WO2006080210A1

WO2006080210A1 - Reagent, method, and kit for improving ionization efficiency and utilization thereof

Info

Publication number: WO2006080210A1
Application number: PCT/JP2006/300554
Authority: WO
Inventors: Shin-Ichiro Nishimura; Noriko Nagahori
Original assignee: National University Corporation Hokkaido University
Priority date: 2005-01-27
Filing date: 2006-01-17
Publication date: 2006-08-03
Also published as: JPWO2006080210A1

Abstract

A protein is modified under mild conditions to improve the ionization efficiency of the protein. A metal complex having a specific functional group and a tridentate ligand is bonded to a target protein. Thus, the protein is detected with high sensitivity regardless of the nature of the protein.

Description

Specification

Reagents, methods and kits for improving ionization efficiency and use thereof

Technical field

[0001] The present invention relates to a reagent, method and kit for improving ionization efficiency in protein mass spectrometry. Specifically, the present invention introduces a reagent to be introduced onto the surface of a protein before ionization, and the reagent. The present invention relates to a method for modifying a protein and a kit comprising the reagent.

Background art

[0002] In the field of analytical chemistry, particularly mass spectrometry, many ionization methods have been developed and used in post-genomic research. However, it is very difficult to measure the interactions between unstable organic compounds, host molecules—guest molecules, and Z or biomolecules by mass spectrometry. In mass spectrometry, a certain amount of energy must be applied to the molecule to be measured by voltage, heat, laser, etc. during the ionization process. As a result, weak interactions such as hydrogen bonds that occur between molecules in the solution are broken, or fragmentation occurs due to cleavage of intramolecular bonds, so the behavior of the original molecules in the solution can be determined using mass spectrometry. It was difficult to observe.

[0003] In recent years, a mass spectrometer (cold spray ionization mass spectrometry (CSI- Mass) system based on a new ionization method improved from the conventional ionization method in order to observe the original molecular behavior. ) Was developed (see Non-Patent Document 1). This instrument is a modification of the conventional electronic spray ionization mass spectroscopy (ESI-Mass) instrument for observing more stable organic molecules and / or intercluster clusters. is there.

[0004] CSI—Mass is very effective for analyzing unstable molecules, and is particularly useful for analyzing molecular dynamics in solution (solution dynamic analysis of molecular species based on non-covalent interactions). It is widely used. [0005] The feature of the CSI-Mass device is that the nebulizing gas used for ionization in the conventional ESI-Mass can be controlled to a low temperature of 15 ° C to -80 ° C, and the ionization can be performed at a low temperature. It is. Originally, ESI-Mass ionizes sample molecules by spraying the sample solution together with a nebulizing gas from the force of the needle tip to which voltage is applied, and evaporating the sprayed droplets in a solvent removal chamber at 250 ° C to room temperature. To promote. CSI_Mass, on the other hand, adopts a new method that promotes ionization by increasing the polarizability of molecules at low temperatures.

[0006] The greatest advantage of CSI_Mass is that there is no need to increase the temperature at the time of ionization, which is suitable for observing weak interactions of sample molecules in the solution. As evidence, Yamaguchi et al. Show the superiority of the device by analyzing the structure of unstable organometallic complexes and / or biomolecules (amino acids, DNA, etc.) and water using CSI_Mass. (See Non-Patent Document 1).

[0007] In order to improve ionization efficiency, attempts have been made to improve mass spectrometers (see, for example, Patent Documents:! To 3). Patent Document 4 describes that the addition of triethylamine to an oligonucleotide sample improves the ionic efficiency of the oligonucleotide and increases the detection efficiency by 20%.

[Patent Document 1]

Japanese Patent Laid-Open No. 2003-28849 (published on January 29, 2003)

[Patent Document 2]

JP 2003-151486 (published May 23, 2003)

[Patent Document 3]

JP 2004-257873 Gazette (released September 16, 2004)

[Patent Document 4]

Japanese Patent Laid-Open No. 2003-04704 (published January 8, 2003)

[Non-Patent Document 1]

J. Mass Spectrometry, 38: 473-490 (2003)

Disclosure of the invention

[0009] However, CSI-Mass has the following drawbacks. First, desolvation during ionization The measurement sensitivity of the sample molecules is low compared to ESI-Mass because the heating and desolvation gas necessary to promote the measurement are not used. Second, the sample molecules are ionized with nonspecific binding to solvent molecules or other molecules, leading to a decrease in the sensitivity of the sample molecules and at the same time It is obstructing observation. In particular, in a polymer sample such as a protein, an effect depending on the characteristics of the sample (molecular weight and / or amino acid composition) appears remarkably.

[0010] Therefore, in order to make protein interaction analysis using CSI_Mass more versatile, it is indispensable to develop a method for efficient ionization without depending on the physical properties of the protein. is there. As a conventional method for improving ionic efficiency in mass spectrometry of proteins, a method in which amidine or guanidine is chemically introduced to the protein surface is known. In such a method, it is necessary to use basic conditions. It was necessary and reaction control was difficult.

[0011] The present invention has been made in view of the above problems, and an object thereof is to realize protein modification under mild conditions such as low temperature and / or neutrality. Specifically, it is intended to provide a reagent capable of improving ionization efficiency in CSI-TOF Mass, and to achieve protein modification under low temperature and neutral conditions using this reagent.

[0012] The method for improving protein ion efficiency according to the present invention is characterized by including a step of introducing an organic residue derivative onto the surface of a target protein.

[0013] In the method for improving protein ion efficiency according to the present invention, the organic residue derivative is preferably a metal complex.

[0014] In the method for improving protein ion efficiency according to the present invention, the ligand of the metal complex is preferably a tridentate ligand.

[0015] In the method for improving the protein ion efficiency according to the present invention, the ligand of the metal complex is preferably terpyridine.

[0016] In the method for improving protein ion efficiency according to the present invention, the metal element of the metal complex is preferably a transition element or a typical element.

[0017] In the method for improving protein ion efficiency according to the present invention, the metal element of the metal complex is Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag, Au, ○ s or I r is preferred.

[0018] In the method for improving protein ion efficiency according to the present invention, the metal complex preferably has a quaternary ammonium.

[0019] In the method for improving protein ion efficiency according to the present invention, the metal complex has the following formula:

[0020] [Chemical 4]

[0021]

Here, R is preferably H or quaternary ammonia, and X is preferably Pt or Cu.

[0022] The method for improving the protein ion efficiency according to the present invention includes a step of coordinating an unshared electron pair of an amino acid side chain present on the surface of a target protein with the metal complex. preferable.

[0023] A kit for improving protein ion efficiency according to the present invention comprises a metal complex having a tridentate ligand.

[0024] In the kit for improving protein ion efficiency according to the present invention, the ligand is preferably terpyridine.

[0025] In the kit for improving the protein ion efficiency according to the present invention, the metal element of the metal complex is preferably a transition element or a typical element.

[0026] In the kit for improving the protein ion efficiency according to the present invention, the metal element of the metal complex is Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag, Au, ○ It is preferably s or Ir.

In the kit for improving the protein ion efficiency according to the present invention, the above gold The genus complex preferably has a quaternary ammonia.

[0028] In the kit for improving protein ion efficiency according to the present invention, the metal complex has the following formula:

[0029] [Chemical 5]

[0030]

Here, R is preferably H or quaternary ammonia, and X is preferably Pt or Cu.

[0031] The metal complex according to the present invention is characterized by having a tridentate ligand.

[0032] In the metal complex according to the present invention, the ligand is preferably terpyridine.

[0033] In the metal complex according to the present invention, the metal element of the metal complex is preferably a transition element or a typical element.

[0034] In the metal complex according to the present invention, the metal element of the metal complex is Pt, Pd, Fe, Co,

Ni, Cu, Cr, Ru, Rh, Ag, Au, Os or Ir is preferable.

[0035] The metal complex according to the present invention preferably has a quaternary ammonium.

[0036] The metal complex according to the present invention has the following formula:

[0037] [Chemical 6]

[0038]

Here, R is preferably H or quaternary ammonia, and X is preferably Pt or Cu.

[0039] The composition according to the present invention includes the metal complex described above, and is characterized by improving ionization efficiency in protein mass spectrometry.

[0040] The composition is preferably used to improve detection sensitivity in mass spectrometry.

[0041] In the method for producing an artificial protein according to the present invention, the functional residue is introduced into the protein by introducing the functional residue into the surface of the protein using the kit, the metal complex, or the composition. It is characterized by providing the function that originates.

[0042] Further, in the protein analysis method according to the present invention, the presence or absence of histidine residues, arginine residues, and / or cysteine residues in the protein is determined using the kit, the metal complex, or the composition. It is characterized by that.

[0043] When the method according to the present invention is used, functional residue introduction into a protein can be realized without being limited by the chemical structure of the residue to be introduced. Furthermore, if the method according to the present invention is used, the protein surface can be modified under mild conditions using a complex formation reaction, so that functional molecules can be introduced without impairing protein function. Furthermore, if the method according to the present invention is used, application to artificial protein creation can be promoted.

[0044] Other objects, features, and advantages of the present invention will be sufficiently understood from the following description. The advantages of the present invention will be apparent from the following description with reference to the accompanying drawings.

Brief Description of Drawings

[0045] [FIG. 1] FIG. 1 shows the modification of GalT with compound (1) (platinum black mouth (2, 2 ,: 6, 2, 2, _terpyridine)) at various desolvation chamber temperatures. It is a graph which shows the result of mass spectrometry at the time of performing.

[FIG. 2] FIG. 2 is a graph showing the result of deconvolution treatment on the spectrum in FIG. 1 showing unmodified GalT and modified GalT.

[Figure 3] Figure 3 shows the unmodified GalT and modified GalT peaks in the graph shown in Figure 2. 6 is a graph showing a relative value when a value measured at 200 ° C. is set to 1.

FIG. 4 is a graph showing the area ratio between the peak area of modified GalT and the peak area of unmodified GalT.

FIG. 5 is a graph showing the results of mass analysis when GalT modification with compound (2) was carried out at various solvent removal chamber temperatures.

[FIG. 6] FIG. 6 is a graph showing the result of deconvolution treatment on the spectrum in FIG. 5 showing unmodified GalT and modified GalT.

[FIG. 7] FIG. 7 is a graph showing the relative values of the peak area of unmodified GalT and modified GalT in the graph shown in FIG. 6 when the value measured at 200 ° C. is 1. It is.

FIG. 8 is a graph showing the area ratio between the peak area of modified GalT and the peak area of unmodified GalT.

FIG. 9 is a graph showing the results of mass spectrometry of GalT modified with Compound (1) or a complex of unmodified GalT and UDP.

FIG. 10 is a graph showing the result of deconvolution processing for the spectrum in FIG.

FIG. 11 is a graph showing the results of mass spectrometry of GalT modified with Compound (1) or a complex of unmodified GalT and UDP-Gal.

FIG. 12 is a graph showing the result of deconvolution processing performed on the spectrum shown in FIG. 11.

BEST MODE FOR CARRYING OUT THE INVENTION

[0046] The present inventors have introduced a metal complex into a lone pair of amino acid side chains on the protein surface, and confirmed that the protein function is not impaired by the complex introduction. In addition, the metal complex produced by the present inventors was able to improve ionization efficiency by improving the functional group.

[0047] The present invention provides reagents, methods and kits for improving ionization efficiency in protein mass spectrometry. These are described in detail below. [0048] [1] Reagent for improving ionization efficiency

The present invention provides a metal complex for improving ionization efficiency in protein mass spectrometry. The ligand possessed by the metal complex according to the present invention may be either a monodentate ligand or a polydentate ligand, but a tridentate ligand is preferred from the viewpoint of the stability of the complex. Examples of tridentate ligands include tenolepyridine, diethylenetriamine, Schiff base, triazacycloalkane, tetrakis (2'-aminoethyl) -1,2-diaminopropane, and octazabisic mouth [6. 6. 6] Terpyridine is preferred as a ligand that is easy to introduce functional groups. As functional group, guanidine (_NH_C (_NH) = NH ⁺ ) is also available

2 2 or amidine (one C (— NH) = NH ⁺ ) (eg arginine), or 4

twenty two

Having a secondary cation (_N (CH) ⁺ ) is preferred, but ionization in mass spectrometry

Three

The quaternary ammonium is particularly preferable because it does not cleave at the stage. The metal complex according to the present invention has one or more of the above functional groups.

[0049] In the metal complex according to the present invention, the metal element of the metal complex is preferably a transition element or a typical element. Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag Au, Os or Ir is more preferable.

[0050] The metal complex according to the present invention has the following formula:

[0051] [Chemical 7]

[0052]

Here, R is preferably H or quaternary ammonia, and X is preferably Pt or Cu.

[0053] The metal complex having the functional group of the present invention can be synthesized by first preparing a ligand having the functional group and then coordinating the ligand to the metal. Example For example,

[0054] [Chemical 8]

When synthesizing the compound (2) shown in [0055], first, a compound (3) in which an amine is introduced at the 4-position of the terpyridine skeleton.

[0056] [Chemical 9]

[0057] In the next step, compound (3) is reduced to give compound (4)

[0058] [Chemical 10]

[0059] The compound (4) is reacted with a metal salt to obtain a terbiridine metal complex having a quaternary ammonium. The obtained metal complex can be identified by nuclear magnetic resonance spectrum, visible ultraviolet absorption spectrum, mass spectrometry and the like.

[0060] The metal complex according to the present invention comprises a lone pair of amino acid side chains on the protein surface. Linking to the protein surface using coordination with gin, arginine, and cysteine). If the metal complex according to the present invention is used, any functional substance (for example, sugar chain) can be mildly applied to the protein surface. Can be introduced under various conditions. Thereby, the target ability and / or hydrolysis resistance can be imparted to the protein.

[0061] It should be noted that the present invention also includes a composition that includes other compounds in addition to the metal complex, and improves ionization efficiency in protein mass spectrometry, such as the metal complex solution. The composition can be suitably used for improving detection sensitivity in mass spectrometry.

[0062] In addition, since the metal complex and the composition can introduce an arbitrary functional residue on the protein surface under mild conditions as described above, the artificial protein imparting a new function to the protein. Can be used to create Therefore, in the present invention, an artificial protein that imparts a function attributable to the functional residue to the protein by introducing a functional residue on the surface of the protein using the metal complex or composition. This manufacturing method is also included. Examples of the functional residue include a force capable of mentioning a sugar chain. The present invention is not limited to this.

[0063] Furthermore, according to the metal complex, the histidine residue, arginine residue, and / or cysteine residue of the protein is modified, and therefore the presence or absence of the amino acid residue in the protein can be determined. it can. Therefore, the present invention also includes a protein analysis method for determining the presence or absence of a histidine residue, an arginine residue, and / or a cysteine residue in a protein using the above metal complex or composition.

[0064] Presence or absence of the amino acid residue is determined by mass spectrometry of the peptide obtained by reacting the metal complex or composition with a protein and directly reacting the protein after the reaction, or by appropriately decomposing the protein with a protease or the like. It can be determined by doing.

[0065] [2] Kit for improving ionization efficiency

The present invention provides a kit including a reagent for improving ionization efficiency in protein mass spectrometry. The kit according to the present invention preferably includes a metal complex as a reagent. As described above, the ligand of the metal complex may be either a monodentate ligand or a polydentate ligand. From the viewpoint of stability, a tridentate ligand is preferable. Three seat Examples of ligands include terpyridine, diethylenetriamine, Schiff base, triazacycloalkane, tetrakis (2'-aminoethyl) 1,2 diaminopropane, and octazabicyclo [6 · 6.6] eicosane. Forces listed Terpiridin is preferred as a ligand that facilitates the introduction of functional groups. Functional groups include guanidine (one NH— C (— NH) = NH ⁺ ) or

twenty two

Midine (one with C (— NH) = NH ⁺ ) (eg arginine), or quaternary

twenty two

Those with on (_N (CH) ⁺ ) are preferred, but at the ionization stage in mass spectrometry

Three

The quaternary ammonium is particularly preferable because it is not cleaved. The reagent (metal complex) provided in the kit according to the present invention has one or more of the above functional groups.

[0066] In the metal complex according to the present invention, the metal element of the metal complex is a transition element or a typical element. S is preferable. Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag Au, Os or Ir is more preferable.

[0067] In the kit for improving protein ion efficiency according to the present invention, the metal complex has the following formula:

[0068] [Chemical 11]

[0069]

Here, R is preferably H or quaternary ammonia, and X is preferably Pt or Cu.

[0070] The kit according to the present invention uses a metal complex coordinated with an unshared electron pair (histidine, arginine, cysteine) on the amino acid side chain on the protein surface as a reagent, and may be any functional substance (for example, , Sugar chain) is introduced under mild conditions. Thereby, the target ability and / or hydrolysis resistance can be imparted to the protein.

[0071] That is, according to the kit of the present invention, human protein having a new function added to a protein is obtained. It can be used to create proteins. Accordingly, the present invention also includes a method for creating an artificial protein by introducing a functional residue on the protein surface using the kit. Examples of the functional residue include sugar chains, but the present invention is not limited to this.

[0072] Further, since the metal complex according to the invention modifies the histidine residue, arginine residue, and / or cysteine residue of the protein, the presence or absence of the amino acid residue in the protein can be determined. . Therefore, the present invention also includes a protein analysis method for determining the presence or absence of histidine residues, arginine residues, and / or cysteine residues in a protein using the above kit.

[0073] [3] Method for improving ionization efficiency

The present invention provides a method for improving ionization efficiency in protein mass spectrometry. The method according to the present invention preferably includes a step of introducing an organic residue derivative onto the target protein surface, and the organic residue derivative is preferably a metal complex. That is, the method for improving the protein ionization efficiency according to the present invention preferably includes a step of coordinating the metal complex with an unshared electron pair of an amino acid side chain present on the target protein surface. ,.

[0074] As described above, the ligand possessed by the metal complex may be either a monodentate ligand or a polydentate ligand, but a tridentate ligand is preferred from the viewpoint of the stability of the complex. Tridentate ligands include terpyridine, diethylenetriamine, Schiff base, triazacycloalkane, tetrakis (2'-aminoethyl) 1,2 diaminopropane, octazabicyclo [6 · 6.6] aicosane Tellpyridine is preferred as a ligand that can easily introduce functional groups. Functional groups include guanidine (one NH— C (— NH) = NH ⁺ ) or amidine (

twenty two

-C (-NH) = NH ⁺ ) (eg arginine) or quaternary cation (one

twenty two

N (CH) ⁺ ) is preferred, but it is cleaved at the ionization stage in mass spectrometry.

Three

It is preferable that it is not. A quaternary ammonia is particularly preferable. The metal complex used in the method for improving ionization efficiency according to the present invention has one or more of the above functional groups.

[0075] In the metal complex according to the present invention, the metal element of the metal complex is a transition element or a compound. Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag, Au, Os or Ir are more preferable.

[0076] In the kit for improving protein ion efficiency according to the present invention, the metal complex has the following formula:

[0077] [Chemical 12]

[0078]

Here, R is preferably H or quaternary ammonia, and X is preferably Pt or Cu.

[0079] The method according to the present invention utilizes the coordination between a lone pair of amino acid side chains on the protein surface (histidine, arginine, cysteine) and a complex of a metal (Pt, Cu, etc.). If the method according to the invention is used, any functional substance (for example, sugar chain) can be introduced onto the protein surface under mild conditions. As a result, the target ability and / or hydrolysis resistance can be imparted to the protein.

[0080] Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples, and various modifications are possible within the scope shown in the claims and the above embodiments. Embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[Example]

(Example 1: Synthesis of metal complex for protein modification)

[0082] [Chemical 13]

[0083] Potassium tetrachromate platinum (II) lOOmg was dissolved in 1.6 ml of distilled water, 2.4 ml of acetic acid was added, and the mixture was stirred at room temperature. To this solution, 0.1 ml of 1,5-cyclooctagen was added and then heated and stirred at 90 ° C. for 30 minutes. As a result, white crystals were precipitated. This solution was concentrated to about 1 ml under reduced pressure, and then crystals were obtained by filtration under reduced pressure. The crystals were washed with distilled water, ethanol, and jetyl ether, and then dried under reduced pressure to obtain 59 mg of platinum dichloro (1,5-cyclooctagen) crystals. In a flask, 50 mg of a white gold dichroic mouth (1,5-cyclooctadine) was dissolved in 4 ml of Ν, Ν'-dimethylformamide. This solution was heated to 50 ° C, then 2, 2 ': 6', 2 "-terpyridine (31. lml) was added and stirred for 15 minutes. The solution was cooled to room temperature, and then solidified by vacuum filtration. The filtrate obtained was concentrated under reduced pressure and then dried to obtain crystals of the compound (1) (platinum black mouth (2, 2,: 6, 2 ''-terpyridine)). · 5 mg was obtained, which was confirmed by ESI-Mass.

(Example 2: Synthesis of platinum complex having quaternary ammonium)

[0085] [Chemical 14]

[0086] 4, —Black mouth—2, 2 ,: 6 ′, 2 ′ ′ — terpyridine lg, potassium hydroxide 280 mg and 1-amino-3-propanol 0 · 32 ml were dissolved in about 5 ml of dimethyl sulfoxide in the flask. I understood. The solution was stirred for 90 minutes at room temperature and then heated and stirred at 55 ° C for 40 hours. It was. This solution was returned to room temperature and 5 ml of distilled water at 0 ° C was added. The solution was concentrated under reduced pressure and the compound (3) was purified using silica gel chromatography.

[0087] [Chemical 15]

[0088] was purified. Identified by NMR and MALDI-TOF Mass. Compound (3) (422 mg) and potassium carbonate (300 mg) were dried in a flask and then dissolved in 15 ml of black mouth form. The gas in the flask was replaced with nitrogen, and the mixture was stirred at room temperature for about 6 hours while adding excess methyl iodide in small portions. The potassium carbonate was removed by filtering the solution under reduced pressure using Celite. The resulting filtrate was concentrated and dried under reduced pressure to give compound (4)

[0089] [Chemical 16]

[0090] was obtained in 381 mg. Compound (4) was dissolved in 20 ml of nitromethane, and 218 mg of potassium tetrachromate platinum (succinate) and 244 mg of silver fluoride were added. The solution was heated to 110 ° C. and stirred for 21 hours, and then filtered under reduced pressure to remove solid substances in the solution. After concentrating the filtrate, the product was precipitated by adding a small amount of jetyl ether, followed by filtration under reduced pressure again to obtain 208 mg of Compound (2).

[0091] (Example 3: Introduction of compounds (1) and (2) into protein, and comparison of ionic efficiency by CSI-TOF Mass) Galactose transferase (TO YOBO) (hereinafter referred to as GalT) was replaced with 20 mM ammonium acetate buffer (PH7) to prepare a 13 μ μ protein solution. To this protein solution 100 _β1 , 1 equivalent of compound (1) or 2 equivalents of compound (2) with respect to protein was added, followed by stirring at room temperature for 4 hours. These solutions were ultrafiltered using Microcon YM-10 (Millipore) to remove unreacted compound (1) or (2). Mass analysis of these protein solutions was performed using MS-T100 CS (JASCO). The measurement conditions are as follows: acceleration voltage—7kV, needle voltage 2kV, orifice voltage 80V, resolution 600 0 (full width at half maximum), gas flow rate 31 / min, solvent removal chamber temperature 200 ° C, 100 ° C, 80 ° C, 50 ° C, 10 ° C

[0092] For the GalT modified with the compound (1), mass spectrometry was performed under the conditions of a solvent removal chamber temperature of 200 ° C, 80 ° C, and 10 ° C (Fig. 1). The resulting spectrum was subjected to deconvolution treatment, and it was confirmed that a part of the protein was modified by the compound (1) (Fig. 2). The obtained data power GalT and modified GalT peak areas were determined. Figure 3 shows a graph when the value measured at 200 ° C is 1. Furthermore, Fig. 4 shows a graph of the area ratio of GalT and modified GalT under each temperature condition.

[0093] For the GalT modified with the compound (2), mass spectrometry was performed under the conditions of solvent removal chamber temperatures of 200 ° C, 100 ° C, 50 ° C, and 10 ° C (Fig. 5). The obtained spectrum was deconvolved, and it was confirmed that a part of the protein was modified with the compound (2) (FIG. 6). Based on the obtained data, the peak areas of GalT and modified GalT were determined. Figure 7 shows a graph when the value measured at 200 ° C is 1. Furthermore, Fig. 8 shows a graph of the area ratio of GalT and modified GalT under each temperature condition.

[0094] From Fig. 2 and Fig. 6, the peak of GalT modified with GalT and one compound (1) or compound (2), respectively, was confirmed. From this result, it was found that the protein can be modified under neutral conditions by using a platinum complex or terpyridine. That is, if various molecules having terpyridine are used, a protein modification method under mild conditions can be provided.

[0095] From FIG. 3 and FIG. 7, the peak area of the protein changes due to changes in the desolvation chamber temperature, the peak area decreases as the temperature decreases, and there is a modification with GalT. It was confirmed that the ratio of the peak area decrease accompanying the temperature decrease differs depending on the absence.

[0096] From FIG. 4 and FIG. 8, it was found that the peak area ratio of the modified protein increases with decreasing temperature. The reason for this may be that the platinum ion used for modification or the force thione of the quaternary amine promotes molecular ionization.

[0097] From Fig. 10 and Fig. 12, it was confirmed that GalT formed a complex with UDP or UDP_Gal regardless of the presence or absence of modification. From this result, it was found that the modified GalT also preserves the enzyme active site of the protein.

[0098] From these results, it was found that the compounds (1) and (2) using the platinum complex can be modified while retaining the activity of the protein under mild conditions. Furthermore, the modified GalT reduced the reduction in ionization efficiency with CSI_Mass compared to the unmodified one. By utilizing modification of these compounds, protein-ligand molecule complexes in the CSI-Mass state can be measured or screened with high sensitivity.

[0099] The specific embodiment or example made in the section of the best mode for carrying out the invention is merely to clarify the technical content of the present invention, and such a specific example. The present invention should not be construed as being limited to a narrow sense, but can be implemented with various modifications within the spirit of the present invention and the scope of the claims set forth below.

Industrial applicability

[0100] The ionization efficiency in protein mass spectrometry often depends on properties specific to individual proteins (for example, the molecular weight and / or isoelectric point of the protein). By introducing a compound having a structure capable of improving the ionization efficiency by the method according to the present invention, it is possible to detect a protein with high sensitivity without depending on the properties of the protein. As a result, protein interactions can be analyzed at high speed and with high sensitivity. In addition, by introducing a functional residue such as a sugar chain to the protein surface, the function of the protein can be modified and / or multifunctional, and a novel artificial glycoprotein can be created.

Claims

The scope of the claims

[1] A method for improving protein ion efficiency, which comprises the step of introducing an organic residue derivative onto the target protein surface.

[2] The method according to claim 1, wherein the organic residue derivative is a metal complex.

[3] The method according to claim 2, wherein the ligand of the metal complex is a tridentate ligand.

[4] The method according to claim 2, wherein the ligand of the metal complex is terpyridine.

[5] The method according to claim 2, wherein the metal element of the metal complex is a transition element or a typical element.

[6] The metal element according to claim 2, wherein the metal element of the metal complex is Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag, Au, Os, or Ir. Method.

[7] The method according to [2], wherein the metal complex strength has a high degree of ammonium.

[8] The metal complex has the following formula:

[Chemical 1]

Indicated by

3. The method according to claim 2, wherein R is H or quaternary ammonium, and X is Pt or Cu.

[9] The method according to claim 2, comprising the step of coordinating the metal complex with a lone pair of amino acid side chains present on the surface of the target protein.

[10] Protein ionization efficiency characterized by comprising a metal complex having a tridentate ligand Kit to improve.

11. Kit according to claim 10, characterized in that the ligand is terpyridine. The kit according to claim 10, wherein the metal element of the metal complex is a transition element or a typical element.

11. The kit according to claim 10, wherein the metal element of the metal complex is Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag, Au, Os or Ir.

11. The kit according to claim 10, wherein the kit has the metal complex strength quaternary ammonium.

The metal complex has the following formula:

[Chemical 2]

Indicated by

11. The kit according to claim 10, wherein R is H or quaternary ammonia, and X is Pt or Cu.

[16] A metal complex having a tridentate ligand.

17. The metal complex according to claim 16, wherein the ligand is terpyridine.

[18] The metal complex according to claim 16, wherein the metal element of the metal complex is a transition element or a typical element.

[19] The metal element according to claim 16, wherein the metal element of the metal complex is Pt, Pd, Fe, Co, Ni, Cu, Cr, Ru, Rh, Ag, Au, Os, or Ir Metal complex.

[20] The metal complex according to claim 16, which has a quaternary ammonia.

[21] The following formula

[Chemical 3]

Indicated by

17. The metal complex according to claim 16, wherein R is H or quaternary ammonium, and X is Pt or Cu.

[22] A composition comprising the metal complex according to any one of [16] to [21], which improves ion efficiency in protein mass spectrometry.

23. The composition according to claim 22, which is used for improving detection sensitivity in mass spectrometry.

[24] The force according to any one of claims 10 to 15, the force according to any one of claims 16 to 21, the metal complex according to claim 1, or the composition according to claim 22,

A method for producing an artificial protein, characterized by imparting a function attributable to the functional residue to the protein by introducing a functional residue into the surface of the protein.

[25] Claim 10 to: Any force of 15 In the kit according to claim 1, In any one of claims 16 to 21, The metal complex according to claim 1, or The composition according to claim 22, A method for analyzing a protein, characterized by determining the presence or absence of a histidine residue, an arginine residue, and / or a cysteine residue.