was slow, believed to surface modification by the thiol acid is performed.

[FePtCu alloy nanoparticles / FePtCu- SO ₃ ^- SALDI-MS using nanoparticles]
<SALDI-MS using FePtCu alloy nanoparticles
>
The SALDI characteristics of FePtCu nanoparticles before surface modification were studied.

FIG. 13 shows angiotensin I (10
fmol) is a diagram showing a SALDI mass spectrum. Citrate buffer was used as the cationizing agent. The synthesis conditions of FePtCu particles were (a) in the absence of the protective agent and (b) in the presence of the protective agent SDS.

Regardless of the synthesis conditions in the presence / absence of the protective agent SDS, proton-added [M + H] ⁺ ions (m / z 1297) could be observed with high ionic strength and resolution. Since the standard sample, angiotensin I, has femtomolar level detection sensitivity, it was found that FePtCu nanoparticles can be used as an inorganic matrix in SALDI-MS. However, when FePtCu nanoparticles synthesized in the presence of the protective agent SDS were used, a peak group considered to be derived from SDS remaining in a range of m / z 400 or less was also observed. This suggests that FePtCu nanoparticles synthesized in the presence of the protective agent SDS are somewhat inferior to the use of low molecular weight compounds in SALDI-MS.

FIG. 14 is a diagram showing a SALDI mass spectrum of insulin human (5 pmol). As nanoparticles, (a) Pt nanoparticles, (b) FePtCu nanoparticles synthesized in the presence of the protective agent SDS, and citrate buffer as a cationizing agent were used. When Pt nanoparticles composed of a single element of platinum were used, a peak derived from the sample could not be observed (see the following non-patent document) FIG. 14a). On the other hand, when FePtCu nanoparticles (synthesized in the presence of the protective agent SDS) are used, [M + H] ⁺ ions (m / z 5806) can be observed (Fig. 14b), which improves the ionization efficiency by alloying. It is suggested.

H. Kawasaki, T. Yonezawa, T. Watanabe, R. Arakawa: J. Phys. Chem C, 11, 16278-16283 (2007)

FIG. 15 is a diagram showing a SALDI mass spectrum of cytochrome C (5 pmol). Below m / z 4000 is blank. FePtCu nanoparticles synthesized in the presence of the protective agent SDS, citrate buffer was used as a cationizing agent. Monovalent [M + H] ⁺ ion
(m / z 12360) and divalent [M + 2H] ²⁺ ions (m / z 6180) could be observed, but their intensity and resolution were low. From this, it was found that ionization of a polymer compound having a molecular weight of 10,000 or more is inferior to MALDI as in SALDI using other metal nanoparticles.

<FePtCu- SO ₃ ^- SALDI-MS using nanoparticles>
The basic low-molecular pharmaceutical compounds as a sample, FePtCu- SO ₃ ^- and SALDI mass spectrum using nanoparticles, was compared with MALDI mass spectra.

16, (MALDI using (a) CHCA, (b) FePtCu- SO 3 - SALDI with nanoparticles) mass spectrum of caffeine (100 pmol) is a diagram showing a. 0.1% NFA was used as the cationizing agent. In the MALDI mass spectrum, proton-added [M + H] ⁺ ions (m / z 195) were observed, but a peak derived from the CHCA matrix agent was also observed nearby (m / z 190), indicating caffeine Ionization was suppressed (Figure 16a). On the other hand, in the SALDI mass spectrum, [M + H] ⁺ ions (m / z 195) were dominantly observed, and the number of interfering ion peaks was significantly smaller than that of MALDI (FIG. 16b). M / z
The peak observed at 345 is derived from NFA used as the cationizing agent.

Figure 17 shows GGH
It is a figure which shows the SALDI mass spectrum of (100 pmol). FePtCu- SO ₃ ^- was used nanoparticles. As the cationizing agent, (a) 0.1% TFA, (b) citrate buffer, and (c) 0.1% NFA were used. FePtCu- SO ₃ ^- was examined cationizing agent in SALDI using nanoparticles, peak NFA can be used similarly to the citrate buffer or TFA as the cationizing agent, derived from the cationizing agent itself It had the characteristic that there were few (Fig. 17).

Next, FIG. 18, (MALDI using (a) CHCA, (b) FePtCu- SO 3 - SALDI with nanoparticles) mass spectrum of amitriptyline hydrochloride (1 pmol) is a diagram showing a. No cationizing agent was used. In the ALDI mass spectrum, [M-Cl] ⁺ ions (m / z 278) in which chlorine was dissociated were observed, but were obstructed by peaks derived from the CHCA matrix agent, making assignment difficult (FIG. 18a). On the other hand, in the SALDI mass spectrum, in addition to [M-Cl] ⁺ ion (m / z 278), [MH ₂ -Cl] ⁺ ion (m / z
276) was also observed, but the interfering ion peak was significantly less compared to MALDI (Fig. 18b).

Finally, FIG. 19, (MALDI using (a) CHCA, (b) FePtCu- SO 3 - SALDI with nanoparticles) mass spectrum of dextromethorphan hydrobromide (1 pmol) shows the It is. No cationizing agent was used. In the MALDI mass spectrum, a peak (m / z 272), which was considered as [M-Br] ⁺ ion with bromine dissociated, was observed. However, m / z 272 can also be considered as a peak derived from the CHCA matrix agent and the assignment is not definitive (Figure 19a). On the other hand, in the SALDI mass spectrum, in addition to [M-Br] ⁺ ion (m / z 272), [MH ₂ -Br] ⁺ ion (m / z
270) was also observed, but the interference ion peak was still significantly smaller than that of MALDI (Fig. 19b). From these facts, FePtCu- SO ₃ ^- SALDI-MS using nanoparticles was found to be effective for the detection of low-molecular pharmaceutical compounds.

[FePtCu- SO ₃ ^- capture experiment LDI-MS of the target molecule using affinity Nanoparticles
<Single Oligopeptide Capture Experiment and SALDI-MS>
Figure 20 is attempted to capture GGYR under different pH conditions FePtCu- SO ₃ ^- is a diagram showing a SALDI-MS spectra of the nanoparticles. Capture conditions are (a) pH 4, (b) pH 6, (c)
pH 8, (d) pH 10. GGYR ([M + H] ⁺
m / z 452) could be detected under the conditions of pH 4, 6, and 8 (Figs. 20a, 20b, and 20c, respectively), but not under the conditions of pH 10 (Fig. 20d). .

21, any FePtCu- SO in pH ₃ ^- illustrates the charge state of the nanoparticles and oligopeptides GGYR, and capture and extraction results oligopeptide GGYR. Since GGYR (pI = 9.35) has a positive charge at pH 4, 6, and 8 below pI, it is considered that it was captured by negatively charged nanoparticles and detected by SALDI-MS. On the other hand, at pH 10 above pI, GGYR has a negative charge, so it is not captured by the negatively charged nanoparticles and cannot be detected by SALDI-MS (FIG. 21).

{SALDI-MS of supernatant collected immediately after capture}
FIG. 22 is a diagram showing a SALDI mass spectrum of the supernatant recovered after the oligopeptide GGYR (1 μM) capture / extraction operation. That is, the SALDI-MS spectrum of the supernatant collected immediately after attempting to capture the above GGYR is shown. Capture conditions are (a) pH 4, (b) pH 6, (c) pH 8, (d)
The pH was 10. GGYR ([M + H] ⁺ , m / z 452) could not be detected under pH 4, 6, and 8 conditions (Figures 22a, 22b, and 22c, respectively), but it was detected under pH 10 conditions. (Fig. 22d).

These show the opposite results to the spectrum of FIG. GGYR remains in the supernatant at pH 10 above pI and is almost 100 at pH 4, 6, 8 below pI.
% Are considered captured, supporting the above considerations.

<Capture experiment of mixed oligopeptide and SALDI-MS>
Fig. 23 shows FePtCu- recovered after capture and extraction of the three mixed oligopeptides (10 μM).
SO ₃ ^- is a diagram showing a SALDI mass spectrum of the nanoparticles. Capture conditions are (a) pH 2.5, (b) pH 8, (c) pH
It was 10. At pH 2.5, DDDD, GGGG, and GGYR ([M + Na] ⁺ , [M + Na] ⁺ , [M + H] ⁺ , m / z, respectively)
501, 269, 452) were all detected, but not at pH10. On the other hand, at pH 8, it was found that only GGYR (m / z 452) was detected in the mixture of three oligopeptides. This is DDDD
This is explained by the difference in isoelectric point (pI) between (pI = 3.2), GGGG (pI = 6.1), and GGYR (pI = 9.4).

Figure 24 is any FePtCu- SO ₃ in pH ^- shows charge states of the nanoparticles and oligopeptides, and capture and extraction results oligopeptide three. In other words, at pH 2.5 below pI, all three oligopeptides have a positive charge, so it is considered that they were captured by negatively charged nanoparticles and detected by SALDI-MS. At pH 10 above pI, all peptides have a negative charge, so they are not captured by negatively charged nanoparticles and are not detectable by SALDI-MS. On the other hand, at pH 8, it is considered that only GGYR having a positive charge was captured and extracted by negatively charged nanoparticles, and only GGYR could be detected from the mixture.

<Capture experiment of protein in serum and MALDI-MS>
The MALDI mass spectrum obtained in the capture experiment of egg white lysozyme (pI = 11) added to human serum (pH 8) will be described. FIG. 25 is a diagram showing a MALDI mass spectrum of egg white lysozyme (100 μM) added to serum. Other than m / z 4500-15000, it is blank. Citrate buffer was used as the cationizing agent. (a) No pretreatment, 100 pmol (b) FePtCu- SO 3 - is after capture and extraction with nanoparticles. FIG. 26 is a diagram showing a MALDI mass spectrum of egg white lysozyme (10 μM) added to serum. Other than m / z 4500-15000, it is blank. A citrate buffer was used as the cationizing agent as the cationizing agent. (a) No pretreatment, 10 pmol (b) FePtCu- SO 3 - is after capture and extraction with nanoparticles.

When serum samples were applied directly without pretreatment, almost no lysozyme was detected (FIGS. 25a and 26a). On the other hand, FePtCu- SO ₃ ^- when performing capture and extraction of egg white lysozyme from serum by nanoparticles, monovalent is protonated, divalent, trivalent ions (each ^{[M + H] +, [} M + 2H] ²⁺ , [M + 3H] ³⁺ , m / z
14304, 7153, 4769) and succeeded in detecting lysozyme (Figs. 25b and 26b). It was possible to detect lysozyme while suppressing the influence of nonspecific adsorption by biological substances such as albumin contained in serum.
SO ₃ ^- shows a high retention-extraction selectivity ability of nanoparticles.

[FePt nanoparticles]

A comparison will be made between Ang I SALDI-MS using FePt nanoparticles and Ang I SALDI-MS using FePtCu nanoparticles.

Ang I (angiotensin I) was used as the sample, and citric acid / ammonium citrate was used as the cationizing agent. As the matrix, FePt nanoparticles (or FePtCu nanoparticles) synthesized without SDS were used. In the case of FePt nanoparticles, it was 5.6 mg / mL and FePtCu nanoparticles were 5 mg / mL.

Ang (10 μM Ang I × 0.5 μL = 5 pmol) was dropped onto the plate and dried. Subsequently, FePt nanoparticles (or FePtCu nanoparticles) (5 mg / mL × 0.5 μL) were added dropwise and dried.

Figures 27 and 28 show Ang with FePt nanoparticles.
It is a figure which shows the SALDI mass spectrum of I and the SALDI mass spectrum of Ang I using FePtCu nanoparticles. In the case of using FePt nanoparticles and the case of using FePtCu nanoparticles, a peak intensity equivalent to a molecular weight of 1296.5 was observed. It was found that the impurity peak in the low molecular weight region was larger when FePt nanoparticles were used than when FePtCu nanoparticles were used.

From these results, similarly to the above-described FePtCu alloy nanoparticles, for example, FePt- SO ₃ ^- when modified with sulfonic acid on the surface of the FePt nanoparticles through a thiol group as nanoparticles, the oligopeptide having a positive charge It is considered that lysozyme and the like can be selectively captured and extracted from the mixed sample and detected by SALDI / MALDI-MS.

In addition, FePt is superior to FePtCu described above in that particles having a uniform chemical composition of Fe and Pt can be synthesized. Similarly, FePt with surface modification is superior to FePtCu with surface modification because there is no problem that thiol surface modification becomes difficult due to oxidation of Cu, and is superior in that surface modification is easy.

[Usage]

Application to proteome analysis and metabolomics analysis is promising. In particular, in the metabolomics analysis that investigates the changes in the living body under certain conditions by comprehensively analyzing the low-molecular compounds contained in the sample, the above-mentioned technique with few interfering ion peaks in the low-molecular region is effective. It is.

In addition, it can be used in combination with a separation operation by SDS-PAGE or two-dimensional electrophoresis. In particular, the separated substance can be mapped by applying or transferring the separated substance on the plate. Since the sample can be efficiently captured and a large difference in sensitivity depending on the place (so-called sweet spot) can be suppressed, it is also effective for a mapping method that can know the position of such a substance. It can also be diverted to AP-LDI, which performs measurements at atmospheric pressure.

[Summary]

The present inventors synthesize FePtCu alloy nanoparticles in the presence / absence of a protective agent, and FePtCu nanoparticles synthesized in the absence of a protective agent are surface-modified with thiolsulfonic acid. I was able to confer a negative charge by (e.g. FePtCu- SO ₃ ^- nanoparticles). FePtCu- SO ₃ ^- by using a nanoparticle, selectively capture and extract such oligopeptides and lysozyme having a positive charge from the mixed sample could be detected by SALDI / MALDI-MS. In addition, FePt alloy nanoparticles were synthesized in the same manner, and the results were fully confirmed that they could be detected by SALDI / MALDI-MS by surface modification of FePt alloy nanoparticles.

[Interpretation of rights, etc.]

The present invention has been described above with reference to specific embodiments. However, it is obvious that those skilled in the art can make modifications or substitutions of the embodiments without departing from the gist of the present invention. That is, the present invention has been disclosed in the form of exemplification, and the contents described in the present specification should not be interpreted in a limited manner. In order to determine the gist of the present invention, the claims section described at the beginning should be considered.

It will also be appreciated that illustrative embodiments of the invention achieve the above objects, but that many modifications and other examples can be made by those skilled in the art. The elements or components of each embodiment described in the claims, specification, drawings, and description may be employed in combination with one or more other elements. The claims are intended to cover such modifications and other embodiments, which are within the spirit and scope of the present invention.

Claims (14)

The surface is modified,
Containing Fe, Pt and Cu,
A sample-trapping alloy characterized by capturing a sample. 2. The sample-trapping alloy according to claim 1, wherein a charge is applied by modifying the surface. 2. The sample-trapping alloy according to claim 1, wherein a thiol group is bonded, and a surface is modified through the thiol group. 2. The sample-trapping alloy according to claim 1, wherein the surface is modified with sulfonic acid. 2. The sample-trapping alloy according to claim 1, wherein the sample-trapping alloy supports ionization of the sample captured by irradiating a laser. 6. The sample trapping alloy according to claim 5, wherein the sample trapping alloy supports ionization of the sample trapped by the surface-assisted laser desorption ionization method. 7. A mass spectrometer equipped with the sample-trapping alloy according to any one of claims 1 to 6. A mass spectrometric method comprising performing mass spectrometry of a sample supplemented with the sample trapping alloy according to any one of claims 1 to 6. A sample capturing method, wherein a sample is captured by an alloy having a modified surface and containing Fe, Pt, and Cu. Containing Fe, Pt and Cu,
The SO ₃ ^- group is attached to the surface via a thiol group,
A sample-trapping alloy characterized by capturing a sample. A method for producing a sample-trapping alloy, comprising surface-modifying an alloy containing Fe, Pt and Cu via a thiol group. Consisting essentially of Fe and Pt,
A sample-trapping alloy characterized by capturing a sample. 13. The sample capturing alloy according to claim 12, wherein the surface is modified. Consisting essentially of Fe and Pt,
The SO ₃ ^- group is attached to the surface via a thiol group,
A sample-trapping alloy characterized by capturing a sample.