WO2015056992A1

WO2015056992A1 - Peptide complex for measuring kinase activity and use thereof

Info

Publication number: WO2015056992A1
Application number: PCT/KR2014/009733
Authority: WO
Inventors: 김성훈; 김병규
Original assignee: 재단법인 의약바이오컨버젼스연구단
Priority date: 2013-10-16
Filing date: 2014-10-16
Publication date: 2015-04-23
Also published as: KR20150044438A

Abstract

The present invention relates to a peptide complex for measuring kinase activity and a use thereof and, more specifically, to a peptide complex for measuring kinase activity and a use thereof, the peptide complex having, combined in order, (i) an immobilization means, (ii) 1 to 10 amino acids for regulating retention time, (iii) a phosphorylation substrate peptide, and (iv) 1 to 5 hydrophilic amino acids having positive charges. The peptide complex for measuring kinase activity, of the present invention, is capable of simultaneously analyzing activity for a plurality of kinase-related enzymes and is effective in screening drugs for regulating the activity of kinase-related enzymes. Accordingly, a method for simultaneously analyzing activity of kinase-related enzymes and screening drugs, using the peptide complex, is highly industrially applicable since the method enables mass screening of drugs and enhances the efficiency of screening by measuring phosphorylation of a target in large quantities.

Description

【Specification】

[Name of invention]

Peptide Complexes and Their Uses for Measuring Phosphorylase Activity

Technical Field

<ι> This application claims the priority of Korean Patent Application No. 2013-0123662, filed October 16, 2013, the entirety of which is a reference of the present application.

<2>

The present invention relates to a peptide complex for measuring kinase activity and a use thereof, and more particularly, to (i) immobilization means, (ii) 1 to 10 retention time amino acids (iii). Simultaneous activity of phosphatase peptides and (iv) peptide complexes for measuring phosphorylation activity in which hydrophilic amino acids having 1 to 5 positive charges are sequentially bound, and kinase-related enzymes using the peptide complex for measuring phosphorylation activity It relates to analytical methods and methods for screening drugs that modulate activity.

<4>

Background Art

Protein kinases form a large family of structurally related enzymes involved in the regulation of various signaling processes in cells. These kinases can be grouped by the substrates they phosphorylate (eg, protein-tyrosine, protein-serine / threonine, lipids, etc.).

Tyrosine kinase can be classified as growth factor receptors (eg EGFR, PDGFR, FGFR and erbB2) or non-receptors (eg c-src and bcr-abl) kinase. Receptor type tyrosine kinase consists of about 20 different subfamily. Non-receptor type tyrosine kinase consists of numerous subfamily. Such tyrosine kinase has a variety of biological activities. Receptor tyrosine kinase crosses the cell membrane and has a huge effect on cell proliferation by having extracellular binding domains for growth factors, transmembrane domains, and intracellular regions that act as kinase to phosphorylate specific tyrosine residues in proteins. It is an enzyme. Abnormal or inadequate protein kinase activity may be responsible for developing disease states associated with this abnormal kinase activity. <7> Therefore, kinase activity analysis may be an important method for the diagnosis and treatment of various diseases.

In the conventional assay for phosphatase, activity can be measured only when the phosphatase is phosphorylated, and it is difficult to analyze using antibodies, and there are disadvantages such as the use of radioactive reagents. Therefore, improved assay methods are needed to detect the presence and activity of enzyme activity, in particular cell signaling components and activators.

<9>

[Detailed Description of the Invention]

[Technical problem]

Accordingly, the inventors of the present invention prepared a peptide complex for measuring phosphorylation activity, which can be analyzed by mass spectrometry, and simultaneously analyzing the activity of kinase-related enzymes, thereby mass screening drugs that control the activity of the enzymes. The method was found and the present invention was completed.

<11>

The object of the present invention is therefore to provide (i) immobilization means, and (ii) 1 to 10 dwell times.

(Retention time) Amino acid for regulating (iii) phosphorylated substrate peptide and (iv) a peptide complex for measuring the phosphorylation activity in which the hydrophilic amino acid having 1 to 5 positive charges are sequentially linked.

<13>

[0014] Another object of the present invention is to provide a method for coating a plate comprising: (a) coating a peptide complex of claim 1 comprising different phosphorylated substrate peptides onto a plate;

(B) contacting the sample to be analyzed with the coated peptide complex; And

(C) The present invention provides a method for simultaneously analyzing the activity of a kinase-related enzyme, which comprises recovering a peptide complex in contact with a sample and analyzing the same by mass spectrometry.

<17>

Another object of the present invention (a) coating the peptide complex of claim 1 on a plate;

(B) treating the drug candidate with the cell;

(C) administering the cell lysate of the cell treated with the drug candidate substance to the plate of step (a) to contact the peptide complex;

(D) Recovering the peptide complex in contact with the cell lysate and separating it by mass spectrometry. It provides a method for screening a drug that modulates the activity of a kinase-related enzyme comprising the step of analyzing.

<22>

Technical Solution

In order to achieve the above object, the present invention (i) immobilization means, (ii) 1 to 10

Provided are temporal complexes for measuring phosphorylation activity in which amino acids (iii) phosphorylated substrate peptides for adjusting retention time of dogs and (iv) hydrophilic amino acids having 1 to 5 positive charges are sequentially linked.

<24>

In order to achieve another object of the present invention, the present invention comprises the steps of: (a) coating the peptide complex of claim 1 comprising a different phosphorylated substrate peptide to the paste;

(B) contacting the sample to be analyzed with the coated peptide complex; And

(C) The present invention provides a method for simultaneously analyzing the activity of a kinase-related enzyme, including recovering a peptide complex in contact with a sample and analyzing the same by mass spectrometry.

<28>

In order to achieve another object of the present invention, the present invention comprises the steps of (a) coating the peptide complex of claim 1 on a plate;

(B) treating the drug candidate material on the cells;

(C) administering the cell lysate of the cell treated with the drug candidate to the plate complex of step (a) to contact the peptide complex;

(D) The present invention provides a method for screening a drug that modulates the activity of a kinase-related enzyme, which includes recovering a peptide complex in contact with a cell lysate and analyzing the same by mass spectrometry.

<33>

Hereinafter, the present invention will be described in detail.

<35>

The present invention relates to a method for (0) immobilization means, (ii) 1 to 10 retention time amino acids (iii) phosphorylated substrate peptides, and (iv) hydrophilic amino acids having 1 to 5 positive charges. Provided is a peptide complex for measuring phosphorylation activity.

<37>

(I) the immobilization means comprises a peptide complex for measuring phosphorylation activity of the present invention It is a means to fix and fix the peptide complex for measuring phosphorylation activity of the present invention by fixing to a support means (polymer resin or plate, etc.), and polymer polymers, organic, inorganic compounds, and peptides having a property of being fixed to the support means. Include. The immobilization means of the present invention may preferably be biotin (biot in).

<39>

Biotin is a compound also known as vitamin H or B7.

(avidin) or streptavidin binds very strongly and specifically. Biotin can also be used as an immobilization means in the present invention because it is chemically bound or tagged to a molecule or protein.

<4i> In the present invention, the term 'biot inylat ion' or biotinylation refers to a protein

(Or peptide, polypeptide) means that the biotin is covalently bonded.

<42>

(Ii) The amino acid for controlling the retention time of 1 to 10 is to enable accurate measurement by preventing the overlapping of the measurement signal when simultaneously analyzing the activity of a number of kinase-associated enzymes, the peptide to be measured. As long as the amino acid can change the mass or charge of the amino acid, there is no particular limitation on the type thereof. Preferably, the amino acid may be one or more amino acids selected from the group consisting of a positively charged hydrophilic amino acid, a hydrophobic amino acid, and a frolin.

The positively-charged hydrophilic amino acid slows down the retention time in the analysis, and may preferably be lysine (10, arginine (R) and histidine (H). The negative charge applied to the substrate peptide is canceled to maintain positive charge ionization of the peptide to be suitable for bipolar mode based mass spectrometry.

The hydrophobic amino acid may slowly adjust the retention time in the analysis and may be preferably phenylalanine (F), leucine (L), isoleucine (1), tryptophan (W) and valine (V).

The proline is a fast control of the retention time in the analysis, and is located in the N-terminal front sequence of the phosphorylated amino acid (i.e., (Hi) phosphorylated substrate peptide) (ie, (Π) 1 to 10 identities). At the C terminus of the peptide consisting of amino acids for time regulation) to increase the production of y ions comprising phosphorylated amino acids. In this case, in relation to the generation of y ions, it may be preferable that phenylalanine (F) is located at the N terminus of proline located at the N-terminal front sequence of the phosphorylated amino acid. Also, if prine is present in the peptide, the C-horse when the peptide is cut into small fragments in the mass spectrometer Since it is mainly cut from the end to the plinin, there is an advantage that can help to check the amino acid sequence information well.

The proline may be preferably composed of 0 to 2, and the number thereof may be adjusted according to the presence or absence of proline in the phosphorylated substrate peptide sequence.

<48>

The (iii) phosphorylated substrate peptides are peptides in which phosphorylation reactions occur by kinase-related enzymes to be analyzed, but the sequence and number thereof are not particularly limited, but preferably 6 to 12 more preferably 9 It can consist of two sequences.

<50>

More preferably, the phosphorylated substrate peptide of the present invention may be a substrate for tyrosine kinase, and the sequence and the number of amino acids are not particularly limited as long as the peptide includes a tyrosine residue, for example, represented by SEQ ID NO: 10. Phosphorylation substrate peptide for EGFR, phosphorylation substrate peptide for STAT3 represented by SEQ ID NO: 11, phosphorylation substrate peptide for VGFRR3 represented by SEQ ID NO: 12, phosphorylation substrate peptide for HER2 represented by SEQ ID NO: 13, sequence Phosphorylation Substrate Peptide for PDGFR Represented by Number 14, Phosphorylation Substrate Peptide for JAK1 Represented by SEQ ID NO: 15, Phosphorylation Substrate Peptide for PI3K Represented by SEQ ID NO: 16, PDGFR Represented by SEQ ID NO: 17 Phosphorylation Substrate Peptides and Phosphorylation Substrate Peptides for FGFR1 Represented by SEQ ID NO: 18 It may be one selected from the group consisting of.

<52>

In this case, the (i i) 1 to 10 amino acid for controlling the retention time can be specifically used by those skilled in the art to selectively select the number and type of amino acids according to the peptide relative hydrophobic information of the (i i). For example, if the phosphorylated substrate peptide of (H i) between the peptide complexes of the present invention to be analyzed simultaneously shows a similar degree of relative hydrophobicity, P, at the N-terminus of the phosphorylated substrate peptide is markedly different. Combinations of various amino acids such as PFP can be attached.

In addition, the amino acids of the above (i i) is preferably made in consideration of the sequence information that is not in the existing database (target protein amino acid sequence information).

<55>

(Iv) Hydrophilic amino acids having 1 to 5 positive charges of the present invention cancel the negative charges applied to the substrate peptides by phosphorylation and are based on bipolar mode mass spectrometry. It serves to maintain the positive charge ionization of the peptide to suitably, preferably may be composed of amino acids selected from the group consisting of lysine 00, arginine (R) and histidine (H). Phosphorylated amino acids (Y, S, etc.) in each peptide have anions, and there are cations (H, K, R) that can counteract this to increase the production of y ions.

<57>

Meanwhile, one to five positively charged hydrophilic amino acids of the present invention may be substituted with isotopes. The amino acid substituted with the isotope means that a specific element constituting the amino acid is substituted with an element (isotope) having a different mass with the same atomic number but different neutron numbers.

The isotope is preferably a stable isotope without radioactive decay, and preferably has an increased mass than a general element. Isotopes of the present invention may be selected from the group consisting of, for example, ¾, ¹³ C and ¹⁵ N. As described above, when mass spectrometry is performed by adding a peptide complex including an amino acid substituted with an isotope as a control, the mass of an experimental group peptide complex not substituted with an isotope can be measured.

<60>

In the present invention, for example, (ii) the amino acid for adjusting the retention time of 1 to 10; (Hi) phosphorylated substrate peptides; And (iv) hydrophilic amino acids having 1 to 5 positive charges may be any one selected from the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 9. In this case, the peptide complex of the present invention may be specifically any one selected from the group consisting of nine peptide complexes whose N—terminals of the polypeptides represented by SEQ ID NOs: 1 to 9 are each biotinylated.

<62>

In addition, the present invention is, for example, of the polypeptide represented by SEQ ID NOS: 1 to 9, respectively

2 selected from the group consisting of EGFR, STAT3, VGFRR3, HER2, PDGFR, JAK1, PI3K, PDGFR and FGFR1, wherein the N-terminus comprises at least two peptide complexes selected from the group consisting of nine biotinylated peptide complexes It provides a composition for simultaneous analysis of the above tyrosine kinase.

In this case, the N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 1 includes a phosphorylated substrate peptide (SEQ ID NO: 10) for EGFR, and a peptide for activity analysis for EGFR. It is a complex. The polypeptide represented by SEQ ID NO: 2 The N-terminal biotinylated peptide complex of Tide contains a phosphorylated substrate peptide (SEQ ID NO: 11) for STAT3, and is a peptide complex for activity analysis for STAT3. The N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 3 includes a phosphorylated substrate peptide (SEQ ID NO: 12) for VGFRR3, and is a peptide complex for activity analysis for VGFRR3. The N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 4 includes a phosphorylated substrate peptide (SEQ ID NO: 13) for HER2, and is a peptide complex for activity analysis for HER2. The N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 5 includes a phosphorylated substrate peptide (SEQ ID NO: 14) for PDGFR, and is a peptide complex for activity analysis for PDGFR. The N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 6 includes a phosphorylated substrate peptide (SEQ ID NO: 15) for JAK1, and is a peptide complex for activity analysis for JAK1. The N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 7 includes a phosphorylated substrate peptide (SEQ ID NO: 16) for PI3K, and is a peptide complex for activity analysis for PI3K. The N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 8 includes a phosphorylated substrate peptide (SEQ ID NO: 17) for PDGFR and is a peptide complex for activity analysis for PDGFR. The N-terminal biotinylated peptide complex of the polypeptide represented by SEQ ID NO: 9 contains a phosphorylation substrate peptide (SEQ ID NO: 18) for FGFR1, and is a peptide complex for activity analysis for FGFR1.

Therefore, those skilled in the art can selectively use the type of peptide complex of the present invention according to the type of phosphorylation enzyme to be analyzed.

<66>

The peptide complex of the present invention can measure the activity of a number of kinase enzymes at one time, and it is easy to measure the phosphorylation activity by mass spectrometry by a hydrophilic amino acid having a positive charge, and includes an amino acid for controlling retention time. Thus, when measuring the activity of multiple phosphorylated enzymes simultaneously, it is possible to avoid overlap of measurement signals. In addition, the peptide complex of the present invention, which is the subject of analysis during the measurement experiment, including the immobilization means, can be easily separated. In addition, using the peptide of the present invention, the Digest i on process that has been previously performed in protein mass spectrometry can be omitted. That is, by omitting the process of making a protein into a peptide with an enzyme (mainly using t ryps in.), Errors occurring during the experiment process can be reduced. Therefore, the peptide complex of the present invention can be effectively used for screening drugs that simultaneously analyze the activity of a number of phosphorylation-related enzymes or modulate the activity of phosphatase-related enzymes.

<69>

Accordingly, the present invention provides a method for coating a peptide complex of the present invention comprising a different phosphorylated substrate peptide on a plate;

<71>^'(b) contacting the coating and the peptide complex the analyte sample; And

<73>

The kinase refers to an enzyme having an activity of adding a phosphate group to a substrate. In the present invention, the term 'kinase-related enzyme' includes both a kinase that is analyte itself, a lower enzyme that is activated by the analyte kinase to phosphorylate a substrate, and an upper enzyme that activates the analyte kinase. In the present invention, the phosphatase-related enzyme preferably refers to a protein (peptide or polypeptide) phosphorylation-related enzyme, and transfers a phosphate group from an phosphate donor such as ATP (adenosine triphosphate) to an amino acid residue of the protein. It includes both enzymes and upper and lower enzymes involved in their activity. The protein phosphorylation-related enzyme is not particularly limited as long as it is a known protein phosphorylation-related enzyme, for example phosphorylase kinase, protein kinase A, protein kinase C,

2+

Serine / threonine-specific protein kinase including Ca / calmodul in-dependent protein kinase, MAP kinase, and Mos / Raf kinase; receptor tyros ine kinase Tyrosine phosphatase (tyrosine 一 speci f ic kinase); histidine kinase (hi st idine—speci f ic kinase); aspartic acid / glutami c acid-speci fic protein kinase But may not be limited thereto.

The protein phosphorylation related enzyme of the present invention may be more preferably tyrosine phosphorylation related enzyme. Tyrosine phosphorylation related enzymes include both tyrosine kinase itself and upper or lower enzymes that modulate and affect its activity.

The tyrosine kinase can be largely divided into receptor tyrosine kinase and non-receptor tyrosine kinase (or cytoplasmic tyrosine protein kinase). As a kind of the receptor tyrosine kinase, for example RTK class I (EGF receptor family or ErbB family), including Herl (EGFR, ErbBl), Her2 (Neu, ErbB2), Her3 (ErbB3), and Her4 (ErbB4); RTK class II (Insulin receptor family); RTK class III (PDGF receptor family); RTK class IV (FGF receptor family); VEGF receptors family (RTK class V); RTK class VI (HGF receptor family); Trk receptor family (RTK class VI I); Eph receptor family (RTK class VIII); RTK class IX (AXL receptor family); LTK receptor family (RTK class X); TIE receptor family (RTK class XI); ROR receptor family (RTK class XII); DDR receptor family (RTK class XIII); RTK class XIV (RET receptor family); RTK class XV (KLG receptor family); RTK class XVI (RYK receptor family); RTK class XVII (MuS receptor family) and the like are known in the art, but are not limited thereto.

<7?> As the type of non-receptor tyrosine kinases, for example, ABL family including ABLl, ARG, etc .; ACK family including ACK1, TNK1 and the like; CSK family including CSK, MATK, etc .; FAK family, including FAK, PYK2, and the like; FES family, including FES, FER, and the like; FRK family, including FRK, BRK, SRMS, and the like; JAK family including JAKl, JAK2, JAK3, TYK2, and the like; SRC family including SRC, FGR, FYN, YESl, BLK, HCK, LCK, LYN and the like; TEC family including TEC, BMX, BTK, ITK, TXK, etc .; SYK family, including SYK, ZAP70, and the like are known in the art and are not limited thereto.

<78>

The signaling system by kinase in cells is usually delivered by kinase cascades of a number of kinase enzymes, thus measuring how phosphorylated substrates of phosphatase are The activity of kinase itself can be measured directly, and the activity of the upper and lower enzymes can be measured indirectly.

<80>

<8i> Hereinafter, a method for simultaneously analyzing the activity of a kinase-associated enzyme of the present invention will be described step by step.

<82>

(A) coating a fabtide complex of the present invention comprising different phosphorylated substrate peptides on a plate

<84> In the step (a), the peptide complex of the present invention comprising different phosphorylated substrate peptides is coated on a plate.

Peptide complexes of the present invention (i) immobilization means, (ii) 1 to 10 retention times

(retent ion t ime) refers to a peptide complex for measuring phosphorylation activity in which the amino acid (iii) phosphorylation substrate peptide for regulation and (iv) hydrophilic amino acids having 1 to 5 positive charges are sequentially linked, as described above.

<87>

In the method of the present invention, two or more phosphate substrates may be used to simultaneously measure the activity of a plurality of enzymes related to phosphatase, and for this purpose, various kinds of peptide complexes including different phosphate-based peptide rolls may be used. It can be prepared and used in combination. The phosphorylated substrate peptide may be different from or different in length from the same phosphorylated substrate peptide to be analyzed simultaneously in the present invention, but preferably, the sequence length may be identical between the phosphorylated substrate peptides to be analyzed simultaneously. In addition, the phosphorylation peptides to be analyzed simultaneously may be controlled to have the same position of phosphorylation (ie, the position in the sequence of the amino acid to be phosphorylated, whether from the c-terminus or the N-terminus).

<89>

The peptide complex of the present invention may be preferably one comprising any one of the amino acid sequences of SEQ ID NOs: 1-9.

<91>

The plate refers to a support means for performing the phosphorylation reaction, the size and the material can be selected and used appropriately by those skilled in the art, preferably the immobilization means of the peptide complex of the present invention can be combined It is preferable that the material that can be bonded or immobilized means of the peptide complex of the present invention is immobilized. The plate is not particularly limited as long as it is a plate for biological sample processing known in the art, and for example, in a typical laboratory environment, a plate made of glass, polypropylene, or polystyrene resin may be used. It is common to be. In the present invention, a plate formed of a polypropylene or polystyrene-based resin may be suitable. Methods of immobilizing materials (polymeric polymers, organic, inorganic compounds, peptides, etc.) to the plates, in particular of biotin, are well known in the art.

<93> The coating is to fix the peptide complex of the present invention on the surface of the plate, according to the kind of immobilization means included in the peptide complex of the present invention can be used by those skilled in the art to selectively change the immobilization method from known techniques The method is not particularly limited, and for example, a ¾-tide composite is prepared by using a ramtide, which is capable of binding to a polyester resin, or the like, and coated on a polystyrene plate, or a biotin is immobilized. The peptide complex may be prepared and coated on a plate coated with streptavidin.

<95>

(B) contacting the sample to be analyzed with the coated fabtide complex;

In step (b), the sample to be analyzed is contacted with the peptide complex coated on the plate. The phosphatase contained in the sample to be analyzed reacts with the peptide complex through the contact to phosphorylate the peptide complex (see Stepl of FIG. 7).

<98>

The sample to be analyzed is a sample containing the kinase to be analyzed, and the type thereof is not particularly limited, and examples thereof include, for example, cell lysate, phosphatase purification, fraction or tissue lysate, blood, It may be a bio-derived substance such as saliva, urine, lymph, plasma, and preferably may be a cell lysate.

<100>

In the step (b), the environment such as temperature, pH, and other enzymes other than kinase-related enzymes may be controlled so that the phosphatase in the sample to be analyzed may phosphorylate the peptide complex. Can be added by selecting. For example, Tr i s with protease and phosphatase inhibitors, phosphate buffered saline (PBS), or sucrose-tr iethanolamine can be used as a homogeneous buffer in the first step of sample preparation.

In addition, in step (c), a phosphate donor component such as ATP may be appropriately selected and added to supply an element (eg, a phosphate group) required for phosphorylation of the peptide complex.

<103>

In order to derive accurate experimental results with respect to the activity of phosphatase, inducing false negat ive and fal se pos it ive results for the phosphatase to be measured during the preparation of the analyte sample (particularly, cell lysate). Potential blockers (protein breakdown Any additional process may be performed to make it unaffected by enzymes, phosphatase, and the like. For example, the cell lysate can be treated with a stabilizer, and can also be treated with a solution containing guanidine or urea for protein denaturation before the enzyme is degraded.

<105>

<| 06> The stabilizer comprises or consists of at least one phosphatase inhibitor. Suitable examples are serine or threonine phosphatase (eg, PPP or PPM family). And / or inhibitors of tyrosine phosphatase (family PTP) For example, inhibitors of PP1 phosphatase include calyculin A, nodular in, NIPP-1, microcystine LR microcystin LR), tautomycin, okadaic acid, and cantharidin. Inhibitors of PP2A include kallikrein A, microcystine LR, okadiic acid, fostriecin, cantharidin, endothal 1, and nodolin. Inhibitors of PP2B include cyclosporin A), FK 506 / immunophylline complexes (FK 506 immunophi 1 in complexes), 1 "cypermethr in, deltamethrin, and fenvalerate. Inhibitors of FTP include bpV (phen), dephostatin, mpV (pic) DMHV, and sodium orthovanadate, so phosphatase inhibitors are known in the art. Commercially available compounds may also be used as stabilizers. Compounds of phosphatase inhibitors, commonly referred to as "phosphatase inhibitor cocktails" by commercial providers of such inhibitors, may also be used as stabilizers. Cocktails "are overall advantageous in that they provide stabilization over the range of proteins of interest; therefore, two or more phosphates They suppress stabilizer containing preparation is desired.

<107>

Furthermore, it may be desirable to include protease inhibitors with phosphatase inhibitors to further enhance protein stability. For example, inhibitors of proteases such as serine proteases, cysteine proteases, tyrosine proteases, aspartic proteases, metal loproteases, thiol proteases, exopeptidases, etc. Include. Among these, non-limiting examples of serine protease inhibitors include antipain aprotinin, chymostat in, elastat inal, phenylmethylsulfo Phenylmethylsulfonyl f luoride (PMSF), APMSF, TLCK, TPCK, leupeptin and soybean trypsin inhibitors. Inhibitors of chrystine protase include, for example, IAAGndoleacetic acid) and E-64. Suitable examples of aspartic proteases include pepstatin and VdLPFFVdL. Non-limiting examples of inhibitors of metalloproteinases include EDTA, as well as 1,10-phenanthroline and phosphoramodon. Inhibitors of exopeptidases include, for example, amastat in, bestatin, diprotin A, and diprotin B. Additional suitable examples of proteases include alpha-2-macroglobulin, soybean or lima bean trypsin inhibitors, pancreat ic proteases inhibitors, egg white ovastatin and egg White cystatin and the like.

<109>

(c) recovering the peptide complex in contact with the sample and analyzing it by mass spectrometry;

<ιιι> In the step (c), the peptide complex contacted with the sample is recovered (see Step 2 of FIG. 7) and analyzed by mass spectrometry.

<112>

<ιΐ3> 'Recovering' of the peptide complex means removing and obtaining the bond between the plate and the peptide complex of step (a), and the recovery method of the peptide complex is a peptide complex coated on the plate. Follows the immobilization means included. The immobilization means has been described above, and methods for removing the bond with the plate according to the characteristics of each immobilization means are known in the art. For example, in the peptide complex of the present invention, when the immobilization means is biotin, it can be separated by affinity with a substance known to bind with biotin (ex. Streptavidin).

<114>

<5> The 'analysis' of the peptide complex may be by any mass spectrometry-based method that enables high-throughput multiplexed analysis. The mass spectrometry is a highly sensitive and accurate technique for separating and specifying molecules. In general, mass spectrometers are used to measure ion sources for ion generation and tnass-to-charge ratio of ions. It has two main components called a mass-selective analyzer. The mass charge ratio of the ions is a measure of the mass of these ions, and is converted into its measurements. Several ionization methods are known in the art and are described herein. Various mass spectrometry methods, such as quadrupole mass spectrometry, ion trap mass spectrometry, and time-of-flight mass spectrometry tandem mass spectrometry Mass spectrometry can be used in various combinations with ion sources and mass analyzers, allowing flexibility in the design of detection protocols for user convenience. In addition, the mass spectrometer can be programmed to send all the ions from this source to the mass spectrometer continuously or simultaneously. The mass spectrometer may also be programmed to select a specific mass of ions to be sent to the mass spectrometer while blocking other ions. The ability to precisely control the movement of ions in a mass spectrometer allows for greater opt ions in the detection protocol, which would be advantageous if large quantities of fragments are being analyzed, for example by multiple experiments. Mass spectrometry is known in the art (Burlingame et al. Anal. Chem. 70: 647R—716R (1998); Kinter and Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry Wiley-Interscience, New York) (2000)). The basic process involved in mass spectrometry is the measurement of the generation of gaseous ions derived from a sample and the mass of silver that is produced. Mass spectrometry techniques exist that can isolate, detect, and quantify thousands of protein species in a single operation.

<116>

Chromatography steps may be preceded before use of the mass spectrometer. A new method based on chromatography is available for identifying proteins contained in complex complexes without the need to separate the mixture into individual protein components. Separation steps can also be used to remove salts, enzymes, or other buffer components. Several methods known in the art, such as chromatography, gel electrophoresis, or precipitation tat ion, can be used to clean up the sample. For example,

Size exclusion chromatography or affinity chromatography can be used to remove salts from the sample. The choice of separation method may depend on the amount of sample. Yes For example, if a small sample is available or a small device is used, a micro-affinity chromatography separation process can be used. In addition, whether the separation process is necessary and the selection of the separation method can be determined according to the detection method to be used. For example, the efficiency of matrix assisted laser desorpt ion / ionizat ion and electrospray ionization can be improved by removing salt from the sample. For example, salt can absorb energy from matrix assisted laser desorption ionization, resulting in a decrease in ionization efficiency.

<119>

In the present invention, the mass spectrometry may be performed by a person skilled in the art as appropriate, various mass spectrometry methods known in the art as described above, and the mass spectrometry method of the present invention is not particularly limited, Preferably it may be ESI / MS / MS (Electrospray Ionization tandem Mass Spectrometry) or MALDI-TOF MSC atr ix assisted laser desorpt ion / ionizat ion t ime-of-f 1ight mass spectrometry (ESI / MS / MS).

<i2i> More preferably, the liquid filled with a solid or a support is used as a fixed phase (distributing agent or adsorbent such as C18 column, silver exchange resin, molecular sieve gel, etc.), and the liquid is used as a mobile phase. Multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) after separation of the mixture into each component using the difference of interaction and separation by liquid chromatography Methods or mass spectrometry equipment or methods to which MS and MS / MS methods can be applied.

Most preferably, the mass spectrometry method of the present invention may use liquid chromatography-tandem mass analysis (LC-MS / MS), and commercially available products include LTQ—Orbitrap, but are not limited thereto. Do not. Currently, one 60-minute LC-MS analysis can list up to 10,000 sequencing runs.

<123>

The present invention is, for example, (a) coating a plate with two or more peptide complexes selected from the group consisting of nine peptide complexes in which the N-terminus of the polypeptides represented by SEQ ID NOS: 1 to 9 are biotinylated. Making; ,

(B) contacting the sample to be analyzed with the coated peptide complex; And

(C) EGFR, STAT3, VGFRR3, HE 2, PDGFR, JAK1, PI3K, PDGFR and FGFR1, comprising the steps of recovering and analyzing the peptide complex in contact with the sample by mass spectrometry It provides a method for the simultaneous analysis of the activity of two or more tyrosine kinase selected from the group consisting of.

<127>

In addition, the present invention

(A) preparing a complex by adding a peptide complex of U terminus containing different phosphorylated substrate peptides to a sample to be analyzed;

(B) it provides a method for simultaneous activity analysis of kinase-related enzymes comprising the step of recovering the peptide complex from the mixture and analyzing it by mass spectrometry. The terms sample, peptide complex, recovery, mass spectrometry and the like described in the above method have the same meaning as described above. In the mixture of step (a), a contact occurs between the sample and the peptide complex to be analyzed, and the additional configuration and any additional process that may be included (or treated) in the complex with respect to the contact are as described above. .

<131>

The present invention is an example

<i33> (a) The mixture is added to the sample to be analyzed by adding two or more peptide complexes selected from the group consisting of nine peptide complexes in which the N-terminal of the polypeptides represented by SEQ ID NOs: 1 to 9 are biotinylated. Manufacturing;

(B) at least two tyrosine selected from the group consisting of EGFR, STAT3, VGFRR3, HER2, PDGFR, JAKl, PI3, PDGFR and FGFRl, comprising the step of recovering the peptide complex from the complex and analyzing it by mass spectrometry; Provides a method for simultaneous analysis of kinase activity.

<135>

Simultaneous analysis of the activity of the kinase-related enzyme of the present invention is a method for simultaneously analyzing whether or not phosphorylation of the phosphate substrate peptides contained in the peptide complexes of the present invention. In addition to analyzing the activity of two or more kinases or their related enzymes simultaneously, both qualitative and quantitative analyzes are available, providing more accurate information about kinase activity.

<137>

Such effects are well illustrated in one embodiment of the specification of the present invention.

In one embodiment of the invention tyrosine kinase EGFR, STAT3, VGFRR3, HER2,

Using tyrosine phosphate peptides of SEQ ID NOs: 10 to 18 for PDGFR, JAKl, PI3K, PDGFR, and FGFRl, respectively, the N-terminus of the peptide is used for Amino acids were combined with arginine to cancel negative charges at the c-terminus to prepare polypeptides of SEQ ID NO: 1 to SEQ ID NO: 9, respectively (Example 1). Nine-terminal of the polypeptide was biotinylated to prepare 9 peptide complexes of the present invention (Example 2).

This was eluted in three solvents through the reaction with streptavidin magnet ic bead and the peptide complex was recovered and compared by mass spectrometry (see FIGS. 3 to 5). As a result, when 5% FA / 70% ACN was used as the solvent, it was confirmed that it showed superior single substance detection ability and separation ability (Example 2).

In addition, in one embodiment of the present invention as a result of performing LC MS / MS analysis using the peptide complex of the present invention, using the peptide complex of the present invention whether or not there is activity of phosphatase (ie, inside the peptide complex As well as qualitative analysis of whether the included phosphorylation substrate is phosphorylated, it can be quantitatively determined how active. Therefore, the analytical method using the peptide complex of the present invention is suitable for measuring phosphatase activity because of excellent detection ability of a single substance, and it is possible to simultaneously measure phosphorylation of a target in large quantities by coating various phosphatase-based peptides on a plate. As a result, simultaneous activity (both qualitative and quantitative) of kinase-related enzymes is possible. In addition, the present invention

(a) coating the peptide complex of claim 1 on a plate;

(b) treating the drug candidate material with the cells;

(c) administering the cell lysate of the cell treated with the drug candidate material to the plate of step (a) to contact the peptide complex;

(d) provides a method for screening a drug that modulates the activity of a kinase-related enzyme comprising the step of recovering the peptide complex in contact with the cell lysate and analyzing it by mass spectrometry. In the present invention, the term 'activity regulation of enzyme' refers to both up-regulat ion in which activity is increased and down-regulat ion in which activity is suppressed. Whether the activity of the enzyme is up-regulated or down-regulated, the desired phosphoric acid The meaning and usefulness of the enzyme can be clearly understood by those skilled in the art depending on the activity state of the enzyme. For example, if a kinase is highly active in a pathological state, it may be understood that the down-regulation of the drug in the present invention may be useful for the treatment of a condition. If so, in the present invention, the up-regulation of the activity of the drug may be obvious to those skilled in the art as useful for the treatment of the condition.

<152>

Hereinafter, a step-by-step description of a drug screening method for controlling the activity of a kinase-associated enzyme of the present invention.

<154>

(A) coating the peptide complex of the present invention on a plate

In step (a), the peptide complex of the present invention is coated on a plate.

The peptide complex of the present invention has been described above.

The method of the present invention can measure the activity of one or more kinase, and may use two or more phosphorylation substrates to simultaneously measure the activity of multiple enzymes associated with the kinase, if necessary. . To this end, various kinds of peptide complexes containing different phosphorylated peptides may be prepared and used in combination.

The peptide complex of the present invention may be preferably one comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 9.

<160>

(B) treating the drug candidate material on the cells;

In step (b), the drug candidate material is treated in the cell. The cells are not particularly limited in origin and type of cells, as long as they contain the desired kinase, and include both normal cells and pathological cells.

<163>

The drug candidate material refers to a material that is expected to be useful for preventing or treating a disease, and the kind thereof is not particularly limited, and may be an anticancer drug candidate. Specifically, the drug candidate material is any material

substance, molecule, element, compound, entity, or combination thereof. For example, but not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleoles Ordinates and the like. It may also be a natural product, a synthetic compound or a chemical compound or a combination of two or more substances, but is not limited thereto.

<165>

In step (C), the cell lysate of the cell treated with the drug candidate material is (a).

It is administered to the plate of step to contact with the peptide complex. By contacting with the phosphatase-coated peptide complex contained in the cell lysate, the peptide complex is phosphorylated.

The cell lysate is obtained by decomposing cells treated with the drug candidate in step (b), and the method of decomposition may be appropriately selected by a person skilled in the art. For example, an optical method such as a laser micropulse, a nanostructure, etc. Acoustic methods such as mechanical methods and ultrasonic waves _ᅤ Electrical methods such as electric fields, and chemical methods such as acids, bases, detergents, and solvents can be decomposed. In addition, the cell lysate includes all of the cells and their fractions and isolates.

In the step (c), the environment such as temperature, pH, and other enzymes other than kinase-related enzymes may be controlled so that the phosphatase in the cell lysate exhibits the activity of phosphorylating the peptide complex. You can add it by selecting. For example, Tris, phosphate-buffered saline (PBS), or sucrose-triethanol amine with protease and phosphatase inhibitors can be used as homogenate in the first step of sample preparation.

In addition, in step (c), a phosphate donor component such as ATP may be appropriately selected and added to supply an element (for example, a phosphate group) required for phosphorylation of the peptide complex.

<171>

In order to derive accurate experimental results with regard to the activity of phosphatase, the phosphatase to be measured at the time of preparation of the cell lysate may be influenced by other factors in the cell (proteinase, phosphatase, etc.) in addition to the effects of drug candidates. Any additional process may be performed to make the subjects unaffected. For example, cell lysates can be treated with stabilizers, or they can be treated with a solution containing guanidine or urea for protein denaturation prior to enzyme degradation. The stabilizer is as described above. <173>

(D) recovering the peptide complex in contact with the cell lysate and analyzing it by mass spectrometry;

In step (d), the peptide complex in contact with the cell lysate is recovered and analyzed by mass spectrometry. 'Recovery ^' and 'analysis' of the peptide complex are as described above.

<176>

<177> The present invention is an example,

(a) The N-terminus of the polypeptide represented by SEQ ID NOS: 1 to 9 is biotinylated.

Coating the plate with at least one peptide complex selected from the group consisting of nine peptide complexes;

(B) treating the drug candidate with the cell;

(c) administering the cell lysate of the cell treated with the drug candidate to the plate of step (a) to contact the peptide complex;

(D) selected from the group consisting of EGFR, STAT3, VGFRR3, HER2, PDGFR, JAK1, PI3K, PDGFR and FGFR1, comprising recovering and analyzing the peptide complex in contact with the cell lysate by mass spectrometry; Provided are methods for screening drugs that modulate the activity of one or more tyrosine kinase related enzymes.

<182>

<183> The present invention also

(A) treating the drug candidate with the cell;

(B) preparing a complex by adding the peptide complex of claim 1 to a cell lysate of the cell treated with the drug candidate material;

(C) provides a method for screening a drug for controlling the activity of a kinase (ki nase) -related enzyme comprising the step of recovering the peptide complex from the complex of step (b) by mass spectrometry do. The terms cell, drug candidate substance, cell lysate, peptide complex, recovery, mass spectrometry, etc. described in the above method have the same meaning as described above. In the complex of step (b), a contact occurs between the sample to be analyzed and the peptide complex, and additional configurations and optional additional steps that may be included (or treated) in the complex with respect to the contact are as described above. .

<187>

<188> The present invention is an example (A) treating the drug candidate material with the cells;

(B) at least one selected from the group consisting of nine peptide complexes in which the N-terminus of the polypeptides represented by SEQ ID NOS: 1 to 9 are biotinylated in the cell lysate of the cell treated with the drug candidate; Preparing a complex by adding a peptide complex;

(C) a group consisting of EGFR, STAT3, VGFRR3, HER2, PDGFR, JAK1, PI3K, PDGFR and FGFR1 comprising recovering the peptide complex from the complex of step (b) and analyzing it by mass spectrometry; It provides a method for screening a drug that modulates the activity of at least one tyrosine kinase related enzyme selected from.

<192>

<193> The drug screening method of the present invention is a method of simultaneously applying the kinase-related enzyme activity of the present invention, and can simultaneously analyze the effects of drug candidates on various kinase activities. Simultaneous analysis of the effects and interrelationships of other kinases as well as the desired kinases can be used to more accurately select drug candidates for the purpose. In addition, the analysis of multiple phosphatase of one drug candidate material is processed in one well of the plate, so that a large number of drugs can be processed by using a folate having a number of wells, thereby analyzing a large number of drug candidate groups. It is easy to

<194>

The effect of the method of the present invention is to simultaneously measure the phosphorylation of the target in large quantities, thereby enabling simultaneous analysis of the activity of kinase-related enzymes (particularly, tyrosine kinase-related enzymes) and mass screening of drugs. To increase the screening efficiency.

<196>

Advantageous Effects

The peptide complex of the present invention is used for measuring the activity of kinase. Also provided are (i) immobilization means, (ii) amino acids for regulating retention time of 1 to 10, (iii) phosphorylated substrate peptide and (iv) hydrophilic amino acids having 1 to 5 positive charges provided in the present invention. The combined peptide complex for measuring phosphorylation activity enables simultaneous analysis of kinase related enzymes and is effective for screening drugs that regulate the activity of kinase related enzymes. Therefore, simultaneous analysis of kinase-related enzymes using the peptide complex and The drug screening method measures the phosphorylation of the target in a large amount, thereby enabling mass screening of the drug to increase the efficiency of the screening.

<198>

[Brief Description of Drawings]

1 is a diagram showing the structure of the peptide complex (Biotin: immobilization means,

Linkerl: amino acids for regulating 1 to 10 retention times, Core motif: phosphorylated substrate peptide, Linker2: hydrophilic amino acid having 1 to 5 positive charges).

<200>

FIG. 2 shows retention times upon mass spectrometry for polypeptides of SEQ ID NOs: 1-9

This figure shows the results of the retention time and relative hydrophobicity in the mass spectrum.

<202>

FIG. 3 is a diagram showing the results of mass spectrometry using a material obtained by elution with H ₂ 0 after reaction of a biotinylated peptide including EGFR sequences and streptavidin magnetic beads.

<204>

FIG. 4 is a diagram showing the results of mass spectrometry using a material obtained by elution with 0.1% FA after reaction of biotinylated peptide and streptavidin magnetic beads containing an EGFR sequence.

<206>

FIG. 5 is a diagram showing the results of mass spectrometry using a material obtained by elution with 5% FA / 70% ACN after reaction of biotinylated peptide including EGFR sequence and streptavidin magnetic beads.

<208>

FIG. 6 is a schematic diagram showing the phosphorylation of the core motif (phosphorylation substrate) portion by enzyme activity when the peptide complex (a) of the present invention comes into contact with the kinase (particularly SEQ ID NO: 1). Tyrosine phosphorylation in the tyrosine phosphorylation substrate in the peptide complex (a) comprising the peptide of (b)).

<210>

FIG. 7 is a schematic diagram showing an overview of a method for simultaneously analyzing the activity of a kinase-related enzyme of the present invention, wherein Stepl mixes the peptide complex of the present invention with a cell lysate. It shows the process of inducing the phosphorylation of the substrate by the action of the kinase in the cell lysate, Step 2 shows the process of obtaining a peptide complex of the present invention from the above-mentioned mixture. Mass spectrometry is carried out with the product obtained in Step 2 above.

<212>

8 shows the mass spectrum of the phosphatase treated group (ppl ~ pp9) and the untreated group (spl ~ sp9) in the peptide complex of the present invention at the same time by mass spectrometry with LTQ-Orbi trap mas s spectrometer (MIX TEST_dilut ion 10 # 1497-6366 RT: 15.56-36.47 AV: 779; NL: 4.90E7 T: FTMS + pNSI ful l ms [350.00-2000.00]).

<214>

9 shows signal intensity (intensi ty) during mass spectrometry according to substrate phosphorylation in the assay method using the peptide complex of the present invention. (A) shows the signal strength when the phosphorylated group treated with blue phosphorus (blue) and untreated group (red) with 1: 1 loaded. (B) shows the change in signal intensity by kinase enzyme treatment in the peptide complex of the present invention (dotted line: reference value measured in FIG. 9A, solid line: actual measured value).

<216>

[Form for implementation of invention]

Hereinafter, the present invention will be described in detail.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<219>

<220> <Example 1>

<22 i> Phosphorylation Substrate Peptide Sequence of Peptide Complex

For tyrosine kinase enzymes of EGFR, STAT3, VGFRR3, HER2, PDGFR, JAK1, PI3K, PDGFR and FGFR1, peptides of SEQ ID NOS: 10 to 18, respectively, were prepared. In the peptides of SEQ ID NOs: 10 to 18, the amino acid for adjusting the retention time of different numbers and types is attached to the N terminus of each sequence, and arginine is attached to the C-terminal as a hydrophilic amino acid having a positive charge, respectively, SEQ ID NO: 1 9 to 9 polypeptides were prepared (see Table 1). For the polypeptides of SEQ ID NOs: 1 to 9, retention time and relative hydrophobicity were confirmed during mass spectrometry. Specifically, the relative hydrophobicity and retention time were determined by the sequence specific retent ion calculator (3. Predicted using the http://hs2.proteome.ca/SSRCalc/SSRCalcX.html) program and based on lOOuM C18 column, 0.1% Formic Acid, Retention time = a + b * hydrophobicity or HI (Hydrophobicity Index) = a + b * measured at a = l, b = l in hydrophobic i ty (LEARN ABOUT NEW HYDROPHOBICITY (HI) SCALES (Krokhin 0V, Spicer V. Anal Chem. 81 (22) 9522-30 (2009), )

<223>

<224> [Table 1]

<226>

The retention time and relative hydrophobicity of the polypeptides of SEQ ID NOS: 1 to 9 are shown in Table 1 and FIG. 2.

Mass spectra (FIG. 2) of the polypeptides of SEQ ID NOS: 1 to 9 confirmed that the polypeptides were well separated and detected at different retention times without overlapping signals.

<229>

<23i> of the biotinylated peptide ^■ Lee and streptavidin magnetic beads and the banong

Mass Spectrometry

<232>

<233> Biotinylation of Polypeptides

<234> Biotinylation of the polypeptides of SEQ ID NOs: 1 to 9 is a peptron (Korea, www.peptron.co.kr) made to order. A total of nine peptide complexes were prepared, with biotin attached to the N-terminus of each polypeptide.

<235>

<236> <2-2> Reaction of Biotinylated Polypeptide and Streptavidin Magnetic Beads

In Example <2-1>, the peptide complex including the sequence of EGFR in the biotinylated polypeptide (stock in 0.001 DMSO) was diluted with PBS. Diluted peptide complex was reacted with streptavidin magnetic beads. Washing was performed three times in the order of PBST, PBS, H ₂ O. I) 0, ii) 0.1% FA (iii), iii)

After eluting with 5% FA / 70% ACN (acetonitrile), each solvent was evaporated, dissolved in 0.1% FA, and analyzed by LC-MS / MS mass spectrometry. In the mass spectrometry, Easy-nlX II (Thermo SCIENTIFIC) was used as LC and LTQ Orbitrap Velos EDT (Thermo SCIENTIFIC) was used as MS.

<238>

I) H ₂ 0, ii) 0.1% FA, iii) 5% FA / 70% ACN Elution in each of three solvents was carried out for the mass spectrometry. [Fig. 3] -¾0, [Fig. 4]- 0.1% FA, [FIG. 5] -5¾> FA / 70% ACN.

<240>

As shown in FIG. 3, FIG. 4, and FIG. 5, FIG. 5 showed a superior detection capability of a single substance as compared with FIG. 3 and FIG. Therefore, 5% FA / 70% ACN was used in later experiments.

<242>

<244> Determination of target kinase activity using peptide complex

<245>

<246> <3-1> Qualitative evaluation

<247> The polypeptide of the biotinylated SEQ ID NO: 1 to 9 prepared in <Example 2>, that is, the phosphorylated form in the nine peptide complexes (indicated by Spl to Sp9 in the order shown in Table 2 below) Ppl-Pp9 was produced by requesting peptron. Sp and Pp prepared at a final concentration of 5 mM were diluted twice for analysis. Transfer the first 5ul (25nmole) to each other tube. Add 995ul of 2% formic acid to this tube and mix well. The concentration of Sp and Pp in each tube was diluted 25 uM All. From the diluted sample, transfer lOul to another tube and place 990ul of 2% formic acid in it. Take 4ul (lpmole) of each sample from the prepared sample and mix in one tube. Finally, the prepared tube contained 72ul of sample, 7ul of which was taken for mass spectrometry. Samples are loaded into EASY-nLC II autosampler, a nano HPLC instrument. For chromatography, EASY-Column (diameter 100 μπι, length 2 cm) and reversed-phase analytical EASY-Column (75 μm diameter, 10 cm length, particle size 3 μιη) are mounted on the Eazy nano LC II autosampler. The nano-bore stainless steel emitter (inner diameter 30 ym) is finally mounted for electrospray ionization. A voltage of 1.9 V is applied while maintaining a flow rate of 300 nl per minute. Activate the Tune file of the LTQ Orbitrap Velos mass spectrometer and start the analysis. The ratio of acetonitrile is gradually increased by 13% '30%, 60%, 90%, and the sample is analyzed from the column for 65 minutes. The first full mass has a resolution of 100,000 in tandem mass (tandom ms / ms) analysis, and the second mass for sequencing uses the CID (colision energy induced dissociation) technique. In the resulting raw file, only the mass values of each peptide are added to confirm the position, peak area and intensity. The results are shown in Tables 2 and 3 below.

[Table 2]

(The -pY- symbol in Table 2 indicates that the tyrosine amino acid residue was phosphorylated.) As a result, as shown in Table 2 and FIG. 6, the signals spl to sp9 and ppl to pp9 overlap signals on the mass spectrum. It was confirmed that the separation well. Thus, using the peptide complex of the present invention, it is possible to clearly distinguish whether or not phosphorylation of the peptide during mass spectrometry.

In addition, the mass produced similarly between the peptide complexes analyzed as shown in Table 1, according to the mass spectrum according to m / z (mass— to— charge rat io of i on) H1 The data appears to be clustered, making it easy to analyze.

<256>

<257> <3 "2> Quantitative Evaluation

When the peak area value at the bottom of the graph was obtained by the mass spectrometry of Example 3-1, the amount of the peptide of the present invention including the phosphorylated substrate was quantitatively measured (see Table 3). In this case, as shown in Table 3, the charge state (z3) was determined to be i ntens i ty as 0, and thus, it was determined that accurate quantitative information could not be provided. As known in the art with respect to protein mass spectrometry it was desirable to obtain quantitative data based on the charge state (z2) and to adjust the pept ide amount. It was calculated based on Spl in this experiment.

<259>

<Table 3> precursor precursor RT

Pep peak area

Lot No. m / z m / z intensity (z2) intensity (z3) peak area (z2) average No. (z3)

(z2) (z3) (min)

13-93201 ppl 828.99864 553.00152 730403625 124677905 22315656967 3848893542 21 .85

13-93202 PP2 802.00364 535.00485 940461754 821501283 26027916123 25797736924 17.94

13-93203 pp3 871.53864 581 .36152 H9603786 781377346 4787670362 34317737489 17.50

13-93204 PP4 821.95864 548.30818 358514649 1917295 10802416212 51044541 26.34

13-93205 pp5 958,55864 639.37485 126348746 414805252 6318441322 23136646191 27.73 pp6 975.54864 650.70152 97403086 21718691 4990744520 1249960171 29.85

13-93207 pp7 936.03364 624.35818 35164509.37 0 2296846036 0 33.30

13-93208 pp8 1007.06364 671 .71 1 52 175400193 76612421 5063615074 2221005449 28.87

13-93209 pp9 943.04364 629.031 52 1230782 0 79923831 0 34.79

13-96501 Spl 788.99864 526.33485 1058555293 1 1056451 9 41231244055 4343722739 22.25

13-96502 Sp2 762.00364 508.33818 347864627 1294574391 10852477701 57706681847 18.80

13-96503 Sp3 831.53864 554.69485 76844967 469024021 2471111167 20668320861 17.91

13-96504 Sp4 781.95864 521 .64152 734088247 2083881 17883190931 43844608 27.20

13-96505 Sp5 918.55864 612.70818 77622524 919205583 3512840765 46541884343 27.10

13-96506 Sp6 935.54864 624.03485 44661 1585 72925419 22516121624 3743887654 29.72

13-96507 Sp7 896.03364 597.69 1 52 92807268 1 166632 5814124778 60234736 34.45

13-96508 Sp8 967.06364 645.04485 542755372 199016604 13379069388 5040402555 29.23

13-96509 Sp9 903.04364 602.36485 15892255 0 879885808 0 36.23 <262><Example4>

<263> Change of Signal Intensity by Substrate Phosphorylation of Peptide Complex of the Present Invention

Each of ¾) and Pp are analyzed first through mass spectrometry to identify basic intensities and peak areas of each peptide (FIG. 9A). Each Sp is added to a cell sample prepared for various situations (cel l lysate) to react and recover. The recovered sample is analyzed by mass spectrometry to identify the phosphorylated peptides. In the case of peptides that are phosphorylated according to a specific situation, quantitative analysis is performed based on the intensity and peak area information of the Sps first identified (FIG. 9B). In addition, information on the degree of phosphorylation is also confirmed through comparison with Sp appearing in the analysis (FIG. 9B).

<265>

Industrial Applicability

As described above, the peptide complex of the present invention is used for measuring the activity of kinase. Also provided in the present invention are (i) immobilization means, (ii) 1-10 amino acids for regulating retention time (iii) phosphorylated substrate peptides, and (iv) hydrophilic amino acids having 1-5 positive charges. This sequentially bound peptide complex for measuring phosphorylation activity can be simultaneously analyzed for a number of kinase-related enzymes, and is effective for screening drugs that regulate the activity of kinase-related enzymes. . Therefore, the simultaneous analysis of kinase-related enzyme activity and drug screening method using the peptide complex measure the phosphorylation of the target in a large amount, thereby enabling the large-scale screening of drugs and thus increasing the screening efficiency. It is highly available.

<267>

Claims

【Scope of Claim】

【Claim 11

(i) immobilization means, (ii) 1 to 10 amino acids for controlling retention time, (iii) phosphorylation substrate peptide, and (iv) phosphorylation activity in which 1 to 5 hydrophilic amino acids with positive charges are sequentially combined. Peptide complex for measurement.

【Claim 2】

The peptide complex according to item U, wherein the 1 to 10 amino acids for controlling the retention time are one or more amino acids selected from the group consisting of hydrophilic amino acids with positive charges, hydrophobic amino acids, and proline.

[Claim 3]

The peptide complex according to claim 2, wherein the hydrophobic amino acid is at least one selected from phenylalanine (F), leucine (L), isoleucine (I), tryptophan (W), and valine (V).

【Claim 4】

The temtide complex according to claim 1 or 2, wherein the hydrophilic amino acid having a positive charge is at least one selected from the group consisting of lysine 00, arginine (R), and histidine (H).

[Claim 5]

The peptide complex according to claim 1, wherein (iv) the hydrophilic amino acid having 1 to 5 positive charges is isotopically substituted.

【Claim 6】

The peptide complex according to claim 1, wherein (i) the immobilization means is biotin.

[Claim 7]

The method of claim 1, wherein (ii) 1 to 10 amino acids for controlling retention time, (Hi) a phosphorylated substrate peptide, and (iv) a parent having 1 to 5 positive charges. A temtide complex, characterized in that the aqueous amino acid is any one selected from the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 9.

【Claim 8】

(a) coating a plate with the peptide complex of item U containing different phosphorylation substrate peptides;

(b) contacting the sample to be analyzed with the coated peptide complex; and

(c) A method for simultaneous analysis of the activity of phosphorylase-related enzymes, including the step of recovering the peptide complex in contact with the sample and analyzing it using mass spectrometry.

【Claim 91

(a) coating the peptide complex of claim 1 on a plate;

(b) treating cells with drug candidates;

(c) administering the cell lysate of cells treated with the drug candidate in step (b) to the plate in step (a) and bringing it into contact with the peptide complex;

(d) A screening method for drugs that regulate the activity of enzymes related to phosphorylase, including the step of recovering the peptide complex in contact with cell lysate and analyzing it using mass spectrometry.

[Claim 10]

(a) preparing a mixture by adding the peptide complexes of claim 1 containing different phosphorylation substrate peptides to the sample to be analyzed;

(b) A method for simultaneous analysis of the activity of enzymes related to phosphorylase, including the step of recovering peptide complexes from the mixture and analyzing them using mass spectrometry.

[Claim 11]

The method of claim 8 or 10, wherein the peptide complex is a complex of two or more peptides selected from the group consisting of nine peptide complexes in which the N-termini of the polypeptides represented by SEQ ID NOs: 1 to 9 are biotinylated. Simultaneous analysis method using.

【Claim 12】 The method of claim 8 or 10, wherein the kinase-related enzymes are two or more tyrosine kinases selected from the group consisting of EGFR, STAT3, VGFRR3, HER2, PDGFR, JAK1, PI3K, PDGFR, and FGFR1. simultaneous analysis method.

[Claim 13]

(a) Processing drug candidates in cells;

(b) preparing a mixture by adding the peptide complex of claim 1 to cell lysate of cells treated with the drug candidate;

(c) A screening method for a drug that modulates the activity of a kinase-related enzyme, comprising the step of recovering the peptide complex from the mixture in step (b) and analyzing it by mass spectrometry.

[Claim 14]

The method of claim 19 or 13, wherein the peptide complex is one or more peptide complexes selected from the group consisting of nine peptide complexes in which the N-termini of the polypeptides represented by SEQ ID NOs: 1 to 9 are biotinylated. Characterized screening method.

【Claim 15】

The method of claim 9 or 13, wherein the kinase-related enzyme is one or more tyrosine kinase-related enzymes selected from the group consisting of EGFR, STAT3, VGFRR3, HER2-PDGFR, JAK1, PI3K, PDGFR, and FGFR1. A screening method characterized in that.