KR100855620B1 - Separating or concentrating method using mesoporous titanium dioxide - Google Patents

Abstract

The present invention relates to a method for separating, concentrating or analyzing a substance including a phosphonate group or a phosphate group, characterized by using a mesoporous titanium dioxide, preferably a mesoporous titanium dioxide structure having a specific structure. The method of the invention is particularly useful for the isolation and / or concentration of DNA / RNA containing phosphorylated protein / peptide or phosphate groups.

Description

Separating or concentrating method using mesoporous titanium dioxide}

1 is a diagram illustrating the binding of mesoporous titanium dioxide and a substance containing phosphate or phosphonate.

2 and 3 are SEM photographs showing the sponge-type mesoporous titanium dioxide structure.

Figure 4 illustrates a competition experiment to confirm the binding of phosphorylated protein and titanium dioxide.

5 is a competition test result confirming the binding of the phosphorylated protein phosvitin (Phosvitin) and titanium dioxide, [Ru] means the amount of Ru derivatives, [PV] means the amount of phosphite.

6 is MALDI-TOF data showing that mesoporous nano titanium dioxide selectively separates and concentrates phosphorylated peptides in a peptide mixed sample.

The present invention relates to a method for separating, concentrating or analyzing a substance containing a phosphonate group or a phosphate group.

Surfaces of many metal-oxide semiconductors have a common feature of high surface oxygen content, so that the chemical reaction patterns on the surface are very similar. Among these reactions, the bonding process in which a carboxyl group (-COOH) or phosphonate group (phosponate, -PO (OH) ₂ ) in a molecule is easily formed through chemical reaction with oxygen on the surface is a surface modification of the semiconductor. It is widely used as a method for modification. In particular, it is known that titanium dioxide (TiO ₂ ), which is one of the most widely used materials among metal oxide semiconductors, can effectively increase the absorbance of visible light in sunlight by fixing dye molecules to the surface using these reactions. Most of these reactions are chemically similar to esterification.

However, it is not known that such titanium dioxide can be used to selectively isolate or concentrate molecules having specific functional groups, more specifically phosphonate groups or phosphate groups, especially those containing phosphorylated protein / peptide or phosphate groups. The technology of selectively separating or enriching DNA / RNA is an important technology that can be widely used or applied in various researches or diagnosis of diseases in the biotechnology field recently, and its ripple effect is very large. Various materials have been developed to isolate and / or concentrate phosphorylated proteins / peptides and phosphate-containing DNA / RNAs.However, due to the selectivity of phosphorylated proteins, inefficiency in using materials, and manufacturing complexity, Methods have been constantly required.

Therefore, the technical problem to be achieved by the present invention is a separation and / or enrichment method having excellent selectivity to a substance having a phosphonate group or a phosphate group, especially a phosphorylated protein / peptide or a phosphate group and a phospho using the method It is to provide a method for analyzing a substance having an nate group or a phosphate group.

In order to achieve the above technical problem, the present invention is a material comprising a phosphonate group or a phosphate group, characterized in that the mesoporous titanium dioxide, preferably using a mesoporous titanium dioxide structure having a specific structure It provides a method of separation or concentration.

More preferably, the present invention provides a separation or concentration method characterized in that the target material is a DNA / RNA containing a phosphorylated protein / peptide or a phosphate group.

The present invention also uses a mesoporous titanium dioxide, preferably a mesoporous titanium dioxide structure having a specific structure to separate and / or concentrate a material comprising a phosphonate group or a phosphate group, and then the material is prepared using MALDI-TOF, Provided is a method for analyzing a phosphonate group or a phosphate group-containing material, preferably a phosphorylated protein / peptide, characterized by mass spectrometry using nanoLC-MS / MS or the like.

Hereinafter, the method for concentrating and / or separating a specific substance using the mesoporous titanium dioxide of the present invention will be described in more detail.

The present invention provides a method for separating or concentrating a substance containing a phosphonate group or a phosphate group, characterized by using mesoporous titanium dioxide, and having a sponge-type pore structure with the mesoporous titanium dioxide, It is preferable to use mesoporous titanium dioxide structures having good resistance.

The surface of a material made of titanium dioxide has a high content of surface oxygen structurally, and thus has a property of easily binding through chemical reaction with materials having a phosphonate group or a phosphate group. Most of these reactions are chemically similar to esterification, and in the case of aqueous solution, the degree of adsorption can be reversibly controlled according to the change in composition (particularly pH).

The present invention is based on the surprising fact that DNA / RNA containing phosphorylated protein / peptide or phosphate group can be easily separated and / or concentrated using mesoporous titanium dioxide, and in particular, mesoporous titanium dioxide having the structural characteristics described below. The construct is based on the fact that DNA / RNA containing phosphorylated protein / peptide or phosphate groups can be isolated or concentrated very efficiently.

The present invention not only overcomes the disadvantages of existing selective separation methods such as phosphorylated proteins / peptides, but also isolates DNA / RNAs containing a new concept of phosphorylated proteins / peptides or phosphate groups by further expanding the application range by making various types of materials. And / or a concentration system.

The present invention also isolates and / or concentrates DNA / RNA containing phosphorylated protein / peptide or phosphate group according to the above-described method, and then mass spectrometry using a device such as MALDI-TOF or nanoLC-MS / MS. Provided are methods for analyzing substances, which may be useful for identifying phosphorylated proteins / peptides, sequencing phosphorylated peptides, and the like.

In particular, the mesoporous titanium dioxide used for the above-mentioned separation and / or concentration has a sponge-like porous structure of several tens of nanometers or more, and it is desirable that the mesoporous titanium dioxide has a structural feature capable of withstanding a certain pressure due to strong sintering between particles. . In other words, the mesoporous titanium dioxide structure for use in the above-described method is preferred to have a strong resistance to withstand the surrounding environment by continuously maintaining a high flow rate even when using a fluid, especially water, and no deformation due to hydraulic pressure. . The mesoporous titanium dioxide structure having such a property may be prepared by, for example, the production method described below, but the separation, concentration, or analysis method of the present invention is only used in the mesoporous titanium dioxide structure produced by the production method described below. It is not limited.

Mesoporous titanium dioxide structure that can be used in the above-described method comprises the steps of preparing (S1) titanium dioxide nanoparticles; (S2) preparing a paste by mixing the titanium dioxide nanoparticles with a nonionic polymer solution; And (S3) heat-treating the paste.

Generally, the smaller the particle size is, the larger the specific surface area is, so it is common to use nanoparticles for separation and concentration.However, unlike microparticles, when the nanoparticles are directly charged to a column, the size of the pores becomes very small. Very little flow In other words, while maintaining the specific surface area increase effect according to the small size of the nanoparticles to form a sponge-like porous structure, the fluid flow through the porous structure is improved, and the use of mesoporous titanium dioxide structure with improved durability when used for this purpose It is more preferred for the separation and / or concentration method of the present invention.

The titanium dioxide nanoparticles in the step (S1) may be prepared by a method well known to those skilled in the art, and commercially available titanium dioxide nanoparticles such as P25 ™ from Deggussa TiO ₂ can be used. In general, it is easier to use commercially available particles in powder form than when nanoparticles are directly synthesized, and when synthesized through a liquid phase reaction such as sol-gel method, a solution containing nanoparticles after presynthesis To be sufficiently concentrated, considering the mass of the included nanoparticles can be applied to the production method.

The nonionic polymer of step (S2) prevents excessive aggregation of the individual nanoparticles and at the same time forms a disordered porous structure between the nanoparticles and is burned after sintering, so that the entire structure is like a cement brick or sponge structure. It can form a mesoporous structure similar to. The nonionic polymer may be easily mixed with a solvent to be described later, may not be rapidly solidified at room temperature, and materials capable of maintaining the dispersibility of titanium dioxide nanoparticles may be used. The size and size of the mesopores can be controlled by adjusting the type and amount of such nonionic polymer.

As the nonionic polymer which plays such a role, polyoxyethylene octyl phenyl ether, polyethylene glycol, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and the like are preferable.

As the polyoxyethylene octyl phenyl ether, which is C ₁₄ H ₂₂ O (C ₂ H ₄ O) _n , a commercially available Triton X ™ series of Union Carbide may be used, but is not limited thereto, and Triton X-100 is preferable. Do. In addition, the polyethylene glycol used in the present invention has a molecular weight of 5,000-30,000 and is preferable for formation of a mesoporous structure, more preferably 10,000-20,000, and hydroxypropyl cellulose or hydroxypropylmethyl cellulose has an average molecular weight thereof. It is preferable that it is 30,000-100,000 in formation of a mesoporous structure, and it is more preferable that it is 50,000-80,000.

Water, ethanol, acetylacetone, and the like may be used alone or in combination as a solvent for preparing the nonionic polymer solution of the step (S2), but is not limited thereto, and the mixed solvent of water and ethanol has a mesoporous structure It is preferable in the formation, the heat treatment process described later, and the like.

It is also possible to modify the crystal structure of the titanium dioxide nanoparticles in the titanium dioxide paste to have a specific surface that is easier to chemically react with the object of separation, analysis or concentration, and to induce structural interconnections between the titanium dioxide nanoparticles or particle groups. In order to heat-treat the paste of step (S2) to induce porosity and fixation of titanium dioxide, the heat treatment is preferably 380 to 500 ℃, more preferably 430 to 470 ℃. Such heat treatment can increase the interconnection and stability between the titanium dioxide forming the nanostructures. In other words, this heat treatment step removes the nonionic polymer that leads to the disordered sponge-like porous structure and at the same time serves to interconnect the randomly distributed nanoparticles.

As will be described later, when the mesoporous titanium dioxide of the present invention has a three-dimensional structure, such as a columnar structure, most of the mesoporous structure is present as a three-dimensional structure rather than a film form, the physical interconnection becomes very unstable or the structure Cracks, such as cracks, are likely to cause structural collapse during use. In order to overcome this problem, the manufacturing method of the present invention may pretreat the structure with a suitable concentration of TiX ₄ (X is halogen) before applying the paste of step (S2) to a specific structure, or heat-treat the step (S3). It is further preferred to further comprise the step of post-treating and drying the titanium dioxide with TiX ₄ (X is halogen). Through this process, a new titanium dioxide buffer layer is formed between the substrate surface or the titanium dioxide porous structure, thereby solving the above problem. As TiX _4, TiCl ₄ is preferable.

The manufacturing method may further include the step of grinding the paste between the step (S2) and (S3).

In the separation, concentration or analysis method of the present invention, the preparation method may further include applying the paste to a structure between the steps (S2) and (S3), and through this step, It is easy to use and can produce a structure for separation or concentration suitable for the purpose to be applied. The structure may be a film, a tip, a column, a filter, a tube, a microchip, or the like, but is not limited thereto.

More specifically, the final form of mesoporous titanium dioxide used in the method of the present invention depends on the type of analyte to be analyzed and the method of analysis, and in various forms, for example, in the form of a thin planar surface film on the substrate surface. -Ti-column in the form of film, pre-column filled with a thin capillary tube, and MEMS-type Ti-MEMS that can be used for lab-on-a-chip analysis type can be used.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1 Preparation of Mesoporous Titanium Dioxide Structure

In the nano titanium dioxide structure having a mesoporous structure, a powder form and a micro column form were made and used for the separation and concentration experiment of phosphate which will be described later.

TiO ₂  Making of paste

Synthesis or preparation of the TiO ₂ nano-particles were well known to a sol-gel method in the conventional synthesis of TiO ₂ nano-particle or nano-particles prepared by buying appropriate. In Example 1 P25 TiO ₂ from Degussa was used.

Making binders: Various kinds of nonionic polymers were dissolved in an appropriate solvent to achieve the proper viscosity. The type of nonionic polymer and solvent may vary slightly depending on the analytical structure to be applied (thin film, capillary, MEMS structure, etc.). For example, in the case of forming a thin porous film on a planar substrate, an appropriate amount of polyethylene glycol may be dissolved in water and ethanol may be added. For example, in Example 1 used in the following evaluation experiment, polyethylene glycol was dissolved in a 1: 6 mixed solution of ethanol: water so as to be 33 wt% and used as a binder.

TiO ₂ Paste Preparation: Mix TiO ₂ nanoparticles with the binder in an appropriate amount until the clusters of nanoparticles are removed from the mortar. For example, in Example 1, P25 TiO ₂ nanoparticles were mixed in a ratio of 0.1 g to 0.6 ml of the binder.

Prepare and Analyze Structures for Analysis TiO ₂  Paste applied

The structure for analysis depends on the type of analyte to be analyzed and the method of analysis, and the Ti-film in the form of a thin flat surface film on the substrate surface, the Ti-colum in the form of a precolumn filled in a thin capillary tube, and lab-on-a. It is divided into Ti-MEMS, which is a MEMS type that can be used for analysis types of chips.

In the case of Ti-film, no special process is required for the preparation of the substrate, but the desired substrate is determined. The TiO ₂ mesoporous structure to be formed into a film on the substrate can be made by applying a conventional doctor-blade method in which a scotch tape is attached to the periphery and a TiO ₂ paste is filled, and the thickness thereof is usually about 10 mm. It is determined through the thickness of.

In the case of Ti-columns, the main parameters are represented by the inner diameter of the capillary tube used and the height of the TiO ₂ mesoporous membrane filled in column form. In particular, the height is appropriately sized according to the amount or type of sample to be analyzed.

In the case of Ti-MEMS, the desired MEMS structure is designed in advance in the form of irregularities on a glass substrate, and then TiO ₂ paste is partially or partially formed in accordance with the purpose of analysis and the type of analyte. Is filled in using the doctor-blade method.

Through heat treatment TiO ₂ Mesoporous membrane  Formation and pretreatment / posttreatment

The TiO ₂ paste (in the form of micro columns and powder in Example 1) applied to the prepared analytical structure was sufficiently dried in air and then heat-treated in a high temperature furnace. Although the heat treatment time and temperature vary slightly depending on the type and solvent of the nonionic polymer used, generally, the temperature at which the crystal structure of the TiO ₂ phase stabilizes to an anatase type is about 450 ° C. and about 30 minutes or more is sufficient. .

Pre-treatment / post-treatment: After all stabilize the porous structure and the substrate in the TiO ₂ porous structure in a well sticking to apply the TiO ₂ paste for the purpose of eager to improve the reliability in the analysis structure before heat treatment the TiO ₂ paste is used to apply as necessary Can be. Treatment with an appropriate concentration of TiCl ₄ and drying to form a new TiO ₂ buffer layer between the substrate surface or the TiO ₂ porous structure.

The structure of the mesoporous titanium dioxide prepared according to Example 1 was confirmed using SEM, and the results are shown in FIGS. 2 and 3.

Experimental Example 1 Confirmation of Phosphorylated Protein and Mesoporous TiO ₂

Experiment using powder form

Mesoporous TiO ₂ powder was suspended in 1% acetic acid and only a portion of this (eg 10 ul) was taken and used in the experiment. The dispersed TiO ₂ was precipitated by centrifugation and the supernatant solution was removed. The analyte dissolved in 1% acetic acid solution was also added to the precipitated TiO ₂ and mixed vigorously. Thereafter, TiO ₂ was precipitated again by centrifugation. The supernatant remaining after precipitation was taken and used in various assays. After washing the TiO ₂ precipitated with distilled water 2-3 times well, the analyte bound to TiO ₂ with an ammonium carbonate solution of pH 10 or higher was separated and used for analysis.

Experiment with micro column

The mesoporous TiO ₂ filled in the micro column was washed 2-3 times with 1% acetic acid to make the column acidic. One end of the microcolumns was immersed in a small amount of solution, and the other end was connected to a syringe or a micro pipette to slowly suck the solution and then push it out again. The analyte dissolved in 1% acetic acid was passed through a micro column and the remaining solution was used for the analysis. After repeated 2-3 times washing with distilled water to remove the remaining analyte was passed through a small amount of ammonium carbonate solution to separate the analyte bound to TiO ₂ . The separated analytes were also analyzed using several methods.

Identification of Phosphorylated Protein Binding Through Competition Experiments

Ru derivatives known to bind TiO ₂ are fluorescent and can be measured by fluorescence measurements. The amount of phosphorylated protein binding to TiO ₂ was indirectly measured using this.

Ru derivatives of known concentration were bound to the mesoporous TiO ₂ powder or micro column prepared above with increasing concentration. The amount of Ru derivative remaining after binding in each process was determined by measuring fluorescence.

The Ti derivative was bound to TiO 2 as much as possible until the Ru derivative could no longer bind. To the phosphorylated protein poseubi tin (phosvitin) little by little added to the TiO ₂ filled with a Ru derivative is a Ru naohneunga derivatives isolated from TiO ₂ were observed through fluorescence measurement. The addition of phosphorylated protein phosbitin increased the fluorescence of the solution, which can be interpreted as the result of the separation of the Ru derivative, a fluorescent substance bound to TiO ₂ by the binding of phosphite and TiO ₂ . . Phosphorylated protein and TiO ₂ binding confirmation process through a competitive experiment is shown in Figure 4, the above-described fluorescence measurement results are shown in FIG.

Experimental Example 2 Isolation of Phosphorylated Peptide

As an experimental method, the same procedure as in Experimental Example 1 was performed, and a phosphorylated peptide having a molecular weight of 905 was added to a peptide fragment mixture prepared by treating bovine serum albumin (BSA) with trypsin as an analyte. The sample contains many types of non-phosphorylated peptides and one type of phosphorylated protein, which is suitable for use in experiments that screen for nonspecific and specific (phosphorylated selective) bonds.

The solution taken in several steps was analyzed by MALDI-TOF to determine which peptide binds to TiO _2, and the results are shown in FIG. 6. In Figure 6 a) is the mass data of the sample before binding to TiO ₂ , b) is a mass analysis of what remains after the sample is bonded to TiO ₂ . C) is the mass data of the peptide separated from TiO ₂ when washed with 50% acetonitrile after binding the sample, and d) is the peptide separated when washed TiO ₂ with ammonium carbonate at pH 10. mass data. From these results, it can be seen that the phosphorylated peptide was finally separated and concentrated by binding to TiO ₂ differently from other peptides.

Experimental Example 3 Confirmation of Concentration of Phosphorylated Peptides Using nanoLC-ESI-MS / MS

After loading TiO ₂ powder on a column filled with C18 resin, a sample containing phosphorylated peptide was passed through a TiO ₂ + C18 resin column to confirm the enrichment effect of the phosphorylated peptide.

Specifically, TiO ₂ powder was loaded on a column filled with C18 resin, and then washed with 0.1% TFA, 80% ACN and 2% NH ₄ OH solution in order. Thereafter, the column was washed again with 0.1% TFA solution, and the sample dissolved in the 0.1% TFA solution was loaded on the column, and the column was washed in the order of 0.2% TFA, 80% ACN, and distilled water. Phosphorylated peptide was re-isolated using 2% NH ₄ OH solution and 80% ACN solution to obtain a concentrated solution of phosphorylated peptide. The concentrated solution was evaporated under reduced pressure and then dissolved again with 6 ul of 0.15% formic acid. 3 ul of samples were analyzed using nanoLC-MS / MS (LTQ) instrument.

Mix two phosphorylated peptides (peptides 1 and 2) and two non-phosphorylated peptides (peptides 3 and 4) with tryptic peptides from BSA to see if the phosphorylated protein is concentrated by the above method. Confirmed. The results are shown in Table 1 below, and it was confirmed that the phosphorylated peptide was concentrated through the above method. Peptides 3 and 4 that would not be phosphorylated either failed to adhere to the column (peptide 3) or fell off in the process of washing the column (peptide 4). Two phosphorylated peptides were obtained at the final stage of enrichment.

Flow-through Washing Elution Peptide 3 -.SYGAPR Peptide 4 -.NSSYFVEWIPNNVK Peptide 1 -.TSTEPQ pY QPGENL Peptide 2 -.DHTGFL pT E pY VATR

In order to examine the effect of enrichment in a real sample, the method according to the present invention was confirmed to lysate (lysate) of Huh7 cells, which is a kind of human-derived hepatocytes. As a result of the experiment, the method of the present invention was able to identify a larger number of phosphorylated peptides compared to the general method (see Table 2) or to identify the phosphorylated peptide (or protein) that was not identified by the general method (Table 3 and 4).

In Tables 2, 3 and 4 below, Y @, S *, T # means phosphorylated tyrosine, phosphorylated serine, and phosphorylated threonine, respectively.

number Control Invention TiO ₂ Protein name scan # Peptide sequence scan # Peptide sequence One kinesin family member C3 [Homo sapiens] 14642 K.LTY@LLQDSLS*GDSK.T 13346 K.LTY@LLQDSLS*GDSK.T 14642 K.LT # YLLQDSLS * GDSK.T 13930 K.LTY@LLQDSLSGDS*K.T 14703 K.LTY@LLQDSLS*GDSK.T 13930 K.LT # YLLQDSLSGDS * K.T 14703 K.LT # YLLQDSLSGDS * K.T 14006 K.LT # YLLQDSLS * GDSK.T 14703 K.LTY@LLQDSLSGDS*K.T 14363 K.LTY@LLQDSLS*GDSK.T 14703 K.LT # YLLQDSLS * GDSK.T 14363 K.LT # YLLQDSLSGDS * K.T 14363 K.LTY@LLQDSLSGDS*K.T 14363 K.LT # YLLQDSLS * GDSK.T 14426 K.LTY@LLQDSLSGDS*K.T 14426 K.LTY@LLQDSLS*GDSK.T 14426 K.LT # YLLQDSLS * GDSK.T 14426 K.LT # YLLQDSLSGDS * K.T 2 ER to nucleus signaling 1 [Homo sapiens] 15355 R.T # EYT # IT # MYDTK.T 14000 R.T#EY@TIT#MYDTK.T 15355 R.T#EY@TIT#MYDTK.T 14000 R.T#EYT#ITMY@DTK.T 14000 R.T # EYT # IT # MYDTK.T 14212 R.T#EY@TIT#MYDTK.T 14212 R.T # EYT # IT # MYDTK.T 14289 R.T # EY @ TITMY @ DTK.T 14979 R.T # EY @ TITMY @ DTK.T 14979 R.T#EY@TIT#MYDTK.T 14979 R.T # EYT # IT # MYDTK.T 14979 R.T#EYT#ITMY@DTK.T 3 adenylyl cyclase-associated protein [Homo sapiens] 14119 K.LSDLLAPISEQIK.E 15242 K.EMNDAAMFY@TNR.V 16205 K.EMNDAAMFYT # NR.V 15303 K.EMNDAAMFY@TNR.V 16205 K.EMNDAAMFY@TNR.V 15364 K.EMNDAAMFY@TNR.V 15613 K.EMNDAAMFYT # NR.V 15613 K.EMNDAAMFY@TNR.V 4 calcium binding atopy-related autoantigen 1; atopy related autoantigen 13597 R.LNSLS * ALAELAVGS * R.W 3596 R.LNSLS * ALAELAVGS * R.W 13597 R.LNS * LSALAELAVGS * R.W 3596 R.LNS * LSALAELAVGS * R.W 13663 R.LNS * LSALAELAVGS * R.W 13464 R.LNS * LSALAELAVGS * R.W 13663 R.LNSLS * ALAELAVGS * R.W 13464 R.LNSLS * ALAELAVGS * R.W 13724 R.LNS * LSALAELAVGS * R.W 13523 R.LNS * LSALAELAVGS * R.W 13724 R.LNSLS * ALAELAVGS * R.W 13523 R.LNSLS * ALAELAVGS * R.W 13585 R.LNS * LSALAELAVGS * R.W 13585 R.LNSLS * ALAELAVGS * R.W

number Protein name scan # Peptide sequence One leucine-rich PPR motif-containing protein [Homo sapiens] 14300 K.MVFINNIALAQIK.N 14527 R.DQMY @ Y @ NLLK.L 16979 K.DLPVTEAVFSALVTGHAR.A 17050 K.DLPVTEAVFSALVTGHAR.A 17059 K.AGY @ PQY @ VSEILEK.V 2 pyrroline-5-carboxylate synthetase; Pyrroline-5-carboxlate synthetase; 11618 R.SWSNIPFIT # VPLSR.T 12166 R.SWSNIPFIT # VPLSR.T 13378 K.FASYLTFSPSEVK.S 14345 K.LIDIFYPGDQQSVTFGTK.S 14434 K.LIDIFYPGDQQSVTFGTK.S 14507 K.LIDIFYPGDQQSVTFGTK.S 15159 K.LIDIFYPGDQQSVTFGTK.S 3 oxygen regulated protein precursors; oxygen regulated protein (150kD) [H 14252 K.AANSLEAFIFETQDK.L 16686 K.AANSLEAFIFET # QDK.L 16715 R.DAVVYPILVEFTR.E 4 phosphatidylinositol transfer protein, cytoplasmic 1 isoform a; retina 13686 K.SAPET # LT # LPDPEK.K 13686 K.S * APET # LTLPDPEK.K 13686 K.S * APETLT # LPDPEK.K 13752 K.S * APET # LTLPDPEK.K 13752 K.S * APETLT # LPDPEK.K 13752 K.SAPET # LT # LPDPEK.K 13812 K.SAPET # LT # LPDPEK.K 13872 K.SAPET # LT # LPDPEK.K 13872 K.S * APETLT # LPDPEK.K 13932 K.SAPET # LT # LPDPEK.K 13932 K.S * APETLT # LPDPEK.K 13932 K.S * APET # LTLPDPEK.K 13992 K.SAPET # LT # LPDPEK.K 14145 K.S * APET # LTLPDPEK.K 14145 K.S * APETLT # LPDPEK.K 5 mitogen-activated protein kinase kinase kinase 6; apoptosis signal-reg 16430 R.Y@LGS*ASQGGYLK.I 16430 R.Y@LGSAS*QGGYLK.I 16430 R.Y @ LGSASQGGY @ LK.I

Number Protein name scan # Peptide sequence 6 zinc finger homeodomain 4 [Homo sapiens] 3578 R.T # TIT # PEQLEILYEK.Y 3578 R.TT # IT # PEQLEILYEK.Y 3578 R.T # T # ITPEQLEILYEK.Y 13346 K.YIYFDYPS * LPLTK.I 7 sperm associated antigen 9 isoform 1; sperm surface protein; JNK / SAPK- 12016 K.YNAPTSHVT # PSVK.K 12677 K.YNAPT # SHVTPSVK.K 12677 K.YNAPTS * HVTPSVK.K 8 solute carrier family 3 (activators of dibasic and neutral amino acid 15256 K.GQSEDPGSLLSLFR.R 15373 K.GQSEDPGSLLSLFR.R 16475 R.LLTSFLPAQLLR.L 16550 R.LLTSFLPAQLLR.L 16678 K.DDVAQT # DLLQIDPNFGS * K.E 9 InaD-like protein isoform 3; protein associated to tight junctions; PD 13930 K.TS * S * S * TSPLEPPSDR.G 13930 K.T # SS * S * TSPLEPPSDR.G 14296 K.T # SS * S * TSPLEPPSDR.G 14296 K.TS * S * S * TSPLEPPSDR.G 14296 K.T # S * SS * TSPLEPPSDR.G 10 insulysin; insulinase [Homo sapiens] 14660 R.NLYVTFPIPDLQK.Y 14739 R.NLYVTFPIPDLQK.Y 15847 K.NEFIPTNFEILPLEK.E 11 tetratricopeptide repeat domain 1 [Homo sapiens] 14664 R.PFGLST # ENFQIK.Q 14724 R.PFGLST # ENFQIK.Q 14724 R.PFGLS * TENFQIK.Q 14784 R.PFGLST # ENFQIK.Q 14829 K.S * NEDVNS * S * ELDEEYLIELEK.N 15258 R.PFGLST # ENFQIK.Q 12 semaphorin 5B [Homo sapiens] 15510 R.FLLEDTWT # TFMK.A 15510 R.FLLEDTWTT # FMK.A 15678 R.FLLEDTWT # TFMK.A 15678 R.FLLEDTWTT # FMK.A 13 dishevelled 3; dishevelled 3 (homologous to Drosophila dsh) [Homo sapi 11999 R.FSSS * TEQS * S * ASR.L 11999 R.FS * SSTEQS * S * ASR.L 12233 R.FSS * S * TEQS * SASR.L 12233 R.FS * S * STEQS * SASR.L

The present invention provides a simple and efficient method for separating and / or enriching or analyzing a substance having a phosphonate group or a phosphate group, in particular a phosphorylated protein / peptide or a DNA / RNA having a phosphate group.

Claims (15)

A method for separating a phosphonate group or a phosphate group-containing material, characterized by using mesoporous titanium dioxide. delete delete The method of claim 1, wherein the substance is a phosphorylated protein or phosphorylated peptide. The method of claim 1, wherein the substance is a DNA or RNA containing a phosphate group. The method of claim 1, wherein the mesoporous titanium dioxide comprises (S1) preparing a titanium dioxide nanoparticle; (S2) preparing a paste by mixing the titanium dioxide nanoparticles with a nonionic polymer solution; (S3) applying the paste to a structure selected from the group consisting of film, tip, column, filter, tube and microchip; And (S4) heat-treating the paste. A method for concentrating a phosphonate group or a phosphate group-containing material, characterized by using mesoporous titanium dioxide. 8. The method of claim 7, wherein the substance is a phosphorylated protein or phosphorylated peptide. The method of claim 7, wherein the substance is a DNA or RNA containing a phosphate group. The method of claim 7, wherein the mesoporous titanium dioxide comprises: (S1) preparing titanium dioxide nanoparticles; (S2) preparing a paste by mixing the titanium dioxide nanoparticles with a nonionic polymer solution; (S3) applying the paste to a structure selected from the group consisting of film, tip, column, filter, tube and microchip; And (S4) heat-treating the paste. A method for analyzing a phosphonate group or a phosphate group-containing material, characterized in that the material is mass spectroscopically separated or concentrated using a mesoporous titanium dioxide. The method of claim 11, wherein the mass spectrometry uses MALDI-TOF or LC-MS / MS. The method of claim 11, wherein the substance is a phosphorylated protein or a phosphorylated peptide. The method of claim 11, wherein the substance is a DNA or RNA containing a phosphate group. The method of claim 11, wherein the mesoporous titanium dioxide is prepared by (S1) preparing titanium dioxide nanoparticles; (S2) preparing a paste by mixing the titanium dioxide nanoparticles with a nonionic polymer solution; (S3) applying the paste to a structure selected from the group consisting of film, tip, column, filter, tube and microchip; And (S4) heat-treating the paste.

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