JP2018145151A - Multifunctional type peptide probe, and selective concentration method of target molecule using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide probes that have no defect/problem possessed by conventional probes and are easy to use for biomolecules.SOLUTION: The inventors of this application studied intensively and the present invention has been completed by finding that a peptide probe having a particular sequence/structure (e.g., CHO substituted C-G-E-N-L-Y-F-Q-G-K-G-X, where X is L-propargyl glycine) increases hydrophilicity to enable the reaction with biomolecules in physiological conditions, and increases options of cutting means at the time of separation and recovery of a target molecule from a resin carrier to enable the cutting means to be selected in consideration of the characteristic of the target molecule, and thereby the separation and recovery of the target molecule from the resin carrier in high yield become possible.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a multifunctional peptide probe mainly used in the fields of proteomics, metabolomics, and glycomics, and a method for selectively enriching target molecules using the same.

As a method of concentrating the target molecule, an azide (N ₃ ) group is introduced into the target molecule, and the probe having the alkyne and the azide group are covalently bonded by a click reaction (cyclization reaction using copper ions as a catalyst). A method of collecting is known (Non-patent Document 1). The probe used here needs to be able to bind to and separate from the resin.

  The feature of the invention described in Non-Patent Document 1 is that 3-ethynylbenzaldehyde is used as a probe. The alkyne group forms a covalent bond with an azide group by a click reaction, and the aldehyde group is shared with a resin having a hydrazine group. The binding and the complex of the probe and the target molecule can be separated and recovered from the resin by treatment with hydroxyamine.

Nishikaze T, Kawabata S, Iwamoto S, Tanaka K .: Reversible hydrazide chemistry-based enrichment for O-GlcNAc-modified peptides and glycopeptides having non-reducing GlcNAc residues. Analyst. 2013 Dec 7; 138 (23): 7224-32.

  The disadvantages and problems of the method described in Non-Patent Document 1 are that 3-ethynylbenzaldehyde has low hydrophilicity and is not easily reacted with biomolecules under physiological conditions, and the means for separating from a resin is hydroxyamine. Since the reaction is limited to the reaction, side reaction occurs and the recovery rate is not good. An object of the present invention is to provide a probe that does not have these problems and is easy to use for biomolecules.

  As a result of earnest research, the inventors of the present application have improved the hydrophilicity by using a peptide probe having a specific sequence / structure, and can react with a biomolecule under physiological conditions, and the target molecule can be removed from a resin carrier. Increasing the choice of cleaving means for separation and recovery, making it possible to select cleaving means in consideration of the characteristics of the target molecule, and found that the target molecule can be separated and recovered from the resin in high yield, completing the following invention did.

(1) A peptide probe having a structure represented by any of the following formulas (I) to (III).
Xa-Xn ₁ -Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₂ -Lys-Xb-Xn ₃ -Xc ... Formula (I)
Xa-Xn ₁ -Lys-Xb-Xn ₂ -Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₃ -Xc ... Formula (II)
Xa-Xn ₁ -Lys-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₂ -Xc ... Formula (III)
(Where
Xn ₁ is n ₁ amino acids (n ₁ = 0 to 10),
Xn ₂ is n ₂ amino acids (n ₂ = 0 to 10),
Xn ₃ is n ₃ amino acids (n ₃ = 0 to 10),
Xa is an amino acid substituted or unsubstituted with a formyl group,
Xb is an amino acid other than proline,
Xc is an amino acid, azide group or -R-azide group (R is a linear or branched alkylene group having 1 to 10 carbon atoms) substituted with a linear or branched alkynyl group having 2 to 10 carbon atoms. It is a substituted amino acid or a specific binding molecule. )
(2) The peptide probe according to (1), wherein the specific binding molecule is avidin, streptavidin, deglycosylated avidin, or biotin.
(3) The peptide probe according to (1), wherein in formulas (I) to (III), Xc is an amino acid substituted with the alkynyl group.
(4) The peptide according to any one of (1) to (3), wherein the alkynyl group is an ethynyl group, a propynyl group, a linear or branched butynyl group, or a linear or branched pentynyl group. probe.
(5) The peptide probe according to any one of (1) to (4), wherein in formulas (I) to (III), n ₂ and n ₃ are 0.
(6) The peptide probe according to any one of (1) to (5), which has a structure represented by formula (I).
(7) The peptide probe according to any one of (1) to (6), wherein Xa is an amino acid substituted with a formyl group.
(8) The structure according to (7), having a structure represented by Xa-Gly-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Lys-Gly-Xc (Xa is cysteine substituted with a formyl group) Peptide probe.
(9) The step of bringing the peptide probe according to any one of (1) to (6) into contact with a target molecule in a liquid sample and binding the target molecule to the C-terminus of the peptide probe;
Binding the N-terminus of the peptide probe onto a resin carrier;
Recovering the resin carrier by solid-liquid separation and washing the resin carrier;
Selective target molecule comprising the step of separating and recovering the target molecule from the resin by digesting the resin carrier-peptide probe-target molecule complex with TEV protease, trypsin or lysyl endopeptidase to cleave the peptide probe Concentration method.
(10) A step of bringing the peptide probe according to (7) or (8) into contact with a target molecule in a liquid sample, and binding the target molecule to the C-terminus of the peptide probe;
Binding the N-terminus of the peptide probe onto a resin carrier;
Recovering the resin carrier by solid-liquid separation and washing the resin carrier;
By digesting the resin carrier-peptide probe-target molecule complex with TEV protease, trypsin or lysyl endopeptidase to cleave the peptide probe, or by reacting with hydroxyamine to dissociate the bond between the resin carrier and the peptide probe A method for selectively concentrating target molecules, comprising the step of separating and recovering the target molecules from the resin.
(11) The peptide probe is an amino acid in which Xc is substituted with a linear or branched alkynyl group having 2 to 10 carbon atoms, the target molecule is a molecule having OGlcNAc, and an azide group is added to OGlcNAc of the target molecule. The method according to (9) or (10), wherein after the introduction, the click reaction is allowed to proceed under a copper catalyst to bind the peptide probe and the target molecule.

  The present invention provides a means for recovering a desired target molecule such as a biomolecule with high selectivity and high yield under physiological conditions. The peptide probe of the present invention has very high specificity for the target molecule, and can separate and recover the target molecule in the liquid sample with high yield. Moreover, since the peptide probe of the present invention is a multi-functional type and there are a plurality of cleavage means for separating and recovering the target molecule from the resin after capturing the target molecule on the resin carrier, the peptide probe may be more suitable depending on the characteristics of the target molecule. A good cutting means can be selected.

It is a figure explaining the manufacturing procedure of the peptide probe of this invention.

  In the present invention, the term “amino acid” typically refers to the 20 common amino acids (alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine found in natural proteins. , Threonine, tryptophan, tyrosine, valine, aspartic acid, glutamic acid, arginine, histidine, lysine).

  A specific binding molecule is a molecule that specifically binds to a specific substance and includes both binding partners. Specific examples include antibodies and their corresponding antigens, aptamers and their target substances, avidin-biotin-based molecules (avidin, streptavidin, deglycosylated avidin, biotin, etc.), nucleic acid binding domains such as zinc fingers and their target nucleic acid sequences, etc. Can be mentioned.

The multifunctional peptide probe of the present invention has a structure represented by any of the following formulas (I) to (III).
Xa-Xn ₁ -Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₂ -Lys-Xb-Xn ₃ -Xc ... Formula (I)
Xa-Xn ₁ -Lys-Xb-Xn ₂ -Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₃ -Xc ... Formula (II)
Xa-Xn ₁ -Lys-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₂ -Xc ... Formula (III)

Where
Xn ₁ is n ₁ amino acids (n ₁ = 0 to 10),
Xn ₂ is n ₂ amino acids (n ₂ = 0 to 10),
Xn ₃ is n ₃ amino acids (n ₃ = 0 to 10),
Xa is an amino acid substituted or unsubstituted with a formyl group,
Xb is an amino acid other than proline,
Xc is an amino acid substituted with a linear or branched alkynyl group having 2 to 10 carbon atoms (hereinafter sometimes referred to as “alkynyl group-substituted amino acid”), azide group or —R-azide group (R is carbon number) It is an amino acid substituted with 1 to 10 linear or branched alkylene groups (hereinafter sometimes referred to as “azido group-substituted amino acid”) or a specific binding molecule.

  In the peptide sequences of formulas (I) to (III), Glu-Asn-Leu-Tyr-Phe-Gln-Gly is a recognition sequence for TEV protease, which is a protease possessed by tobacco mosaic virus. Can be cut with. Lys-Xb on the C-terminal side in formula (I) and on the N-terminal side in formula (III) is provided as a cleavage site by Lys-C peptidase, and can also be cleaved by trypsin. In the formula (III), N-terminal Lys-Glu can be cleaved with Lys-C peptidase or trypsin. Xa is a residue mainly used for binding to the resin carrier. Xc is a structural part for capturing a target molecule.

The number of amino acids Xn ₁ , Xn ₂ , and Xn ₃ is not particularly limited, but a small number is preferable from the viewpoint of synthesis cost and the like. Accordingly, n ₁ , n ₂ and n ₃ are each 0 to 10, preferably 0 to 7, more preferably 0 to 5, and further preferably 0 to 3, for example 1 or 0. For example, n ₁ can be 1, n ₂ and n ₃ can be 0.

  The amino acid of Xa is not particularly limited. The amino acid may be substituted with a formyl group or an amino acid not substituted with a formyl group. In the case of an amino acid substituted with a formyl group, it is sufficient that at least one, for example, one formyl group is introduced into the amino acid molecule. The side chain functional group may be converted to a formyl group, or any hydrogen atom in the molecule may be replaced by a formyl group. Further, structures other than the formyl group may be introduced into the N-terminal residue together with the formyl group.

  Xb is an amino acid other than proline so that the Lys-Xb part can be cleaved by Lys-C peptidase.

  In the alkynyl group-substituted amino acid in Xc, the alkynyl group preferably has 2 to 7 carbon atoms, more preferably 2 to 5 carbon atoms. Moreover, as an alkynyl group, a linear thing is preferable. Specific examples of the alkynyl group include ethynyl group, propynyl group (also referred to as propargyl group), linear or branched, preferably linear butynyl group, or linear or branched, preferably linear pentynyl group. It can mention, but it is not limited to these. The amino acid may be any amino acid.

  In the azide group-substituted amino acid of Xc, it is generally preferred that the carbon number of R in the -R-azido group is small, for example, the carbon number is preferably 1-7, more preferably 1-5, and still more preferably 1- 3. The amino acid may be any amino acid.

  The specific binding molecule of Xc is preferably an avidin-biotin-based molecule such as avidin, streptavidin, deglycosylated avidin, and biotin.

  Xc is preferably an alkynyl group-substituted amino acid.

Among formulas (I) to (III), a peptide probe having the structure of formula (I) can be preferably used. Particularly preferred peptide probes among the formula (I) include peptide probes having the following structure.
Xa-Gly-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Lys-Gly-Xc ・ ・ ・ Formula (I ')
(In Formula (I ′), Xa is cysteine substituted with a formyl group, and Xc is an amino acid substituted with the alkynyl group)

In formula (I ′), in formula (I), Xn ₁ is Gly, Xn ₂ and Xn ₃ are not present (n ₂ and n ₃ are 0), Xb is glycine, Xc is the alkynyl group. It is a peptide probe structure which is a substituted amino acid. Preferred examples of the alkynyl group-substituted amino acid of Xc are the same as in formula (I). As one of the preferred specific examples of such peptide probes, CHO-substituted Cys-Gly-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Lys-Gly-L-propargylglycine synthesized in the following Examples is used. (SEQ ID NO: 1) can be mentioned. The CHO-substituted Cys is cysteine substituted with a formyl group, and examples thereof include amino acid derivatives in which the —SH group of the Cys side chain is replaced with an —CHO group. Xc is not limited to propargylglycine, and preferred examples of alkynyl group-substituted amino acids of Xc include, for example, amino acids substituted with propynyl groups and glycine substituted with alkynyl groups as defined above. Can do.

  In the production of the peptide probe of the present invention, a peptide moiety is first synthesized, and a formyl group (—CHO) may be introduced into the N-terminal residue as necessary. When Xc is an alkynyl group-substituted amino acid or an azide group-substituted amino acid, these amino acids may be used as the C-terminal amino acid residue in peptide synthesis. When Xc is a specific binding molecule, the specific binding molecule may be bound to the C-terminal residue after peptide synthesis.

  The peptide moiety may be synthesized by a solid phase synthesis method. The solid-phase synthesis method itself is a well-known conventional method (for example, “Peptidichi” written by Katayama Laboratory, Graduate School of Engineering, Kyushu University, published in October 2014; “Solid-phase synthesis handbook” edited by B. Dorner, P. White. "See Merck, May 2002, etc.). First, a C-terminal amino acid whose Nα amino group or side chain is protected with a suitable protecting group is introduced onto a suitable solid phase resin, the Nα amino group is deprotected, and then the next protected amino acid is introduced. What is necessary is just to repeat deprotection of an amino group and introduction of a protected amino acid. When the amino acid at the N-terminal is introduced, the side chain is deprotected and the peptide is cleaved from the resin.

  Fmoc and Boc groups can be used to protect the Nα amino group, but in the production of the peptide probe of the present invention, the deprotection conditions are mild (deprotection with a weak base such as piperidine) provides high quality and yield. A method using the obtained Fmoc group can be preferably used. Protecting groups used for side chain protection are also well known. In the case of the Fmoc method, t-butyl alcohol-based protecting groups (such as tBu) can be used. Since the Nα group Fmoc-modified amino acid whose side chain is protected with an appropriate protecting group for use in the Fmoc method is commercially available, such a commercially available product can be preferably used.

  For the introduction of a formyl group at the N-terminus of a peptide, an appropriate method may be used depending on the type of amino acid residue at the N-terminus. When the N-terminus is Cys, the sulfhydryl group of the N-terminus Cys can be formylated in high yield using 3- (4-formylphenyl) propiolonitrile. If the N-terminal is an amino acid other than Cys, dehydration condensation of a molecule having a formyl group and a carboxylic acid (such as 4-formylbenzoic acid) in the molecule is performed on the N-terminal of the peptide before cleaving from the solid phase synthetic resin. Thus, a formyl group can be introduced at the N-terminus. In this case, a structure other than the formyl group is also introduced into the N-terminal residue, and such an amino acid derivative is also included in the term “amino acid substituted with a formyl group”.

  When Xc is a specific binding molecule, the specific binding molecule is bound to the C-terminal residue of the synthesized peptide. This step is usually performed prior to the introduction of the formyl group at the peptide N-terminus. When the specific binding molecule is a protein, both can be bound using the functional group of the C-terminal residue of the peptide and the functional group of the specific binding molecule. In the case of biotin, various reagent kits for labeling a desired molecule with biotin are commercially available, and such commercially available products can be preferably used in the present invention.

  The peptide probe of the present invention can be used for selectively separating and concentrating various desired target molecules. Although not particularly limited, it can be preferably used for selective concentration of glycoproteins / glycopeptides. According to the peptide probe of the present invention, a target glycoprotein / glycopeptide in a sample can be selectively separated and concentrated in a state suitable for analysis by liquid chromatography or mass spectrometry.

  In the method for selectively enriching a target molecule using the peptide probe of the present invention, first, the peptide probe of the present invention is brought into contact with the target molecule in a liquid sample, and the X-terminal structure is used to target to the C-terminus of the peptide probe. Bind molecules. The target molecule in the liquid sample binds directly or indirectly to the peptide probe C-terminus. Prior to this binding, the target molecule is usually modified according to the structure of the Xc portion so that the target molecule can bind to the structure of the Xc portion at the C-terminal end of the probe.

  After binding the target molecule to the C-terminal of the peptide probe, the N-terminal of the peptide probe is bonded on the resin carrier.

When a formyl group (—CHO) is present at the N terminal of the probe, the formyl group may be used to bind to the resin carrier. When a hydrazine-modified resin is used, a covalent bond (hydrazide bond) is formed between NH-NH ₂ on the resin and -CHO of the peptide probe, and the peptide probe-target molecule complex is captured on the resin carrier. The Such resins are known and commercially available products exist.

When no formyl group is present at the N-terminal of the probe, an appropriate means may be employed depending on the type of N-terminal residue. For example, when the N-terminus is cysteine, a resin having an Iodoacetyl group may be used and bonded to the resin via the SH group of cysteine. When the N-terminal is a residue other than cysteine, a resin having a carboxylic acid activated with 4-Hydroxysuccinimidyl may be used and bonded to the resin via NH _{2 of the} N-terminal residue. Both resins are known and commercially available products exist.

  After capturing the complex on the resin carrier, the resin carrier is recovered by solid-liquid separation and washed to remove unreacted molecules.

  Next, the target molecule is separated and recovered from the resin carrier by digesting the complex of the resin carrier-peptide probe-target molecule with TEV protease, trypsin or lysyl endopeptidase to cleave the peptide probe. When a probe having a formyl group at the N-terminus is used to bind to a hydrazine-modified resin by hydrazide bond, in addition to digestion with these enzymes, reaction with hydroxyamine dissociates the bond between the resin carrier and the peptide probe. It is also possible to select means for cutting out the target molecule. TEV protease, trypsin, or lysyl endopeptidase may be selected from one or more appropriate ones that do not cleave the target molecule, depending on the amino acid sequence of the target molecule. A combination of bond dissociation with hydroxyamine and enzymatic cleavage may be performed in combination. As described above, the desired target molecule in the liquid sample can be selectively concentrated and recovered.

  Hereinafter, a click reaction between alkyne and azide (copper-catalyzed azide alkyne cycloaddition reaction (CuAAC)), which is one of the preferred embodiments of the selective concentration method according to the present invention, is used. The selective enrichment that binds the peptide probe and the target molecule will be described. CuAAC has the advantage that it proceeds in water at room temperature, and even if many other molecules coexist, the reaction is not hindered and the reaction proceeds smoothly. It seems to be widely used in various fields including life science. It is becoming.

  A preferred example of the target molecule in this embodiment is a molecule having OGlcNAc (O-linked N-acetylglucosamine). OGlcNAc is a sugar that is O-glycosidically bonded to the hydroxyl groups of Ser and Thr residues in protein molecules. A molecule having OGlcNAc is digested with trypsin or the like to digest a glycoprotein having an O-linked sugar chain into a peptide, and one residue of OGlcNAc (one residue of fucose is bound to this OGlcNAc). It may be a glycopeptide obtained by cleaving the sugar chain, leaving After the glycoprotein is fragmented into peptides, it is reacted with endoglycosidases such as Endo F1, Endo F2, Endo F3, Endo M, Endo H, etc., so that one residue of OGlcNAc (or one residue of OGlcNAc + 1 residue) The sugar chain can be cleaved leaving fucose. When this selective concentration method is performed as a pretreatment for liquid chromatography or mass spectrometry, the molecule having OGlcNAc is typically such an OGlcNAc-binding peptide.

  In order to use the click reaction, it is necessary to use a peptide probe having a C≡C bond in the Xc portion, that is, one in which Xc is an alkynyl group-substituted amino acid, and an azide group on the target molecule side. . If there is an OGlcNAc terminus on the protein / peptide, GalNAz (N-galactosamine azide) can be bound to OGlcNAc by the GalT (Y289L) enzyme (β-1,4-galactosyltransferase). An azido group can be introduced.

  After introducing an azide group into OGlcNAc of the target molecule, a click reaction (CuAAC) is caused under a copper catalyst to covalently bond the alkynyl group at the C-terminal of the peptide probe and the azide group of the target molecule. The subsequent steps such as binding to the resin carrier, solid-liquid separation, and cleavage of the peptide probe may be performed as described above. When the liquid sample containing the target molecule is a sample prepared by trypsinizing an O-linked glycoprotein as described above, the target molecule has already been subjected to trypsin digestion, so trypsin is also used for cleavage of the peptide probe. Can be used without inconvenience.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Synthesis of peptide probes
CHO-substituted Cys-Gly-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Lys-Gly-X (X = L-propargylglycine) (SEQ ID NO: 1) was synthesized by the following procedure (FIG. 1). .
(1) Swell resin Wang resin (hydroxy resin) with dichloromethane overnight. N-Fmocylated L-propargylglycine and dicyclohexylcarbodiimide (DCC), N, N-dimethyl-4-aminopyridine (DMAP) dissolved in dimethylformamide (DMF), added to the swollen resin and added at 4 ° C for 30 minutes By stirring, N-Fmoc-L-propargylglycine is introduced onto the resin. Unreacted hydroxy groups are benzoylated with benzoic anhydride. Wash the resin with DMF or the like.
(2) Add DMF solution of piperidine and shake at room temperature for 15 minutes to deprotect Fmoc. The resin is washed with DMF.
(3) N-Fmoc-Gly is dissolved in a condensing agent cocktail (*) and added to L-propargylglycine-Wang resin together with diisopropylethylamine (DIEA). Shake at room temperature for 15 minutes to obtain N-Fmoc-Gly-L-propargylglycine-Wang resin. Unreacted amino groups are acetylated with acetic anhydride.
(*) Condensation agent cocktail: 1,1,3,3-tetramethyluronium hexafluorophosphate (HTBU) and 1-hydroxy-1H-benzotriazole dissolved in DMF.
(4) Add DMF solution of piperidine and shake for 15 minutes at room temperature to deprotect Fmoc. The resin is washed with DMF.
(5) Dissolve ^α N-Fmoc and ^ε N-Boc-Lys in a condensing agent cocktail and add it together with DIEA to Gly-L-propargylglycine-Wang resin. Shake at room temperature for 15 minutes to obtain ^α N-Fmoc, ^ε N-Boc-Lys-Gly-L-propargylglycine-Wang resin. Unreacted amino groups are acetylated with acetic anhydride.
(6) Similarly, Fmoc-Gln (Trt), Fmoc-Phe, Fmoc-Tyr (PO (Obzl) OH), Fmoc-Leu, Fmoc-Asn (Trt), Fmoc-Glu (OtBu), Fomc-Gly, Fmoc-Cys (Trt) -Peptide-extending peptide, Fmoc-Cys (Trt) -Gly-Glu (OtBu) -Asn (Trt) -Leu-Tyr (PO (Obzl) OH) -Phe-Gln (Trt)- Gly- ^ε N-Boc-Lys--L-propargylglycine-Wang resin.
(7) Add DMF solution of piperidine to de-Fmoc, Cys (Trt) -Gly-Glu (OtBu) -Asn (Trt) -Leu-Tyr (PO (Obzl) OH) -Phe-Gln (Trt)- Gly- ^ε N-Boc-Lys--L-propargylglycine-Wang resin.
(8) Add TFA to deprotect and deresin the side chain.
(9) Formylate N-terminal Cys sulfhydryl using 3- (4-formylphenyl) propiolonitrile.

Peptide probes of other sequences were synthesized in the same procedure (SEQ ID NOs: 2 to 9, X = L-propargylglycine). For peptides whose N-terminus is not Cys, dehydration condensation of a molecule having a formyl group and a carboxylic acid (for example, 4-formylbenzoic acid) in the molecule to the N-terminal residue before deprotection and deresining of the side chain Thus, a formyl group can be introduced at the N-terminal of the peptide. It was confirmed that all synthesized probes were correctly synthesized by measuring using MALDI-TOF-MS (autoflex, Bruker).
CHO-substituted CGENLYFQGKGX (SEQ ID NO: 1)
CHO-substituted GENLYFQX (SEQ ID NO: 2)
CHO substitution GENLYFQGX (SEQ ID NO: 3)
CHO-substituted CGENLYFQGKX (SEQ ID NO: 4)
CHO-substituted CGENLYFQGX (SEQ ID NO: 5)
CHO-substituted CGENLYFQX (SEQ ID NO: 6)
CHO-substituted CRRRR-Pentynoic acid (SEQ ID NO: 7)
CHO-substituted CGGKG-Pentynoic acid (SEQ ID NO: 8)
CHO-substituted CGGK-Pentynoic acid (SEQ ID NO: 9)

2. Selective enrichment of OGlcNAc-binding peptides using peptide probes
OGlcNAc peptide preparation (H-Thr-Ala-Pro-Thr- (O-GlcNAc) Ser-Thr-Ile-Ala-Pro-Gly-OH) was purchased from Thermo. Add this 1 pmol OGlcNAc peptide preparation to 100 pmol bovine albumin trypsin digest and selectively azide label the OGlcNAc peptide preparation using Thermo's Click-IT® O-GlcNAc Enzymatic Labeling System did. Excess reagent was removed with a reverse phase column. To this, 45 μL of t-butyl alcohol / water 1: 1 containing 2.5 μg of CHO-substituted CGENLYFQGKGX (SEQ ID NO: 1) was added to dissolve the azide-labeled O-GlcNAc peptide preparation. To this, tris [(1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl] amine, which is a monovalent copper chelating material, and copper sulfate were added to 500 nM and 100 nM, respectively. The azide-alkyne Huisgen cycloaddition (click reaction) was started by adding 5 μL of 100 mM ascorbic acid to reduce divalent copper to produce monovalent copper. After 4 hours, 1 mg of benzenesulfonyl azide functionalized silica gel was added to the excess CHO-substituted CGENLYFQGKGX and adsorbed on the resin. The supernatant containing the azide-labeled OGlcNAc peptide preparation is mixed with 50 μL of hydrazide resin, and left at room temperature for 4 hours. Fixed. The resin was washed 3 times with 1 mL of distilled water to remove bovine albumin trypsin digest, then treated with 50 μL 50 mM ammonium bicarbonate containing 10 ng trypsin / LysC Mix (Promega) (37 degrees Celsius, The target OGlcNAc peptide preparation was cut out. It was confirmed by nanoLC / MS / MS measurement that the target OGlcNAc peptide preparation could be purified from digested bovine albumin trypsin.

Claims (11)

A peptide probe having a structure represented by any of the following formulas (I) to (III).
Xa-Xn ₁ -Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₂ -Lys-Xb-Xn ₃ -Xc ... Formula (I)
Xa-Xn ₁ -Lys-Xb-Xn ₂ -Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₃ -Xc ... Formula (II)
Xa-Xn ₁ -Lys-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Xn ₂ -Xc ... Formula (III)
(Where
Xn ₁ is n ₁ amino acids (n ₁ = 0 to 10),
Xn ₂ is n ₂ amino acids (n ₂ = 0 to 10),
Xn ₃ is n ₃ amino acids (n ₃ = 0 to 10),
Xa is an amino acid substituted or unsubstituted with a formyl group,
Xb is an amino acid other than proline,
Xc is an amino acid, azide group or -R-azide group (R is a linear or branched alkylene group having 1 to 10 carbon atoms) substituted with a linear or branched alkynyl group having 2 to 10 carbon atoms. It is a substituted amino acid or a specific binding molecule. )   The peptide probe according to claim 1, wherein the specific binding molecule is avidin, streptavidin, deglycosylated avidin, or biotin.   The peptide probe according to claim 1, wherein in formulas (I) to (III), Xc is an amino acid substituted with the alkynyl group.   The peptide probe according to any one of claims 1 to 3, wherein the alkynyl group is an ethynyl group, a propynyl group, a linear or branched butynyl group, or a linear or branched pentynyl group. In the formula (I) ~ (III), n 2 and n ₃ are 0, peptide probe according to any one of claims 1 to 4.   The peptide probe according to any one of claims 1 to 5, which has a structure represented by the formula (I).   The peptide probe according to any one of claims 1 to 6, wherein Xa is an amino acid substituted with a formyl group.   The peptide probe according to claim 7, which has a structure represented by Xa-Gly-Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Lys-Gly-Xc (Xa is cysteine substituted with a formyl group). Contacting the peptide probe according to any one of claims 1 to 6 with a target molecule in a liquid sample, and binding the target molecule to the C-terminus of the peptide probe;
Binding the N-terminus of the peptide probe onto a resin carrier;
Recovering the resin carrier by solid-liquid separation and washing the resin carrier;
Selective target molecule comprising the step of separating and recovering the target molecule from the resin by digesting the resin carrier-peptide probe-target molecule complex with TEV protease, trypsin or lysyl endopeptidase to cleave the peptide probe Concentration method. Contacting the peptide probe according to claim 7 or 8 with a target molecule in a liquid sample, and binding the target molecule to the C-terminus of the peptide probe;
Binding the N-terminus of the peptide probe onto a resin carrier;
Recovering the resin carrier by solid-liquid separation and washing the resin carrier;
By digesting the resin carrier-peptide probe-target molecule complex with TEV protease, trypsin or lysyl endopeptidase to cleave the peptide probe, or by reacting with hydroxyamine to dissociate the bond between the resin carrier and the peptide probe A method for selectively concentrating target molecules, comprising the step of separating and recovering the target molecules from the resin.   The peptide probe is an amino acid in which Xc is substituted with a linear or branched alkynyl group having 2 to 10 carbon atoms, the target molecule is a molecule having OGlcNAc, and after introducing an azide group into OGlcNAc of the target molecule The method according to claim 9 or 10, wherein the click reaction proceeds under a copper catalyst to bind the peptide probe and the target molecule.

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