Peptide microarray

Abstract

To provide a base peptide microarray that has array-purpose capture polypeptide useful for detecting stomach pre-cancerous lesions mounted on a substrate.SOLUTION: A peptide microarray is a specific amino acid sequence that has a hydrophilic amino acid residue and a hydrophobic amino acid residue on a substrate, and includes a polypeptide part in which even the hydrophilic amino acid residue and hydrophobic amino acid residue do not continue respectively three pieces or the like. 20 kinds or more of fluorescent label polypeptides exhibiting a α-helix structure are immobilized in a spot-like way as a capture molecule for each fluorescent label polypeptide.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a peptide microarray based on a novel principle, and particularly to a peptide microarray useful for the examination and discrimination of precancerous lesions of the stomach.

Background Art

[0002] Gastric cancer often occurs when chronic gastritis caused by Helicobacter pylori infection becomes intestinal metaplasia and the intestinal metaplasia progresses to gastric cancer. The cause of intestinal metaplasia has not been fully elucidated. It is beneficial to be able to determine whether or not canceration occurs at the precancerous lesion stage of the stomach, that is, at the stage of non-disease, because early treatment or prevention of gastric cancer can be performed. Heretofore, methods for detecting precancerous lesions of the stomach using body fluids as specimens are well known, and substances such as prostaglandins (PGI, PGII) have been examined using serum samples, but no samples have been directly taken in the gastric environment. Atrophic gastritis can be predicted to some extent, but intestinal metaplasia cannot be examined.

[0003] On the other hand, the present applicant has previously developed a method of using a peptide microarray in which a large number of chemically synthesized peptides having a desired three-dimensional structure are immobilized on a substrate, reacting a test sample with the peptide microarray, converting a change in a signal generated by the interaction between the components in the test sample and each peptide into a color pattern, and characterizing the nature of the test sample according to the resulting color pattern, and has obtained a patent (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Gastric cancer is a disease with a high incidence rate among cancers. However, the treatment methods are relatively well-established, and it is known that early detection and early treatment can lead to a cure. In recent research, it has become possible to detect early gastric cancer associated with risks from pathological findings. Gastric cancer does not suddenly become cancerous but develops with gastritis as the initial symptom. Gastritis can be divided into acute gastritis and chronic gastritis, and chronic gastritis can be further divided into non-atrophic gastritis and atrophic gastritis. The latter is regarded as a precancerous lesion with a high probability of carcinogenesis. In atrophic gastritis, gastric mucosal cells gradually decrease, the ability to produce gastric acid declines, and further progression leads to intestinal metaplasia. Intestinal metaplasia is a state in which gastric mucosal epithelium changes to intestinal mucosal epithelium during the process of gastric mucosal repair, and the mucosa thickens and is considered the "birthplace of gastric cancer development." For the diagnosis of atrophic gastritis and intestinal metaplasia, in addition to the conventional examination using a gastric endoscope, biopsy of the gastric mucosa is required. However, biopsy for pathological diagnosis places a great burden on patients, and tissue sampling by endoscopy is contraindicated for patients taking anticoagulants due to heart or cerebrovascular diseases because of the concern of bleeding. Therefore, a simple and rapid diagnostic classification with less burden on patients without collecting pathological specimens is desired by both patients and clinicians.

[0006] Therefore, an object of the present invention is to provide a peptide microarray having capture polypeptides useful for detecting precancerous lesions of the stomach on a substrate (a peptide immobilized on a chip substrate used for examination is referred to as a capture molecule, and a substrate loaded with these various peptides is referred to as a peptide microarray).

Means for Solving the Problems

[0007] As a result of intensive research, the inventors of the present application have found a combination of polypeptides useful for detecting precancerous lesions at the stage of intestinal metaplasia, which is a precancerous lesion of the stomach, based on the method described in Patent Document 1, and completed the present invention.

[0008] That is, the present invention provides the following. (1) On a substrate, a general formula [Ia] or [Ib]: Flu-Gly-X 1 X 2 X 3 X4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 -Gly-Cys-NH 2 [Ia] Ac-Cys-Gly-X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 -Gly-Flu [Ib] (In general formulas [Ia] and [Ib], Flu is a fluorescent label, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 are, independently of each other, a hydrophobic amino acid residue or a hydrophilic amino acid residue, and the hydrophobic amino acid residues are not arranged continuously in three or more numbers, and the hydrophilic amino acid residues are not arranged continuously in three or more numbers. In general formula [Ib], Ac is an acetyl group, and the fluorescently labeled polypeptides represented by general formulas [Ia] and [Ib] adopt an α-helix structure) A peptide microarray in which 20 or more types of fluorescently labeled polypeptides represented by are immobilized as capture molecules in a spot-like manner for each fluorescently labeled polypeptide. (2) The hydrophobic amino acid residue is a residue of an amino acid selected from the group consisting of leucine, isoleucine, glutamine, phenylalanine, and valine, and the hydrophilic amino acid residue is a residue of an amino acid selected from the group consisting of lysine, glutamic acid, serine, and arginine. The peptide microarray according to (1). (3) In the general formula [I], X 1 , X 4 , X 5 , X 7 , X 8 , X 11 , X 12 and X 14 are hydrophobic amino acid residues, and X 2 , X 3 , X 6 , X 9 , X 10 and X 13 are hydrophilic amino acid residues. The peptide microarray according to (1) or (2). (4) In the general formula [I], X 2 , X 3 and X 13 are, independently of each other, residues of glutamine, glutamic acid, or serine. The peptide microarray according to any one of (1) to (3). (5) In the general formula [I], X 7 and X 14 are, independently of each other, residues of isoleucine, leucine, phenylalanine, or valine. The peptide microarray according to any one of (1) to (4). (6) In the general formula [I], X 1 , X 4 , X 5 , X 8 , X 10 and X 11 are, independently of each other, residues of leucine, isoleucine, or phenylalanine. The peptide microarray according to any one of (1) to (5). (7) In the general formula [I], X 6 , X 9 and X 10The peptide microarray according to any one of (1) to (6), wherein the residues are lysine or arginine residues and are independent of each other. (8) X in the general formula [I] 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 The peptide microarray according to any one of (1) to (4), having 34 kinds of fluorescently labeled polypeptides, wherein the amino acid sequences of the portions of are represented by SEQ ID NOs: 1 to 34. (9) The fluorescently labeled polypeptide is represented by the general formula [Ia], and the peptide microarray according to any one of (1) to (8). [Advantages of the Invention]

[0009] According to the present invention, it has become possible to simply diagnose the state of gastritis and the presence or absence of signs of gastric canceration of the gastritis using a very small amount of gastric juice as a specimen. Further, the present invention also shows that the structure and type of the peptide used for the microarray are important for classification, and it is expected that the microarray with adjusted peptide types can be applied to various diseases. [Brief Description of the Drawings]

[0010]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0011] The inventors of the present application focused on the fact that various proteins are contained in body fluids. The interaction between proteins occurring in vivo is, from a microscopic perspective, the interaction between peptides constituting the proteins. Therefore, the inventors of the present application designed peptides that recognize structures based on the amino acid sequences of regions that recognize the structures of proteins, and developed a technique called peptide fingerprinting for detecting proteins (Patent Document 1). In this technique, designed and fluorescently labeled structure-recognizing peptides are arranged in an array, and the change in fluorescence intensity when a specimen binds is captured to detect the specimen. It is a technique similar to an antibody chip, but is characterized by not being a 1:1 correspondence detection. Since it is not limited to 1:1 correspondence and the change in fluorescence intensity before and after adding the specimen is observed and detection is performed by patterning, it has the advantage of being able to detect regardless of the type of specimen. In the present invention, this technique is applied to gastric juice, and a peptide structure as a capture molecule is provided that is immobilized on a substrate that contributes to the diagnosis of gastric precancerous lesions without relying on tissue sampling, which was a problem.

[0012] The structural peptides used in the production of the peptide microarray in the present invention are those that form an α-helix structure among those represented by the above formula. The α-helix structure forms a helical structure and has hydrophilic and hydrophobic regions. Contributing to the formation of these hydrophilic and hydrophobic regions are the characteristics of the amino acid residues constituting the peptide. In the structural peptide of the present invention, hydrophilic amino acids and hydrophobic amino acids are alternately linked to form an α-helix as shown in Fig. 1.

[0013] The inventors of the present application considered that the difference in the hydrophilic-hydrophobicity of the constituent amino acids is important for applying the technique described in Patent Document 1 to the diagnosis using gastric juice as a specimen. They prepared a peptide microarray by arraying 552 types of the α-helix libraries they possessed, assayed it with gastric juice specimens with clear pathological conditions, and classified them based on the obtained fluorescence intensity changes, thus arriving at the present invention.

[0014] The peptide microarray of the present invention is one in which 20 or more, preferably 30 or more, fluorescently labeled polypeptides represented by the above general formula [I] are immobilized on a substrate. There is no particular upper limit to the number of types of fluorescently labeled polypeptides to be immobilized. However, as the number of types of fluorescently labeled polypeptides increases, it becomes more laborious and costly to manufacture the peptide microarray. Therefore, the fluorescently labeled polypeptides are usually 50 or less, preferably 40 or less. The fluorescently labeled polypeptides to be immobilized may be a mixture of those represented by the general formula [Ia] and those represented by the general formula [Ib], but it is preferable that all are represented by the general formula [Ia].

[0015] In the general formulas [Ia] and [Ib], Flu is a fluorescent label. The fluorescent label is not particularly limited, but TAMRA (5(6)-carboxytetramethylrhodamine) is preferable from the viewpoints of long-term stability, measurement on the long wavelength side, reproducibility, and sensitivity.

[0016] X 1 、X 2 、X 3 、X 4 、X 5 、X 6 、X7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 are each independently any hydrophobic amino acid residue or hydrophilic amino acid residue. Here, the hydrophobic amino acids are leucine, isoleucine, glutamine, phenylalanine, valine, glycine, alanine, proline, methionine, and tryptophan. The hydrophilic amino acids are lysine, glutamic acid, serine, arginine, aspartic acid, histidine, threonine, cysteine, asparagine, glutamine, and tyrosine.

[0017] From the viewpoint of detecting precancerous lesions of the stomach with high sensitivity and high specificity, as the hydrophobic amino acids, leucine, isoleucine, glutamine, phenylalanine, and valine are preferred, and as the hydrophilic amino acids, lysine, glutamic acid, serine, and arginine are preferred.

[0018] Also, from the viewpoint of detecting precancerous lesions of the stomach with high sensitivity and high specificity, in general formula [I], X 1 , X 4 , X 5 , X 7 , X 8 , X 11 , X 12 and X 14 are hydrophobic amino acid residues, and X 2 , X 3 , X 6 , X 9 , X 10 and X 13 are preferably hydrophilic amino acid residues. In particular, in general formula [I], X 2 , X 3 and X 13 are preferably, independently of each other, residues of glutamine, glutamic acid, or serine. Also, in general formula [I], X 7 and X 14 are preferably, independently of each other, residues of isoleucine, leucine, phenylalanine, or valine. Also, in general formula [I], X 1 , X4 , X 5 , X 8 , X 10 and X 11 are preferably residues of leucine, isoleucine or phenylalanine, independently of one another. Further, in general formula [I], X 6 , X 9 and X 10 are preferably residues of lysine or arginine, independently of one another.

[0019] The above-described fluorescently labeled polypeptide has an α-helix structure. The fact that it has an α-helix structure can be confirmed by measuring circular dichroism (CD spectrum). It has also been confirmed by CD spectrum that all 34 kinds of fluorescently labeled polypeptides immobilized on a substrate in the following examples also have an α-helix structure.

[0020] In the following examples, the amino acid sequences (one-letter notation) of X 1 to X 14 of 34 kinds of fluorescently labeled polypeptides immobilized on a substrate are as follows. Note that "A0126" etc. are the code names assigned to each fluorescently labeled polypeptide.

[0021] A0126 LKKLIEILKKLIEI (SEQ ID NO: 1) A0134 LQQLIKILKKLIQI (SEQ ID NO: 2) A0135 LQQLLKILKKLLQI (SEQ ID NO: 3) A0136 LQQLIKILQQLIKI (SEQ ID NO: 4) A0138 LKKLISILKKLISI (SEQ ID NO: 5) A0139 LKKLLSILKKLLSI (SEQ ID NO: 6) A0140 LSSLIKILKKLISI (SEQ ID NO: 7) A0172 LKKLFRFLKKLFRF (SEQ ID NO: 8) A0173 LKKLLRFLKKLLRF (SEQ ID NO: 9) A0183 LKKFFEFLKKFFEF (SEQ ID NO: 10) A0185 LKKLLEFLKKLLEF (SEQ ID NO: 11) A0187 LEEFFKFLKKFFEF (SEQ ID NO: 12) A0188 LEELFKFLKKLFEF (SEQ ID NO: 13) A0189 LEELLKFLKKLLEF (SEQ ID NO: 14) A0197 LKKLLQFLKKLLQF (SEQ ID NO: 15) A0198 FQQFFKFFKKFFQF (SEQ ID NO: 16) A0199 LQQFFKFLKKFFQF (SEQ ID NO: 17) A0200 LQQLFKFLKKLFQF (SEQ ID NO: 18) A0201 LQQLLKFLKKLLQF (SEQ ID NO: 19) A0202 FQQFFKFFQQFFKF (SEQ ID NO: 20) A0203 LQQFFKFLQQFFKF (SEQ ID NO: 21) A0204 LQQLFKFLQQLFKF (SEQ ID NO: 22) A0205 LQQLLKFLQQLLKF (SEQ ID NO: 23) A0206 FKKFFSFFKKFFSF (SEQ ID NO: 24) A0207 LKKFFSFLKKFFSF (SEQ ID NO: 25) A0254 LKKLVKVLKKLVKV (SEQ ID NO: 26) A0255 LKKLLKVLKKLLKV (SEQ ID NO: 27) A0258 LKKLVRVLKKLVRV (SEQ ID NO: 28) A0259 LKKLLRVLKKLLRV (SEQ ID NO: 29) A0261 LRRLLKVLKKLLRV (SEQ ID NO: 30) A0270 LKKLVQVLKKLVQV (SEQ ID NO: 31) A0272 LQQLVKVLKKLVQV (SEQ ID NO: 32) A0277 LKKLLSVLKKLLSV (SEQ ID NO: 33) A0279 LSSLLKVLKKLLSV (SEQ ID NO: 34)

[0022] The above fluorescently labeled polypeptides used in the present invention can be chemically synthesized by well-known methods such as the Fmoc solid-phase synthesis method using a commercially available peptide synthesizer (see the following examples).

[0023] In the peptide microarray of the present invention, each of the above fluorescently labeled polypeptides is immobilized on a substrate in a spot shape. For example, it can be immobilized on an amorphous carbon substrate via the sulfhydryl group of a Cys residue. This can be carried out by a well-known method (see the following examples).

[0024] Note that the material of the substrate is not particularly limited, but a substrate made of amorphous carbon is preferred.

[0025] When performing a gastric fluid test using the peptide microarray of the present invention, it can be carried out as follows. That is, gastric fluid separated from a subject or a dilution thereof is brought into contact with the peptide microarray. The contact can be carried out at room temperature and for about 15 minutes to 1 hour, preferably about 20 minutes to 40 minutes. Then, each fluorescently labeled peptide immobilized on the substrate recognizes various components in the gastric fluid, and the fluorescence intensity of the capture molecule at each spot changes. Note that it is not possible to know which component in the gastric fluid binds to which fluorescently labeled peptide, nor is it necessary to know. The fingerprint method described in Patent Document 1 does not measure the 1:1 binding between a specific component in the test sample and a specific polypeptide immobilized on the substrate, but classifies the binding between various components in the test sample and various polypeptides on the substrate based on the pattern of changes in the fluorescence intensity of each spot. That is, if it is such a pattern, it is highly likely to be a precancerous lesion, and if it is such a pattern, it is determined that the possibility of being a precancerous lesion is low.

[0026] More specifically, based on the change in the fluorescence intensity of each spot, the determination can be made as follows. Gastric fluid (including normal, gastritis, and gastric precancerous lesions) that has already been diagnosed by a doctor through diagnosis, pathological examination, etc. is used as a specimen, and fluorescence intensity change data is obtained using the peptide microarray composed of the peptide probes of this patent. This is used as a comparison database and compared with the fluorescence intensity change data of the gastric fluid specimen to be diagnosed. The data used for comparison is analyzed for the specimen by cluster analysis. In the comparison database, the specimens are classified as clusters for each symptom, and in the diagnosis, the diagnosis is made by confirming which cluster the gastric fluid specimen to be diagnosed belongs to. Once the comparison database is created, it can be used to examine unknown gastric fluid specimens.

[0027] Note that the test results using the peptide microarray of the present invention can enable real-time on-site diagnosis by using a network, and thus are also useful for telemedicine and home healthcare.

[0028] Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

[0029] Example 1 Synthesis and Preparation of Peptide Microarray As the main reagents used in peptide synthesis, amino acid derivatives, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and fluorescent dyes, 5(6)-carboxyfluorescein (FAM) and 5(6)-carboxytetramethylrhodamine (TAMRA), ε-maleimidocaproic acid (EMCA) were obtained from HiPep Laboratories (Kyoto, Japan). The solid support used was TentaGel® S-RAM (Rapp Polymere GmbH, Tubingen, Germany). Other reagents and solvents were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and used. Water was prepared by a Milli-Q® apparatus (Merck-Millipore, Tokyo, Japan). For the quality evaluation of the peptides used in this study, online LC-MS (LC: Agilent 1100, Agilent Technologies Inc., MS: HCTultra, Bruker Japan K.K., Yokohama) was used. A 384-well microtiter plate (Thermo Fisher Scientific K.K., Yokohama, Japan) was used for the preparation of the peptide solution for peptide microarray fabrication. A structured peptide library was synthesized by the Fmoc solid-phase synthesis method using an automatic synthesizer PSSM-8 (Shimadzu Corporation) and a manual synthesizer PetiSyzers® (HiPep Laboratories), and cut out from the resin. After purification with a preparative column (50 i.d.×250 mm), 1 mg of the synthesized fluorescently labeled peptide was dissolved in 180 μL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / H2O / AcOH = 1 / 1 / 1 (v / v / v), and dimethyl sulfoxide (DMSO) was added to make the total volume 400 μL to prepare a stock solution. The concentration of the stock solution was determined by UV measurement using a NanoDrop® (Thermo Fisher Scientific).The stock solution was diluted 200-fold with methanol, and the absorbance at 450 - 600 nm was measured at 25 °C. From the absorbance at the maximum absorption wavelength of TAMRA (545 nm) and the absorption coefficient of TAMRA (ε = 91000), the concentration of the fluorescently labeled peptide in the stock solution was calculated. The prepared stock solution was stored at -80 °C until use in the array.

[0030] For the preparation of the microarray, a microarray layer NanoPrint LM60 (product name, Arrayit Corp., CA, USA) was used, and a stealth pin 946MP4 (product name, spot diameter 160 μm, Arrayit Corp.) was used as the pin for the array. The fluorescently labeled peptide used for the array was prepared by diluting 1 mM of the fluorescently labeled peptide 10-fold with 1% acetic acid. The prepared peptide solution was dispensed into a 384-well microtiter plate (product number) and set on NanoPrint LM-60 (product name) for arraying. An amorphous carbon substrate derivatized with three cover glass-sized regions was used for the array. After arraying, the substrate was washed three times each with a 2-propanol / H2O = 1 / 1 solution and ultrapure water, and spin-dried. The prepared substrate was observed on the surface with a fluorescence detection device (PepTenCam (product name), HiPep Laboratories), and it was confirmed that the spot state was normal and used for the assay.

[0031] Example 2 Assay with Specimen 10 μL of PBS was applied to each of the three blocks on the peptide microarray, covered with a cover glass, spread over the entire array, and incubated at room temperature for 30 minutes in the dark. After incubation, a fluorescence image was measured with a fluorescence detection device and digitized by Arraypro analyzer (product name) to obtain I 0 . After measurement, the substrate was washed three times each with a 2-propanol / H2O = 1 / 1 solution and ultrapure water, and spin-dried. Next, the gastric juice specimen solution was treated in the same manner as PBS, a fluorescence image was measured, and digitized to obtain I 1 . The I 0 and I 1The value was used to calculate the fluorescence intensity change using an equation, and multivariate analysis was performed using the statistical analysis software "R" (an open-source statistical analysis language and its execution environment).

[0032]

Number

[0033] Example 3 Classification of Gastric Juice Specimens The data on the fluorescence intensity change for the gastric juice specimens obtained in Example 2 was converted into CSV format and read into R, a statistical analysis program, for analysis. Since the raw data differed for each specimen, data standardization was performed for statistical analysis. Next, analysis by the silhouette method was carried out to determine the number of clusters suitable for classification, and cluster analysis was performed based on the obtained number of clusters. The results of the cluster analysis are shown in Figure 2. In Figure 2, "C1", "A6", "B12", etc. described at the bottom of the figure are the codes of each subject. Codes starting with A are healthy subjects, codes starting with B are patients with chronic gastritis, among the codes starting with C, C2 are gastric cancer patients, and the others are intestinal metaplasia.

[0034] Thus, by combining and analyzing the fluorescence intensity change data obtained from peptide probes of an array suitable for the specimen, it is possible to classify diseases without marker molecules.

[0035] The present invention diagnoses precancerous lesions of the stomach using gastric juice as a specimen, but by changing the combination of structural peptides used, it can be applied to the diagnosis of various diseases. For example, based on the peptide structure information (information regarding the hydrophilicity and hydrophobicity of the amino acids constituting the peptide) of the present invention, by combining structural peptides such as β-Sheet and β-Loop, it is expected to lead to the diagnosis of diseases without diagnostic markers and early diagnosis before the onset of the disease.

Claims

1. On a substrate, 20 or more types of fluorescently labeled polypeptides represented by the general formula [Ia] or [Ib]: Flu-Gly-X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 -Gly-Cys-NH 2 [Ia] Ac-Cys-Gly-X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 -Gly-Flu [Ib] (In general formulas [Ia] and [Ib], Flu is a fluorescent label, X 1 、X 2 、X 3 、X 4 、X 5 、X 6 、X 7 、X 8 、X 9 、X 10 、X 11 、X 12 、X 13 、X 14 are, independently of each other, a hydrophobic amino acid residue or a hydrophilic amino acid residue, the hydrophobic amino acid residues are not arranged continuously in three or more, the hydrophilic amino acid residues are not arranged continuously in three or more, Ac in general formula [Ib] is an acetyl group, and the fluorescently labeled polypeptides represented by general formulas [Ia] and [Ib] adopt an α-helix structure.) A peptide microarray in which each of the fluorescently labeled polypeptides is immobilized as a capture molecule in a spot-like manner for each fluorescently labeled polypeptide.

2. The hydrophobic amino acid residue is a residue of an amino acid selected from the group consisting of leucine, isoleucine, glutamine, phenylalanine, and valine, and the hydrophilic amino acid residue is a residue of an amino acid selected from the group consisting of lysine, glutamic acid, serine, and arginine. The peptide microarray according to Claim 1.

3. In the general formulas [Ia] and [Ib], X 1 X 4 X 5 X 7 X 8 X 11 X 12 and X 14 are hydrophobic amino acid residues, and X 2 X 3 X 6 X 9 X 10 and X 13 are hydrophilic amino acid residues, the peptide microarray according to claim 1 or 2.

4. In the general formulas [Ia] and [Ib], X 2 , X 3 and X 13 are, independently of one another, residues of glutamine, glutamic acid or serine, the peptide microarray according to claim 3.

5. In the general formulas [Ia] and [Ib], X 7 and X 14 are, independently of one another, residues of isoleucine, leucine, phenylalanine or valine, the peptide microarray according to claim 4.

6. In the general formulas [Ia] and [Ib], X 1 , X 4 , X 5 , X 8 , X 10 and X 11 are, independently of one another, residues of leucine, isoleucine or phenylalanine, the peptide microarray according to claim 5.

7. In the general formulas [Ia] and [Ib], X 6 , X 9 and X 10 are, independently of one another, residues of lysine or arginine, the peptide microarray according to claim 6.

8. X in the general formulas [Ia] and [Ib] 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 The peptide microarray according to claim 1 or 2, which has 34 types of fluorescently labeled polypeptides in which the amino acid sequences of the portions of are represented by SEQ ID NOs: 1 to 34.

9. The fluorescently labeled polypeptide is represented by the general formula [Ia]. The peptide microarray according to Claim 1 or 2.

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