JPWO2018061593A1 - Method for concentration and purification of alkyne-containing molecules - Google Patents

Abstract

The present invention provides an adsorbent for concentration and purification of a terminal alkyne-containing molecule in a liquid, which comprises an insoluble carrier in which silver ions are supported via pKa 2-9 functional groups such as carboxyl groups; Condensing and purifying a terminal alkyne-containing molecule comprising contacting a liquid containing the alkyne-containing molecule and capturing a terminal alkyne-containing molecule by a silver acetylide forming reaction, and eluting with a halide ion, acid or sulfur containing reagent On the way.

Description

  The present invention relates to a method for concentrating and purifying an alkyne-containing molecule, and a silver-loaded carrier and device or apparatus therefor, and in particular, for concentrating and purifying a biorelated molecule or synthetic molecule containing an alkyne as a partial structure. Methods and suitable carriers and devices or apparatus therefor.

  Methods for concentrating and purifying molecules with specific chemical structures include immunoprecipitation using antigen-antibody reaction, a method using avidin-biotin interaction, a method using interaction between His tag and Ni-immobilized solid phase Has been used since ancient times. However, His tag and biotin are large size tags, and it is not desirable to introduce them as tags into low molecular weight compounds such as physiologically active compounds and metabolites. Also, while antibodies are effective against large molecules such as proteins, it is not generally easy to generate antibodies against small functional groups or small molecule compounds, including alkynes, and the specific lows for which antibodies have already been generated. Except for molecular compounds, it can not be a general method for concentration and purification of low molecular weight compounds. Furthermore, for a group of compounds that do not dissolve in aqueous solution and need to be handled in organic solvents, such as lipids and their metabolites, proteins such as antibodies and avidin can not be used for denaturation in organic solvents.

  From such a background, in recent years, small tags such as alkynes and azides have been introduced into low molecular weight compounds, and subsequently, biotin and fluorous tags etc. are introduced as secondary tags by copper-catalyzed azide-alkyne cycloaddition reaction. Came to be This solved the problem of tag size. However, there is a problem that the simplicity and the efficiency of the whole process are lowered by adding the steps of the introduction reaction of the tag using the copper catalyst and the purification after the reaction.

  Therefore, the method of directly concentrating and purifying alkynes can be a method of solving the above problems. To date, methods have been reported in which a dicobalt octacarbonyl complex is immobilized on phosphine-modified silica gel or a polymer carrier, and a complex formation reaction of alkyne and cobalt is used to concentrate and purify alkyne-containing lipids, peptides, and proteins. (Non-patent documents 1 to 3). In the methods of Non-Patent Documents 1 and 2, it is possible to use either an aqueous solution or an organic solvent by not using a protein. On the other hand, the method of Non-Patent Document 3 is not limited to the use in organic solvents, and further, because it requires an oxidation treatment at the stage of eluting the captured alkyne, it can not be applied to a group of compounds weak to oxidation treatment. The Further, the methods of Non-Patent Documents 1 and 2 can be used in an aqueous solution and can be eluted with an acid, but are methods applicable only to propargyl carbamate structures. In addition, as common problems, the formation reaction of alkyne-cobalt complex is slow, instability of zero-valent cobalt, weak interaction between cobalt and phosphine on solid phase, two for one cobalt There is a problem that the complexing ability is lost when the above phosphines are coordinated.

  Also, in the field of steroid analysis in the 1960s, steroids containing alkynes are captured by forming silver acetylide on silver nitrate-treated florisil or silica gel, and can be eluted by the decomposition reaction of silver acetylide by ammonium chloride treatment. It reports (nonpatent literature 4 and 5). This method has very mild conditions, and passes the sample through the column packed with the carrier to capture ethynyl steroid (synthetic steroid hormone agent containing alkyne) in the sample and pass the solvent containing ammonium chloride. It is possible to carry out convenient and rapid concentration and purification of recovering ethynyl steroids. However, this method can be used only in organic solvents, and there is no example applied to samples other than steroids, and citations since 1980 are only a review in the analytical field regarding silver ion chromatography (non-patent literature) 6). Also, in 1979, an example was reported in which silver ions were supported on a sulfonic acid-modified cellulose carrier, ethynyl steroids were captured, and excess hexine or acetylene was eluted (Non-Patent Document 7). It is only used in. Although this paper is also rarely cited in the field of silver ion chromatography, it has not been recognized in the life science field such as chemical biology. Silver ion chromatography is a method of separating low molecular weight compounds according to the difference in the degree of unsaturation, utilizing the coordination ability of silver ions and pi electrons of unsaturated bonds.

  The concentration purification method of the alkyne using the silver ion carrying | support solid phase in said nonpatent literature 4, 5, and 7 is performed in the organic solvent, The object was also narrowed down to the steroid. Under conditions including water and a buffer, it is known that silver-supported florisil and silica gel can not stably hold silver ions, and silver ions are eluted. In particular, when applied to biological samples such as lipid metabolites and peptides, these molecules themselves have the ability to coordinate to silver ions, and elution of silver ions makes it difficult to concentrate and purify them. Therefore, as in the prior art, sulfonic acid modified cellulose was used, it was necessary to fix silver ions on the carrier more firmly. However, as represented by strongly acidic cation exchange resins etc., sulfonic acid-modified carriers are widely used for the adsorption of cationic compounds or basic compounds which are easily protonated even under neutral conditions and have a positive charge. There is. That is, there is a problem that the cationic compound and the basic compound are adsorbed to the carrier with or without the alkyne, which causes an obstacle to selective concentration and purification of the alkyne.

  As a method of supporting and trapping metal ions, there is also a method of using phosphine as a ligand as used in the example of the alkyne-cobalt complex. In addition, carriers modified with thiols, thioamides, carboxylic acids and amines, which are commercially available as scavenger resins, are generally used for the purpose of trapping and removing metal ions in the solution. However, the influence of the functional group acting as a ligand of these silver ions on the acetylide formation reaction of silver ions has not been scrutinized. Also in the above non-patent documents 4, 5 and 7, there is no description or suggestion regarding the relationship between the effect of the functional group on the support as a ligand and the efficiency of the silver acetylide forming reaction, these supports are non-patent mentioned above It is also unclear whether it can be used as well as Florisil, silica gel and sulfonic acid modified cellulose described in Documents 4, 5 and 7.

  As a related prior art, there has been reported a process of removing acetylene contained in a gas using a carboxylic acid-modified polymer loaded with silver ions (Patent Document 1). However, it was applied only to the removal of acetylene contained in low polarity gas, and it was unclear whether this carrier could be used in a solution. It is reported that the filter after use removes acetylene gas and silver ion by treating with dilute nitric acid, and it is possible to support silver ion again to be reusable, and thus strongly acidic and oxidative It is easily expected that the conditions can not be used for biomolecules etc.

  Thus, although the concentration purification method of the alkyne which used the silver ion loading solid phase was researched from 1960 to 1970 in the field of steroid analysis and removal of acetylene in gas, the object of application is the steroid in organic solvent Or it was limited to acetylene in the gas. Also, from the chemical point of view, systematic optimization has not been carried out, and no scrutiny has been made with respect to application scope such as solvent and functional group tolerance.

  Patent Document 2 describes a silver-supported silica gel characterized in that silver is supported by ion exchange, and the silver-supported silica gel is particularly intended to separate terpenes and unsaturated fatty acids. Patent Document 2 makes no mention of alkynes such as acetylene. The silver-supported silica gel is not modified with sulfonic acid or carboxylic acid, and it is described that silver is ion-exchanged with hydrogen to be bound to surface silanol groups.

U.S. Pat. No. 3,273,314 Unexamined-Japanese-Patent No. 5-317699

Egami H, Kamisuki S, Dodo K, Asanuma M, Hamashima Y & Sodeoka M (2011) Catch and release of alkoxy-tagged molecules in water by a polymer-supported cobalt complex. Org Biomol Chem 9: 7667-7670. Miyazaki A, Asanuma M, Dodo K, Egami H & Sodeoka M (2014) A 'Catch-and-Release' Protocol for Alkyne-Tagged Molecules Based on a Resin-Bound Cobalt Complex for Peptide Enrichment in Aqueous Media. Chem. Eur. J. 20: 8116-8128. Milne SB, Tallman KA, Serwa R, Rouzer CA, Armstrong MA, Marnett LJ, Lukehart CM, Porter NA, and Brown HA. (2010) 205-207. Ercoli A, Vitali R & Gardi R (1964) Adsorbents for detection, isolation and evaluation of ethynyl steroids. Steroids 3: 479-485. Kulkarni BD & Goldzieher JW (1969) Isolation of 17α-ethynyl steroids by column chromatography on silver impregnated florisil. Steroids 13: 467-475. Williams CM & Mander LN (2001) Chromatography with silver nitrate. Tetrahedron 57: 425-447. Sjovall J & Axelson M (1979) General and selective isolation procedures for GC / MS analysis of steroids in tissues and body fluids. Journal of Steroid Biochemistry 11: 129-134.

  An object of the present invention is to provide a method for rapidly and efficiently concentrating and purifying a wide range of alkyne-containing molecules under mild conditions in a wide range of solvent systems including aqueous solution systems.

  In order to solve the above problems, it is necessary to support silver ions strongly so that silver ions are not eluted by a solvent or buffer without impairing the reactivity of silver ions for capturing alkynes as silver acetylide on a carrier. It is. From such a point of view, the present inventors first examine the ability to support silver ions and the ability to form silver acetylide on the support using variously functionalized supports, and the function to support the support and silver. Group optimization was performed. As a result of intensive studies, it was found that a strong silver ion supporting ability is insufficient for the purpose of concentrating and purifying an alkyne-containing molecule, and that a carrier suitable for this purpose exists. Based on this finding, we will establish a selective concentration and purification method for alkyne-containing molecules that can be used not only in organic solvents but also in aqueous solutions and applicable to lipid metabolites and peptides derived from cells etc., and complete the present invention It came to

That is, the gist of the present invention is as follows.
(1) An adsorbent for concentration and purification of a terminal alkyne-containing molecule in a liquid, containing an insoluble carrier in which silver ions are supported via a functional group of pKa 2-9.
(2) The adsorbent according to the above (1), wherein the functional group having a pKa of 2 to 9 is a carboxyl group, a phosphoric acid group or a sulfonamide group.
(3) The adsorbent according to (1), wherein the insoluble carrier is a silica gel or organic polymer carrier on which silver ions are supported via a carboxyl group.
(4) An apparatus or apparatus for concentration and purification of a terminal alkyne-containing molecule using the adsorbent according to any one of (1) to (3) above.
(5) The apparatus or the device according to (4), wherein the silica gel in which silver ions are supported via a carboxyl group and the silica gel modified with thiol or thiourea are used in a two-layer structure.
(6) The above (4) or (5) having a plurality of layers, at least one layer containing the adsorbent according to any one of the above (1) to (3), and at least one layer containing a reverse phase carrier. The apparatus or device as described in 4.).
(7) The device or device according to any one of (4) to (6), wherein the device or device is a solid phase extraction column, kit, filter or chip.
(8) a step of bringing a liquid containing a terminal alkyne-containing molecule into contact with the adsorbent according to any one of the above (1) to (3) to capture the terminal alkyne-containing molecule, and halide ion, acid or sulfur A method for concentrating and purifying a terminal alkyne-containing molecule, comprising the step of eluting with a containing reagent.
(9) The method according to (8), wherein the device or device according to any one of (4) to (7) is used.
(10) The method according to (8) or (9) above, wherein the solvent in the liquid containing the terminal alkyne-containing molecule is an organic solvent or an aqueous solvent.

  According to the present invention, concentration purification of a terminal alkyne-containing molecule in a liquid is possible under mild conditions and a practical yield and convenience. A sample that has been subjected to indirect purification by copper-catalyzed azide-alkyne cycloaddition reaction or the like can be directly concentrated and purified. In particular, concentration purification using antibodies or avidin-biotin can not be applied, and it is a very effective method for lipid samples that are weak to acids and oxidation conditions.

  In addition to comprehensive analysis of metabolites generated by metabolism from precursors containing terminal alkynes, identification of proteins to which alkinated low molecular weight compounds bind, and their binding sites, alkynes selectively react with specific functional groups By using the probe, it is possible to carry out concentration purification and analysis of an endogenous molecule group having a specific functional group.

  It can also be used as a purification method for terminal alkyne-containing molecules. It is possible to selectively isolate and purify terminal alkyne-containing molecules, even when purification is difficult by general separation methods such as ordinary normal phase / reverse phase chromatography and size exclusion chromatography. Therefore, it is useful not only for analytical applications, but also for the purpose of preparing terminal alkyne-containing molecules, such as purification of products in organic synthesis and enzymes or synthetic products using organisms.

FIG. 1 shows an example of the method for concentration and purification of terminal alkyne-containing molecules and apparatus or apparatus of the present invention. FIG. 2 shows functional group tolerance. FIG. 3 shows a comparison of carboxylic acid modified silica gel (COOH silica gel) and sulfonic acid modified silica gel (SO ₃ H silica gel) in concentration purification of terminal alkyne-containing peptide. FIG. 4 shows the results of concentrated purification from cellular lipid extracts of terminal alkyne-containing phospholipids. FIG. 5 shows the results of concentration and purification of metabolites of terminal alkyne-containing choline in cells. FIG. 6 shows the results of concentrated purification from cultured cell enzymatic digests of terminal alkyne-containing peptides. FIG. 7 shows the results of concentration and purification of the alkynated peptide using a spin column combining a silver-supported carrier and a reverse phase carrier.

  A compound having a terminal alkyne structure, that is, an ethynyl group (H-C≡C-) reacts with metal ions such as silver ions, copper ions and gold ions to form metal acetylides. Among metal acetylides, silver acetylide is low in reactivity, so applications (reactions) in organic synthesis are limited, but it rapidly forms in a liquid, is stable, and can be isolated.

  The present invention uses the above-mentioned properties of silver acetylide to immobilize silver ions on a solid phase carrier and capture terminal alkyne-containing molecules as silver acetylide on the solid phase, and silver acetylide is a halide ion, acid or acid. It utilizes the property of easily reacting with a sulfur-containing reagent to regenerate a terminal alkyne-containing molecule, and elution is carried out by regenerating the terminal alkyne-containing molecule from acetylide, and the terminal alkyne-containing molecule is concentrated and purified.

  The present invention provides an adsorbent for concentration and purification of terminal alkyne-containing molecules in a liquid, which contains an insoluble carrier in which silver ions are supported via a functional group of pKa 2-9.

  When the pKa of the functional group used to support silver ions is less than 2, a basic compound that is likely to be positively charged is strongly adsorbed and difficult to elute, while when the pKa of the functional group exceeds 9 The effect of supporting silver ions is reduced.

  In the present specification, “pKa” means “the inverse logarithm of acid dissociation constant Ka” defined in Chemical Handbook (II) (revised 4th edition, 1993, edited by The Chemical Society of Japan, Maruzen Co., Ltd.) Point to.

  Examples of functional groups having a pKa of 2 to 9 include, for example, a carboxyl group (carboxylic acid group) (pKa: about 3 to 5), a phosphoric acid group (pKa: about 2 to 7), and a sulfonamide group (pKa: about 4 to 9) And are preferably pKa3-5 functional groups.

The insoluble carrier used in the present invention is not particularly limited as long as it can be modified with a functional group having a pKa of 2 to 9, for example, a carboxyl group (carboxylic acid group), a phosphoric acid group or a sulfonamide group. for the chromatography, there can be used those which are commercially available, include for example silica gel, activated alumina, activated carbon, cellulose, resins (e.g., Amberlite ^TM, polystyrene, styrene-divinylbenzene copolymer, methacrylate copolymer) is Be It is preferable to use a highly versatile carrier that can be used for both water and organic solvents, and examples of highly versatile carriers include silica gel.

The silica gel modified with a functional group PKa2～9, carboxylate modified silica gel (e.g., InertSep ^TM CBA (manufactured by GL Sciences Inc.)), propyl carboxylic acid modified silica gel, Chromatorex COOH (manufactured by Fuji Silysia Chemical Ltd.), ISOLUTE ^TM CBA (Biotage), 3-carboxypropyl silica gel ^{(Aldrich), SiliaMetS TM TAAcOH (SiliCycle)} , SiliaBond TM carboxylic acid (SiliCycle) carboxylic acid-modified silica gel such as; Disodium ethyl / butyl phosphonate silica (Aldrich) phosphoric acids such as Modified silica gel; 4-Ethylbenzene sulfoamide-f nctionalized silica gels (Aldrich) sulfonamide modified silica gel and the like.

Using an insoluble carrier other than silica gel, (Bio-Rad) as the polymeric carrier modified with functional groups PKa2～9, for example, a carboxylic acid modified porous polymeric carrier MacroPrep ^TM CM, CM Sepharose ^TM Fast Flow (GE Healthcare Co., ^Ltd.), (manufactured by Pall Technologies, ^Inc.) Ceramic HyperD TM CM, include Toyopearl TM CM-650S (Tosoh Co., Ltd.).

  The form of the insoluble carrier used in the present invention may be any form such as spherical, crushed, monolith, fibrous and the like. The particle size, pore size and specific surface area may be selected depending on the application, but those having an average particle size of 3 μm to 1 mm and a pore size of 30 Å to 5000 Å can be used. For general use, those having a pore diameter of 60 Å to 150 Å are usually used. The average particle size is usually 5 μm to 10 μm for high performance liquid chromatography, and 50 μm to 100 μm for column chromatography.

  The carboxyl group density of the carboxylic acid modified silica gel used in the present invention is usually 0.2 to 2 mmol / g, preferably 0.6 to 1.5 mmol / g.

  The silver ion is supported on the carboxylic acid-modified silica gel by using an aqueous solvent such as water or a solvent in which one or more of ethanol, methanol, propanol, acetonitrile and dioxane are mixed with water, ammonium acetate, ammonium carbonate, etc. After conversion of the carboxylic acid in the carboxylic acid-modified silica gel into an ammonium form or a sodium form, followed by contacting with a silver compound solution.

  The silver compound is not particularly limited as long as it is water-soluble and anions in the compound can be easily removed after supporting silver, and for example, silver nitrate, silver chlorate, silver nitrite and the like can be used, but it is particularly stable Silver nitrate, which has high character and high water solubility, is preferred.

  From the viewpoint of preventing elution of silver ions by a solvent, particularly an aqueous solvent or buffer, it is preferable to combine a carboxylic acid-modified silica gel and a thiol or thiourea-modified silica gel in a two-layer structure.

The thiol-modified silica gel, for example, 3-mercaptopropyl silica gel (Biotage ISOLUTE ^TM Si-Thiol and Tokyo Kasei 3-Mercaptopropyl silica gel), 2,4,6- trimercaptotriazine silica gel (Biotage ISOLUTE ^TM Si-TMT) and the like.

The thiourea-modified silica gel, for ^example, SillaMetS TM Thiourea (SilliCycle) is.

  In the case where the mixing of silver ions is not a problem, or in the case where a buffer or the like is not used in a nonpolar solvent, it is not necessary to use a silica gel modified with thiol or thiourea.

In the case of mass spectrometric analysis after desalting treatment using a reverse phase carrier, such as a peptide mixture obtained by decomposing cell extract and the like with a protease, the step of concentration purification of the alkinated peptide and thereafter desalting reverse phase carriers performed by a spin column, for suppressing the loss of the sample due to adsorption, silver loaded support (for example, MacroPrep ^TM CM manufactured by Bio-Rad) and reverse-phase carrier (e.g., GL it is preferable to combine Science Co. MonoSpin ^TM C18) and a two-layer structure.

  The alkyne-containing molecules targeted by the present invention are terminal alkyne-containing molecules in a liquid. Thus, internal alkyne-containing molecules that do not form acetylides are excluded from the subject matter of the present invention.

  Sulfonic acid-modified silica gel (pKa: about -3 to -2) is strongly negatively charged and has a strong cation exchange action, so a basic compound that is likely to be positively charged is strongly adsorbed and can not be eluted. The adsorbent of the present invention using an insoluble carrier modified with 9 functional groups such as a carboxyl group (carboxylic acid group), a phosphoric acid group or a sulfonamide group is weak in cation exchange activity and low in acidity, so that it is basic Compounds applicable to, for example, amines and guanidines, amino acid derivatives (for example, amino acids having alkynes, amino acids having alkynes, basic groups such as guanidine and imidazole and / or one or more polar groups described later), It is also suitable for application to samples of biological origin such as lipid metabolites, water-soluble metabolites, complex lipids such as peptides, phospholipids and glycolipids

  The alkyne-containing molecule to which the present invention is applied is not particularly limited, and in addition to the above-mentioned basic compounds (eg, amines, guanidines), carbonyl group, carboxyl group, hydroxyl group, cyano group, nitro group, sulfonamide It can apply also to the compound which has polar groups, such as a group.

  The solvent in the liquid containing the terminal alkyne-containing molecule is not particularly limited, and is not limited to the organic solvent, and an aqueous solvent such as water or one or more of ethanol, methanol, propanol, acetonitrile and dioxane mixed with water Any solvent may be used, and a mixed solvent of water and a water immiscible organic solvent can also be used.

  The present invention is not particularly problematic as long as the functional group present in the object is highly tolerant of the alkyne-containing molecules and contaminants, and is a functional group other than the thiol group that strongly bonds to metals, and the alkyne-containing molecules or contaminants The present invention is also applicable to the case where the substance has an acidic functional group such as a carboxyl group, a basic functional group such as an amine and guanidines, and a thioether group. It is applicable by performing beforehand the alkylation process by an iodoacetamide etc. also with respect to the sample containing thiol groups, such as a peptide.

  However, it is preferable to avoid acid conditions (pH <5) when the liquid containing the terminal alkyne-containing molecule contains a halide ion.

  The invention also relates to an apparatus or device for the concentration purification of terminal alkyne-containing molecules using the adsorbent of the invention. The device or device of the present invention is not particularly limited as long as it is a device or device for concentrating and purifying terminal alkyne-containing molecules, and examples include a solid phase extraction column, a kit, a filter, a chip and the like. One example of the device of the present invention is a chip in which the layer containing the adsorbent of the present invention is separably or non-removably fixed inside. The chip has a bottom that holds the liquid sample that has passed through the layer. The chip may be of a size and shape that can be used as it is as a chip for a centrifuge.

  An outline of a method for producing a silver-supported solid phase extraction column of the present invention and a method for concentrating and purifying an alkyne-containing molecule using the silver-supported solid phase extraction column is shown in FIG.

  For example, a small amount of cotton wool, glass wool, a filter, etc. is packed in a narrow part of columns such as glass Pasteur pipette, plastic syringe tip, spin column etc., a solvent (eg methanol) is added, and thiourea is added there A suspension of modified silica gel in a solvent (eg, methanol) is added, a carboxylic acid modified silica gel is similarly added, silica gel is stacked in two layers, and a small amount of cotton wool, glass wool, a filter, etc. is spread on the silica gel layer, Wash with solvent (eg, methanol). Then, the aqueous solvent contains a silver compound after conversion of the carboxylic acid in the carboxylic acid-modified silica gel to an ammonium form or a sodium form by treatment with an aqueous solvent containing ammonium acetate such as ammonium acetate or ammonium carbonate or sodium hydroxide. After being brought into contact with the solution, it is washed with a solvent (eg, methanol) to prepare a silver-supported solid phase extraction column.

  In the method for concentrating and purifying an alkyne-containing molecule of the present invention, the adsorbent of the present invention is brought into contact with a liquid containing a terminal alkyne-containing molecule to capture a terminal alkyne-containing molecule by a silver acetylide forming reaction, The terminal alkyne-containing molecule can be concentrated and purified by elution with an acid or a sulfur-containing reagent.

  Sources of halide ions include chloride (eg, ammonium chloride, sodium chloride), bromide (eg, ammonium bromide, sodium bromide), iodide (eg, ammonium iodide, sodium iodide). In addition, it is also possible to elute with an acid such as trifluoroacetic acid (TFA) for a sample such as a peptide that is usually handled under acidic conditions. Furthermore, when the above-mentioned reagent is not preferable, it is possible to elute with a sulfur-containing reagent such as sodium sulfide, thiourea, dithiothreitol, mercaptoethanol and the like.

  When ammonium chloride is used as a reagent used for elution, volatile salts are sufficient, and therefore, the eluate can be directly introduced into the mass spectrometer for measurement.

  When an aqueous solvent is used as the solvent, both capture and elution of the alkyne-containing molecule can be performed under neutral conditions such as in an ammonium acetate buffer or an ammonium carbonate buffer (pH 7 to 8). In addition, depending on the purpose, concentration purification of the terminal alkyne-containing molecule in the liquid becomes possible under mild conditions of room temperature and non-oxidation conditions.

  Alkyne-containing molecules eluted with ammonium acetate, ammonium carbonate, ammonium chloride, TFA and the like can be analyzed directly with an analytical instrument such as LCMS. In this method, unlike the method using click chemistry or biotin, the step of desalting or removing the excess reagent can be omitted. This point contributes to the efficient recovery of the alkyne-containing molecule.

One aspect of the device or apparatus of the present invention is an embodiment having a laminated structure, at least one layer containing the adsorbent of the present invention, and at least one layer containing a reverse phase carrier. When the side to which the sample is injected is upstream, it is preferable that the layer containing the adsorbent of the present invention is disposed more upstream than the layer containing the reverse phase carrier. According to the apparatus or device of the present embodiment, the alkyne-containing molecule in the sample is adsorbed to the adsorbent of the present invention and concentrated, and then, the concentrated alkyne-containing molecule is present more downstream in elution from the adsorbent The desalting treatment and the like can be performed by passing the layer containing the reverse phase carrier. For example, a peptide mixture obtained by degrading a cell extract with a protease (for example, trypsin) needs to be desalted before analysis such as mass spectrometry. According to the apparatus or device of the present embodiment, concentration purification of the alkinated peptide and desalting treatment required for mass analysis can be simultaneously performed, so that effective sample preparation even if the amount of sample is small Is possible. There is no particular limitation on the reverse phase carrier that can be used in combination with the adsorbent of the present invention. For example, a reverse phase carrier or the like in which the surface of a silica gel or organic polymer insoluble carrier is modified with a nonpolar group (such as a long chain alkyl group such as _C18 ) can be used. The reverse phase carrier may also be porous. An example of a device of the embodiment, the silver-supporting carrier (e.g., Bio-Rad Co. MacroPrep ^TM CM carboxyl group anion (COO ^-) silver supported carrier carrying a silver ion) and a layer containing a reversed phase carrier (e.g., GL Science Co. MonoSpin ^TM C18) is a chip having a layered structure having a layer containing therein. The liquid sample that has passed through the laminated structure can be held on the more downstream side of the laminated structure.

  EXAMPLES The present invention will be described in more detail by way of the following examples, but the scope of the present invention is not limited to the scope of the following examples.

Example 1: Example of preparation and use of a silver-supported solid phase extraction column
An example of preparation of an alkyne concentration purification column using a glass Pasteur pipette is shown (see FIG. 1).

A small amount of cotton wool was packed in the narrowed portion of a glass Pasteur pipette, and the tip was cut with a glass cutter. Methanol was added 500μL Pasteur pipette, 20 mg of thiourea modified silica gel (SiliCycle Co. SiliaMetS ^TM Thiourea) was added which was suspended in methanol 500μL there. The silica gel adhering to the wall was removed with 500 μL of methanol, and 60 mg of carboxylic acid-modified silica gel (CHROMATOREX COOH, MB100-75 / 200 manufactured by Fuji Silysia Chemical Ltd.) was similarly added, and silica gel was stacked in two layers. Similarly, the silica gel adhering to the wall was removed, and a small amount of absorbent cotton was spread on the silica gel layer and washed with 500 μL of methanol. The carboxylic acid was converted to ammonium form by adding 400 μL of 100 mM ammonium acetate-90% methanol aqueous solution. After washing with 500 μL of methanol, 200 μL of 10 mM silver nitrate-90% methanol aqueous solution was added. Finally, the alkyne concentration purification column was completed by washing with 500 μL of methanol.

  Thiourea-modified silica gel is used to prevent elution of silver ions, but it can be omitted if mixing of silver ions is not a problem or if a buffer or the like is not used in a nonpolar solvent.

  Next, the specific procedure of alkyne concentration purification is shown (refer FIG. 1).

  After washing with 500 μL of a solvent in which the sample to be used is dissolved (for lipid samples shown in the following examples, isopropanol-methanol-chloroform (4: 2: 1) using 7.5 mM ammonium acetate is used) Then, 1 mL of the sample was passed through the column, and the flow-through fraction was collected. After washing with the same solvent, 220 μL of a solvent to which 10 mM ammonium chloride is added is passed, and then 780 μL of the solvent is poured to collect 1 mL of eluted fractions. In the present example, all fractions are made 1 mL, but if it is desired to elute in a higher concentration state, the amount can be appropriately reduced.

In the Raman signal from silica gel treated with Ag and alkyne, the frequency (2120 cm ^-1 ) of the alkyne itself is not detected, and the signal is detected at almost the same frequency (2026 cm ^-1 ) as silver acetylide prepared separately. From the results, it was confirmed that the alkyne was trapped as silver acetylide.

[Example 2: Comparison of Carriers]
In order to screen a carrier suitable for alkyne concentration purification, a column was prepared in the same manner as in Example 1 using various types of silica gel etc., and alkyne-containing model compound (N-propargyldansilamide) was dissolved in 50% isopropanol aqueous solution The sample was used to examine the silver loading capacity and the concentration purification efficiency of the alkyne. The amount of model compound in each fraction is quantified by fluorescence detection on thin layer chromatography, elution of silver ions into each fraction is stained with 4-dimethylaminobenzylidenerhodanine on thin layer chromatography I checked it out. The examination results are shown in Table 1.

  The details of the solid phase carrier shown in Table 1, and the silver loading ability, the concentration purification efficiency of the alkyne, and the criteria for the buffer / polar solvent resistance are as follows.

(Solid phase carrier)
Normal silica gels: unmodified silica gel; silica gel 60 N (particle diameter 100 to 210 μm) manufactured by Kanto Chemical Co., Ltd.
C8 (octyl): octyl group modified silica gel; Wakogel ^TM 50 C 18 (broken, particle size 40-63 μm) manufactured by Wako Pure Chemical Industries, Ltd.
SO ₃ H: sulfonic acid-modified silica gel; manufactured by Fuji Silysia Chemical Ltd. _{CHROMATOREX SO 3 H, MB100-75 / 200} ( particle size 75-200Myuemu, mean pore diameter 100 Å) Cyano: cyanopropyl modified silica gel; SiliCycle Co. SiliaBond ^TM Cyano (Particle diameter 40-63 μm, average pore diameter 60 Å, specific surface area 500 m ² / g, modification density 1.38 mmol / g)
Diol: CHROMATOREX Diol manufactured by Fuji Silysia Chemical Ltd., MB 100-75 / 200 (particle diameter 75-200 μm, pore diameter 100 Å)
NH ₂ : aminopropyl modified silica gel; Kanto Chemical Co., silica gel 60 (spherical) NH ₂ (particle diameter 40-50 μm, average pore diameter 64 Å, specific surface area 730 m ² / g)
-NHCH ₂ CH ₂ NHCH ₂ CH ₂ NH ₂ : Kanto Chemical Co., Ltd. R-Cat-Sil TA (Modified density: 1.0 mmol / g)
Celite 545: Biotage ^Celite TM 545
Florisil: US Silica Co. Florisil COOH: carboxylic acid-modified silica gel; manufactured by Fuji Silysia Chemical Ltd. CHROMATOREX COOH, MB100-75 / 200 (particle size 75-200Myuemu, mean pore diameter 100 Å) Thiourea: SiliCycle Co. SiliaMetS ^TM Thiourea (particle size 40-63 μm, average pore diameter 60 Å, modified density 1.0 mmol / g)
Thiol: 3-Mercaptopropyl silica gel (0.5-0.8 mmol / g) manufactured by Tokyo Chemical Industry Co., Ltd.
Imidazole: SiliCycle Co. SiliaMetS ^TM Imidazole (particle size 40-63Myuemu, average pore diameter 60 Å, modified density 1.2 mmol / g
Piperazine: Aldrich 3- (1-Piperazino) propyl functionalized silica gel (40-75 μm, 0.8 mmol / g)
Phosphine: 2-Diphenylphosphinoethyl-functionalized silica gel (40-75 μm, 0.7 mmol / g) manufactured by Aldrich
TAAcOH (EDTA type): SilliaMetS ^TM TAAcOH manufactured by SiliCycle (particle diameter 40-63 μm, average pore diameter 60 Å, modification density 0.4 mmol / g)

(Judging standard of silver carrying capacity)
Strong: There is no clear color reaction of 4-dimethylaminobenzylidenerhodanine in the pass-through fraction when silver ions are supported.
Medium: A coloring reaction of 4-dimethylaminobenzylidenerhodanine is observed in the pass-through fraction when silver ions are supported.
Weak: A clear color development of 4-dimethylaminobenzylidenerhodanine is observed in the flow-through fraction when silver ions are supported.
Absence: Clear color development of 4-dimethylaminobenzylidenerhodanine is observed in the flow-through fraction when silver ion is supported, and no color development is observed in subsequent washing with a buffer (silver ion is not supported) If that is suspected).

(Judgment criteria of concentration purification efficiency of alkyne)
Good: Alkinated dansylamide is recovered at 5% or less in the pass-through and the wash fractions and 90% or more in the eluted fractions.
Acceptable: Alkinated dansylamide is detected at 5% or more in the pass-through and the wash fraction, but 40% or more is recovered in the eluted fraction (the alkinated dansylamide elutes when switching to the elution conditions) ing).
Impossible: Other than the above. While the alkinated dansylamide is detected in the flow through and the wash fraction, no behavior is observed that the elution starts by switching to the elution conditions.

(Judging criteria for buffer and polar solvent tolerance)
Strong: No clear color reaction of 4-dimethylaminobenzylidenerhodanine with silver ion is observed in the pass-through and wash fractions.
Medium: A color reaction of 4-dimethylaminobenzylidenerhodanine is observed in the pass-through and washed fractions.
Weak: A color reaction of 4-dimethylaminobenzylidenerhodanine is strongly observed in the pass-through and washed fractions, and silver ions are eluted rapidly.

  As a result of the investigation, it has been revealed that, in the carboxylic acid-modified silica gel, it is possible to achieve both good silver ion retention and good alkyne concentration purification efficiency. Sulfonic acid-modified silica gel also strongly retains silver ions, and concentration and purification of alkynes are also good, but it is undesirable in that it can not be used as a compound having high acidity of the carrier and being weak to acids. In addition, sulfonic acid modified silica gel is strongly negatively charged and has a strong cation exchange action. Therefore, it was found that basic compounds that tend to be positively charged are strongly adsorbed and can not be eluted. On the other hand, carboxylic acid modified silica gel is weak in cation exchange action and low in acidity, so it is suitable for application to samples of biological origin.

  It was found that carriers such as non-modified silica gel, florisil and celite had a low loading amount of silver ions, and that silver was easily eluted by polar solvents and buffers (such as ammonium acetate). Moreover, although various amine modification | denaturation silica gel has the high carrying capacity of silver ion, it became clear that the capture efficiency of an alkyne is low. Similarly, it was found that silica gel having a sulfur-containing functional group had high silver ion loading ability, but low alkyn trapping ability.

Example 3 Enrichment and Purification of Alkynated Amino Acid from Amino Acid Derivative Mixture Having Various Functional Groups
The example which concentrated-purified the alkyne containing amino acid is shown from the mixture of the amino acid derivative (Fmoc protected body) which has various functional groups (refer FIG. 2). As a sample, one dissolved at 25 μM in a 50% isopropanol-water mixed solvent containing 7.5 mM ammonium acetate is used, passed through the column for alkyne concentration purification described above, and after washing with the same solvent, 10 mM iodine Elution was performed with sodium chloride. Detection was performed using reverse phase liquid chromatography and a UV detector. In addition, the method used for elution can select suitably what does not influence subsequent analysis.

  The solution before concentration and purification, the flow-through fraction and the wash fraction from the column were analyzed, and amino acid derivatives other than propargylglycine (PrgGly) having an alkyne were observed in the flow-through and wash fraction, and the elution fraction It was confirmed that PrgGly was concentrated and purified in good yields for one minute. In particular, amino acid derivatives having functional groups such as imidazoles, thioethers, amines, and guanidines capable of coordinating to metal ions were also washed without problems, and it was shown that the functional group tolerance was high. However, as a result of further examination, it turned out that amino acid derivatives having a disulfide bond are also easily washable, but it is also evident that washing is difficult because of the very strong binding to silver ions with respect to thiols. It became. From this result, when the method of the present invention is used for a sample that may contain thiols, such as a peptide or a water-soluble metabolite, it is treated in the state of forming a disulfide bond or capped with iodoacetamide or the like. There is a need. In mass spectrometry of proteins, since such capping treatment is usually performed, it is considered that there are no major problems in applications in the field of chemical biology.

[Example 4: Application example to concentration purification of alkynated peptide]
In the above examples, it was shown that the alkyne-containing molecule can be concentrated and purified also in the presence of various functional groups that can be contained in the peptide. In this example, an example in which the alkyne-containing model peptide is concentrated and purified is shown.

  As shown in FIG. 3, a peptide derived from a tryptic digest of bovine serum albumin is used as a model peptide, a terminal alkyne structure-introduced peptide is prepared as an alkynated model peptide, and the alkynized peptide is selectively concentrated from a mixture thereof We examined whether it could be refined. Both peptides were dissolved in a 25% isopropanol / water mixed solvent containing 7.5 mM ammonium acetate at a concentration of 1 μM, passed through the solid phase extraction column packed with silver ion-supported carboxylic acid modified silica gel prepared in Example 1, After washing with a solvent, it was eluted with the same solvent containing 0.1% trifluoroacetic acid (TFA) and analyzed by liquid chromatography. As a result, as shown in FIG. 3, the peptide not containing the terminal alkyne structure was recovered in the pass-through / washed fraction, and the alkyne-containing peptide was almost quantitatively recovered in the eluted fraction. From this result, it was shown that the present invention is also applicable to peptides. In general, peptides are generally handled as a solution containing highly soluble TFA or formic acid, and elution with a similar concentration of TFA means that buffer exchange and desalting treatment can be omitted after concentration and purification. It is an advantage of being able to As described above, whether to use a halide salt or an acid for elution can be selected according to the sample.

Example 5 Enrichment and Purification of Alkinated Phospholipids from Cellular Lipid Extracts
In this example, it is shown that the method of the present invention can concentrate and purify alkynated phospholipid (Hexynoyl PE, from avanti polar lipids) from complex lipid mixtures (lipid extracts of cultured cells).

  Extraction of total cellular lipids was performed according to the method reported by Matyash et al. (J Lipid Res 49: 1137-1146, 2008). The lipid extract of the extracted cells is dissolved in a 7.5 mM ammonium acetate-isopropanol-methanol-chloroform (4: 2: 1) mixed solvent (hereinafter referred to as AmAc-IMC), and the alkinated phospholipid Hexynoyl PE is dissolved. A final concentration of 1 μM was added. One mL of this solution is passed through the alkyne concentration purification column prepared by the method of Example 1, and after washing twice with 1 mL of AmAc-IMC, AmAc-IMC (300 μL) containing 10 mM ammonium chloride and then AmAc-IMC (700 μL) And the combined 1 mL was collected as an elution fraction.

  Each fraction collected was subjected to direct mass spectrometric analysis. Specifically, 5 μL of sample was measured in negative mode with LTQ-Orbitrap XL (Thermo Fischer Scientific) using nanoSprayTip (HUMANIX). Identification of lipid species was performed using LipidXplorer software from MS / MS spectra measured by data-dependent acquisition mode (Genome Biol. 12: R8, 2011). The spectrum obtained is shown in FIG.

  Complex lipids derived from cells are mainly observed in the m / z 600-900 range, and among them, phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), etc. Lipid species with various functional groups are included (part of the main peaks indicated the identified lipid species). The fatty acid chain constituting the lipid mainly includes fatty acids having 14 to 22 carbon atoms, and the number of double bonds also mainly includes fatty acids of about 0 to 5. In the spectrum of the sample before concentration and purification, a number of lipid species are observed in addition to the peak (m / z 784.55) of the model lipid Hexynoyl PE. In the pass-through fraction in which this is passed through an alkyne-concentrated purification column, this peak is not observed because Hexynoyl PE is trapped on the column. On the other hand, in the eluted fraction, the peak of Hexynoyl PE is strongly detected, and it can be confirmed that most of the other lipid groups are removed. Thus, by using the present invention, concentration and purification of the alkinated model phospholipid can be efficiently performed even in the presence of a lipid having various functional groups.

[Example 6: Enrichment purification / analysis of alkinated choline metabolite]
In this example, there is shown an example in which intracellular metabolites of alkynated molecules are concentrated and purified using alkynes which are metabolically incorporated into the metabolites. Due to the small size of alkynes, molecules into which they are introduced are often metabolized by intracellular metabolic enzymes as well as endogenous molecules. Therefore, it is possible to administer the precursor of the metabolite into which the alkyne has been introduced to cells and the like, concentrate and purify the metabolite by this method, and analyze the structure by a method such as mass spectrometry. Here, an example of performing concentration purification of a metabolite of choline, which is a lipid precursor, is shown.

  HEK 293 cells were treated with propargyl choline bromide (300 μM) for 24 hours for lipid extraction. The extracted lipid was dissolved in AmAc-IMC solvent and passed through an alkyne concentration column to obtain a flow through fraction. The column was washed 3 times with 1 mL of the same solvent (washed fraction), and the eluted fraction was obtained by flowing AmAc-IMC (220 μl) to which 10 mM ammonium chloride was added, followed by AmAc-IMC (780 μl). The fractions obtained were measured in positive mode on a LTQ-Orbitrap XL mass spectrometer. As metabolites of propargyl choline, propargyl phosphatidyl choline and propargyl sphingomyelin are mainly expected. Therefore, to clearly show that propargyl choline-derived lipid metabolites can be concentrated and purified, targeted measurements were performed on these choline-containing lipids. Specifically, by using a precursor ion scan method (in the present mass spectrometer, it is possible by combining parent ion mapping method, PQD fragmentation, and detection with a linear ion trap), phosphatidylcholine and Selective detection of sphingomyelin was performed. The results are shown in FIG.

  The left row in FIG. 5 shows the results of selective detection of endogenous cholinelipid (detection of phosphocholine fragment of m / z 184.1), and the right row shows selective choline lipid derived from propargyl choline. It is the result of detection (detection of fragment of m / z 208.1). In the sample before concentration and purification, propargylcholine-derived choline lipids accounted for about 44% of the total choline lipids. In the pass-through fraction, only about 1% of propargyl choline lipid is detected, which indicates that the column is efficiently captured. Then, in the eluted fraction, only a little amount of an alkyne-free choline lipid is detected (about 1%), and it can be seen that about 99% of the lipid derived from propargyl choline is occupied. As described above, it was clearly shown that the metabolite derived from the alkynated precursor can be concentrated and purified by using the present invention.

[Example 7: Enrichment purification from cell enzyme digest of alkynated peptide]
In order to elucidate the mechanism of action of a physiologically active substance, it is important to clarify which protein group the physiologically active substance binds to and which site on the protein it acts upon. is there. In addition, post-translational modifications such as acetylation and lipid modification are known to play an important role in protein functions, and analysis of post-translational modifications is important for elucidating protein functions. Usually, mass spectrometry is used for identification of a binding protein group, identification of a binding site of a compound, and identification of a post-translational modification site. As a general flow, protein and peptide sequences are obtained by digesting a sample such as a cell extract with an enzyme such as trypsin to form peptide fragments, and collating mass-charge ratios and fragmentation patterns of the obtained peptide fragments with a database. Identify In such cases, complex mixtures such as cell extracts and the like are often used for such analysis, but in complex mixtures, ionization suppression and the like occur, so identification of peptide proteins and proteins in small amounts is difficult. The present invention makes it possible to selectively purify alkyne-bearing peptides from complex mixtures, and helps to solve problems such as ionization suppression. In this example, a tryptic digest of a cell extract is mixed with an alkynated peptide, and a typical experiment is performed to purify the alkynated peptide.

HEK 293 cells (3 × 10 ⁷ cells) were suspended in 50 mM HEPES-KOH buffer (pH 7.5) and sonicated under ice cooling. After disruption, centrifugation (10000 g, 5 minutes) was carried out, and the supernatant was recovered. The resulting cell extract (total protein 100 μg equivalent, 50 μL) was mixed with the alkynated model peptide (BSA 66 alk, 0.1 μg), and 50 μL of trifluoroethanol (TFE) and dithiothreitol (DTT, 10 mM) were added. After heating at 60 ° C. for 1 hour, iodoacetamide was added to 40 mM and incubated at room temperature for 1 hour. Thereafter, DTT was again added to a final concentration of 20 mM and diluted 10-fold. 1 μg of lysyl C endopeptidase was added and incubated at 37 ° C. for 12 hours. Furthermore, 1 μg of trypsin was added and incubated for 6 hours. A 1/10 volume of a 3% TFA aqueous solution was added to make it acidic, and desalted with SepPak C18 (Millipore). The solution eluted from SepPak C18 with 0.1% formic acid-65% acetonitrile solution was subjected to solvent removal using a Concentrator plus (Eppendorf) to obtain an enzymatic digest of cells.

  40 mg of carboxylic acid modified silica gel (manufactured by Fuji Silysia Chemical Ltd., CHROMATOREX COOH, MB 100-75 / 200) was taken in a microtube and suspended in 80% aqueous methanol solution containing 200 mM ammonium acetate. After centrifugation, the supernatant was removed and washed with 400 μL of methanol. 180 μL of methanol and 20 μL of 200 mM aqueous solution of silver nitrate were added and mixed, and the supernatant was removed after centrifugation. After washing again with methanol, the silver ion-supported carboxylic acid-modified silica gel was transferred to the filter unit of a spin column (Microbiospin manufactured by Bio-Rad, using a filter unit mounted on a microtube). By centrifuging, the silver ion-carried carboxylic acid-modified silica gel remained on the filter, and the solution was dropped into the lower microtube. The solution was discarded and the filter unit was transferred to another microtube. A solution of the enzyme digest prepared above in 40 mM ammonium carbonate-5% acetonitrile buffer was added to the silica gel with a micropipette, and the silica gel was suspended by repeating aspiration and discharge. The silica gel and the solution were centrifuged, and the solution was collected as a flow-through fraction. The filter unit was transferred to a new microtube and washed 4 times with the same procedure using 40 mM ammonium carbonate-5% acetonitrile buffer. After washing, it was transferred to a new microtube, and 200 μL of the above buffer containing 10 mM ammonium chloride was added to silica gel and suspended. The solution obtained by centrifugation was taken as an elution fraction. Elution was repeated twice.

  To each of the obtained fractions, an equal amount of BSA 66 peptide was added as an internal standard to each fraction, desalted with GLtip SDB (manufactured by GL Sciences Inc.), and subjected to analysis by mass spectrometry. The analysis result (whole image and enlarged view around BSA 66 alk) by the MALDI-TOF mass spectrometer (AutoflexSpeed by Bruker Daltonics) is shown in FIG. In the sample before purification with the alkyne purification column, many peaks derived from the cell extract digest were observed. Although the peak derived from the added alkinated peptide (BSA 66 alk) is observed around the mass-to-charge ratio of 1145.6, the signal-to-noise ratio was low due to the influence of ionization suppression and peak overlap. In the pass-through fraction of the alkyne purification column, no peptide derived from BSA 66 alk was observed. In the eluted fraction, the peak derived from the cell digest was significantly reduced, and the peak derived from BSA66alk was observed as the strongest peak excluding the internal standard peak. Furthermore, it was confirmed from analysis by LC-MS / MS using LTQ-Orbitrap XL (Thermo Scientific) that the alkynated peptide is relatively 100-fold more concentrated than the other peptides.

[Example 8: Concentrated purification of alkynated peptide using spin column combining silver supported carrier and reverse phase carrier]
Usually, a peptide mixture obtained by decomposing a cell extract or the like with a protease such as trypsin is subjected to desalting treatment using a reverse phase carrier and then analyzed by mass spectrometry. When handling a small amount of sample, adsorption to containers such as microtubes can be a problem as the number of processes increases. For such problems, it is effective to reduce the loss of the sample due to the adsorption by performing the step of concentration and purification of the alkynated peptide and the subsequent desalting treatment of the reverse phase carrier in one spin column. In this embodiment, an example of concentration and purification of an alkyne-containing molecule using a spin column in which a silver-supported carrier and a reverse phase carrier have a two-layer structure is shown. Also, it is shown that not only a silica gel type carrier but also a carboxylic acid-modified polymer type insoluble carrier can be used as a carrier carrying silver. As a representative experiment example, an experiment example in which the alkynated peptide was concentrated and purified from a peptide mixture obtained by trypsin-digesting a HeLa cell extract and LC-MS / MS analysis is shown.

HeLa cells (1 × 10 ⁷ cells) were suspended in TNE buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0) and sonicated under ice cooling. After disruption, centrifugation (15000 g, 20 minutes) was performed, and the supernatant was recovered. 50 μL of TFE and DTT (10 mM) were added to the obtained cell extract (equivalent to 100 μg of total protein, 50 μL) and mixed. After heating at 60 ° C. for 1 hour, iodoacetamide was added to 40 mM and mixed, and incubated for 1 hour at room temperature under light shielding. The resulting reaction solution is diluted with 100 μL of a 10 mM aqueous solution of ammonium bicarbonate (5% TFE-10 mM AmBic) containing 5% TFE, and washed beforehand with an aqueous solution containing 400 μL of 20% methanol Amicon Ultra-0.5 10 K filter It was transferred to a unit (manufactured by Millipore) and centrifuged (14000 g, 20 minutes). The filtrate was discarded, 200 μL of 5% TFE-10 mM AmBic was added to the filter unit, and centrifugation was performed under the same conditions. Again, 200 μL of 5% TFE-10 mM AmBic was added to the filter unit, and centrifugation was performed under the same conditions. Add 400 μL of 5% TFE-10 mM AmBic onto the filter, add 1 μg of Lysyl C endopeptidase and incubate at 37 ° C. for 12 hours. Furthermore, 1 μg of trypsin was added and incubated for 6 hours. The filtrate was collected by centrifugation (14000 g, 20 minutes) to obtain an enzymatic digest of the cells. To this digested product, the alkinated model peptide BSA66alk was added to a final concentration of 100 nM (400 ppm by weight ratio to the amount of digested peptide), and used for alkinated peptide concentration and purification experiments.

MonoSpin ^TM C18 centrifugation by the addition of 400μL of isopropanol reversed-phase monolithic silica gel filter (GL Science Inc.) (2000 g, 2 min), porous polymer carrier is a carboxylic acid-modified MacroPrep ^TM CM (Bio-Rad Co. ) Was added onto a 100 μL filter. After centrifugation under the same conditions, 300 μL of methanol was added and centrifuged again. 200 μL of a 90% aqueous solution of 100 mM silver nitrate in methanol was added, mixed by pipetting, and incubated for 3 minutes at room temperature. After centrifugation (1000 g, 1 min), wash twice with 300 μL of methanol (centrifugation at 1000 g for 1 min), alkyne-concentrated and purified spins in which the silver-supported solid phase carrier and the reverse phase solid phase carrier have a two-layer structure The column was prepared.

  To the prepared spin column, a peptide mixture containing 90 μL of alkinated peptide was added, mixed by repeated inhalation and discharge, and centrifuged (300 g, 3 minutes). After that, the plate was washed 6 times with 400 μL of 10 mM ammonium bicarbonate in 50% isopropanol aqueous solution (the solution was added to 1000 g, centrifugation for 2 minutes), followed by washing 6 times with 400 μL of 10 mM ammonium bicarbonate 5% isopropanol aqueous solution. Subsequently, the plate was washed twice with 400 μL of 80% aqueous solution of 10 mM ammonium bicarbonate in isopropanol and once with 400 μL of 5% aqueous solution of 10 mM ammonium bicarbonate in isopropanol. Next, 400 μL of a 5% aqueous solution of 10 mM ammonium bicarbonate in 50 mM ammonium chloride was passed through the column three times (500 g for 3 minutes) and washed once with 400 μL of a 5% aqueous solution of isopropanol. Finally, 50 μL of 80% aqueous acetonitrile solution containing 0.1% formic acid and 0.0017% dodecyl maltoside was added, and the solution obtained by centrifugation (5000 g, 2 minutes) was recovered. 0.0045 pmol / μL of BSA66 peptide as an internal standard peptide was added to this solution, the solvent was removed by a centrifugal evaporator, and then dissolved in a 5% aqueous acetonitrile solution containing 0.1% trifluoroacetic acid, reverse phase liquid chromatography-tandem mass It was subjected to analysis by analytical method (LC-MS / MS).

  The liquid chromatography was carried out using Ultimate 3000 (manufactured by Dionex), TipColumn (manufactured by Nikkei Technos) as a column and an ionization part, and LTQ-Orbitrap XL (manufactured by ThermoFisher) as a mass spectrometer. Measurement is performed in data-dependent acquisition mode, and peptide identification is performed using SearchGUI and PeptideShaker software. It was done using Tandem and MS-GF + algorithm. In addition, quantification of each identified peptide fragment was performed using Dinosaur software.

  FIG. 7 shows the results of LC-MS / MS analysis of the sample prior to treatment with the alkyne concentration purification column and the sample subjected to the above alkyne concentration purification treatment. In the sample before the alkyne concentration purification procedure, the added alkinated peptide BSA66alk only accounted for only 0.38% of the ion amount, but after concentration and purification, it was concentrated and purified to 86% of the identified peptide ions. And was able to concentrate the alkinated peptide with a concentration of 226-fold. In addition, it was confirmed that the recovery rate of the BSA 66 alk peptide was as high as 77%.

  In the field of chemical biology / life science research, there is a need for a technology for concentrating and purifying alkynes. At present, a method of introducing biotin into an alkyne by a copper-catalyzed azide-alkyne cycloaddition reaction and purifying it using its biotin tag has become a standard method. Kits for such purposes are commercially available and widely used. The present invention enables concentration and purification of alkyne-containing molecules in a shorter step, and is applicable to both aqueous solution systems and organic solvent systems, and is useful. It may also be used in the purification of alkyne-containing compounds in the organic synthesis of pharmaceuticals and fine chemicals.

Claims (10)

  An adsorbent for concentration and purification of terminal alkyne-containing molecules in a liquid, containing an insoluble carrier in which silver ions are supported via a functional group of pKa 2-9.   The adsorbent according to claim 1, wherein the functional group having a pKa of 2 to 9 is a carboxyl group, a phosphoric acid group or a sulfonamide group.   The adsorbent according to claim 1, wherein the insoluble carrier is a silica gel type carrier or an organic polymer type carrier on which silver ions are supported via a carboxyl group.   An apparatus or apparatus for concentration purification of terminal alkyne-containing molecules using the adsorbent according to any one of claims 1 to 3.   5. The apparatus or device according to claim 4, wherein the silica gel supported by silver ions via a carboxyl group and the silica gel modified with thiol or thiourea are used in a two-layer structure. The apparatus or device according to claim 4 or 5, comprising a plurality of layers, at least one layer containing the adsorbent according to any one of claims 1 to 3 and at least one layer containing a reverse phase carrier.
  The device or device according to any one of claims 4 to 6, wherein the device or device is a solid phase extraction column, a kit, a filter or a tip.   A step of bringing a liquid containing a terminal alkyne-containing molecule into contact with the adsorbent according to any one of claims 1 to 3 to capture the terminal alkyne-containing molecule, and eluting with a halide ion, an acid or a sulfur-containing reagent A method for concentrating and purifying terminal alkyne-containing molecules, comprising the steps of   A method according to claim 8, wherein the device or device according to any one of claims 4 to 7 is used.   The method according to claim 8 or 9, wherein the solvent in the liquid containing the terminal alkyne-containing molecule is an organic solvent or an aqueous solvent.

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