KR101296743B1 - Biomarkers for diagnosing cardiac hypertrophy and method for diagnosis using the same - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing myocardial hypertrophy and a method for diagnosing myocardial hypertrophy using the same. According to one embodiment, a biomarker for diagnosing myocardial hypertrophy, a diagnostic kit including the same, and a method for diagnosing myocardial hypertrophy using the same, may be useful for determining myocardial hypertrophy from biological samples, and as a tool for identifying the pathogenesis of myocardial hypertrophy. Can be used.

Description

Biomarker for diagnosing myocardial hypertrophy and method for diagnosing myocardial hypertrophy using the same {{Biomarkers for diagnosing cardiac hypertrophy and method for diagnosis using the same}

The present invention relates to a biomarker for diagnosing myocardial hypertrophy and a method for diagnosing myocardial hypertrophy using the same.

Heart disease is thought to be the leading cause of death worldwide. Heart disease is often caused by abnormalities in heart function, which often begins with myocardial hypertrophy, in which the size of heart cells increases, the sarcomeric organization and the synthesis of proteins increase. Myocardial hypertrophy can be caused by constant physical pressure or functional load caused by the heart being hypertensive, myocardial infarction, cardiac arrhythmia, abnormal endocrine system, and genetic factors. Hypertrophic reactions occur initially to increase heart function, but if myocardial hypertrophy persists, myocardial dysfunction may occur and myocardial infarction may die. Myocardial hypertrophy caused by various stimuli increases cell size, protein synthesis, and induces the expression of various genes.

Examples of substances that induce myocardial hypertrophy in the body include endothelin-1, angiotensin II, insulin-like growth factor-I. Endothelin-1 can transmit signals through the G protein-coupled receptor (GPCR), which binds to GPCR to activate phospholipase C, producing IP ₃ (inositol triphosphate), which is called endoplasmic reticulum or intracellular calcium storage. It activates the calcium channel in the store to release calcium from the myoplasmic reticulum, increasing the concentration of calcium in the cytoplasm. Increased calcium binds to calmodulin to dephosphorylate calcineurin and dephosphorylated calcineurin activates transcription factor NFAT to activate transcription of various myocardial hypertrophy related genes (FIG. 1).

Based on this, the present inventors can detect the myocardial hypertrophy by discovering proteins overexpressed or underexpressed by endothelin-1 by observing and analyzing changes in protein profiles in the HL-1 myocardial cell line by endothelin-1 using proteomics techniques. Biomarkers and methods of diagnosing myocardial hypertrophy have been established.

One embodiment provides a biomarker for diagnosing myocardial hypertrophy, a diagnostic kit including the same, and a method for diagnosing myocardial hypertrophy using the same.

One aspect provides a biomarker for diagnosing myocardial hypertrophy, comprising one or more proteins selected from the following group, wherein expression is relatively increased in cells that develop myocardial hypertrophy:

PDZ and LIM domain proteins having the amino acid sequence of SEQ ID NO: 1;

ADP-ribosylhydrolase having the amino acid sequence of SEQ ID NO: 2;

Peptidyl-prolyl cis-trans isomerase E having the amino acid sequence of SEQ ID NO: 3;

Translation elongation factor 1 delta isoform b of eukaryotic cells having the amino acid sequence of SEQ ID NO: 4;

Ran-specific GTPase-activating protein having the amino acid sequence of SEQ ID NO: 5;

ATP synthase subunit beta, mitochondrial precursor having the amino acid sequence of SEQ ID NO: 6; And

Isoform of Astrostatic Phosphoprotein PEA-15 Having the Amino Acid Sequence of SEQ ID NO: 7

Another aspect provides a biomarker for diagnosing myocardial hypertrophy, comprising one or more proteins selected from the following group, wherein expression is relatively reduced in cells with developing myocardial hypertrophy:

Isoform C protein of Lamin-A / C having the amino acid sequence of SEQ ID NO: 8;

A 75 kDa heat shock protein having the amino acid sequence of SEQ ID NO: 9;

Heat shock 70 kDa protein 14 having the amino acid sequence of SEQ ID NO: 10;

FK506-binding protein 4 having the amino acid sequence of SEQ ID NO: 11;

Poly (ADP-ribose) glycohydrolase ARH3 having the amino acid sequence of SEQ ID NO: 12;

Ubiquitin thioesterase protein OTUB1 having the amino acid sequence of SEQ ID NO: 13; And

Isoform short 14-3-3 protein β / α having the amino acid sequence of SEQ ID NO: 14.

According to one embodiment, the biomarker may include one or more proteins selected from the following group, and may include a biomarker for diagnosing myocardial hypertrophy in which expression is relatively increased in cells in which myocardial hypertrophy has developed.

PDZ and LIM domain proteins having a trypsin digested sequence of SEQ ID NOs: 15-21 and having a molecular weight of 34-37 kDa and a pi value of 6.3-6.5;

ADP-ribosylhydrolase having a trypsin digested sequence of SEQ ID NOs: 22 to 31 and having a molecular weight of 38 to 41 kDa and a pi value of 5.5 to 5.7;

Peptidyl-prolyl cis-trans isomerase E having a trypsin digested sequence of SEQ ID NOs: 32 to 39 and having a molecular weight of 32 to 35 kDa and a pi value of 5.3 to 5.5;

Translation elongation factor 1 delta isoform b of eukaryotic cells having a trypsin digested sequence of SEQ ID NOs: 40-47 and a molecular weight of 30-33 kDa and a pi value of 4.9-5.1;

Ran-specific GTPase-activating protein having a trypsin digested sequence of SEQ ID NOs: 48 to 50 and having a molecular weight of 22 to 25 kDa and a pi value of 5.4 to 5.6;

ATP synthase subunit beta, mitochondrial precursor, having a trypsin digested sequence of SEQ ID NOs: 51 and 52, having a molecular weight of 55 to 58 kDa and a pi value of 5.1 to 5.3; And

Isoform 1 of the astrogenic phosphoprotein PEA-15 having a trypsin digested sequence of SEQ ID NOs: 53-55 and having a molecular weight of 13-16 kDa and a pi value of 4.8-5.0.

According to one embodiment, the biomarker may include one or more proteins selected from the following group, and may include a biomarker for diagnosing myocardial hypertrophy in which expression is relatively reduced in cells in which myocardial hypertrophy has developed.

Isoform C protein of Lamin-A / C having a trypsin digested sequence of SEQ ID NOs: 56-61 and having a molecular weight of 64-67 kDa and a pi value of 6.3-6.5;

A 75 kDa heat shock protein having a trypsin digested sequence of SEQ ID NOs: 62 to 85 and having a molecular weight of 78 to 81 kDa and a pi value of 6.2 to 6.4;

Heat shock 70 kDa protein 14 having a trypsin digested sequence of SEQ ID NOs: 86-97 and having a molecular weight of 54-57 kDa and a pi value of 6.3-6.5;

FK506-binding protein 4 having a trypsin digested sequence of SEQ ID NOs: 98-100, having a molecular weight of 50-53 kDa and a pi value of 5.4-5.6;

Poly (ADP-ribose) glycohydrolase ARH3 having a trypsin digested sequence of SEQ ID NOs: 101 to 108 and having a molecular weight of 38 to 41 kDa and a pi value of 4.8 to 5.0;

Ubiquitin thioesterase protein OTUB1 having a trypsin digested sequence of SEQ ID NOs: 109-118 and having a molecular weight of 30-33 kDa and a pi value of 4.8-5.0; And

Isoform short 14-3-3 protein β / α having a trypsin digested sequence of SEQ ID NOs: 119 to 124 and a molecular weight of 26 to 29 kDa and a pi value of 4.7 to 4.9.

As used herein, the term “trypsin digested sequence” refers to the amino acid sequence of a polypeptide fragment produced by digesting the protein with trypsin for MALDI-TOF or MS / MS analysis. In addition, the molecular weight and pI are interpreted to include generally accepted experimental error ranges as values confirmed on two-dimentional electrophoresis.

Another aspect provides a myocardial hypertrophy diagnostic kit comprising an antibody that specifically binds to said protein or fragment of said protein.

A fragment of the protein can be, for example, an immunogenic fragment, which refers to a fragment of a biomarker protein having one or more epitopes that can be recognized by an antibody against a singular biomarker protein. According to one embodiment, the antibody may be a polyclonal antibody or a monoclonal antibody, but it is most preferred to use a monoclonal antibody.

The polyclonal antibody can be prepared by injecting a biomarker protein or fragment thereof as an immunogen into an external host according to conventional methods known to those skilled in the art. The external host can use mammals such as mice, rats, sheep, rabbits. The immunogen can be administered in combination with an adjuvant to increase antigenicity when injected by intramuscular, intraperitoneal or subcutaneous injection methods. Thereafter, blood may be collected periodically from an external host to collect serum showing enhanced titers and specificity for the antigen or to separate and purify the antibody therefrom.

The monoclonal antibodies can be prepared by the technology of generating immortalized cell lines by fusion known to those skilled in the art. Briefly, a method for producing a monoclonal antibody may be purified to immunize an appropriate amount (about 10 µg) in a Balb / C mouse or synthesize a polypeptide fragment of the protein and bind it to bovine serum albumin. After immunization, antigen-producing lymphocytes isolated from mice are fused with myeloma in humans or mice to produce immortalized hybridomas, and only the hybridoma cells that produce the desired monoclonal antibody are selected using the ELISA method. After propagation, monoclonal antibodies can be isolated and purified from the culture.

Myocardial hypertrophy diagnostic kit according to one embodiment may be prepared by conventional manufacturing methods known to those skilled in the art, and may typically include a freeze-dried form of the antibody and buffers, stabilizers, inert proteins and the like. The antibody can be labeled by radionuclides, fluorescors, enzymes, and the like.

Meanwhile, the monoclonal antibody according to one embodiment may be variously used in an immunoassay kit (eg, ELISA, antibody coated tube test, lateral-flow test, potable biosensor), and also has higher specificity and sensitivity. It can also be used to develop protein chips with various myocardial hypertrophy detection spectrum through the development of antibodies.

Another aspect is that the expression of one or more of the proteins having an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 7 from the biological sample suspected of myocardial hypertrophy is increased relative to normal cardiomyocytes, or the SEQ ID NO: 8 to SEQ ID NO: Detecting whether expression of at least one of the proteins having an amino acid sequence of 14 is reduced relative to normal cardiomyocytes; And determining whether my biological sample is myocardial hypertrophy from the detection result.

Such biological samples include, but are not limited to, for example, proteome extracted from mammalian myocardial tissue, myocardial cells, myocardial tissue or cardiomyocytes. Biomarker proteins having the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 14 are all mice ( Mus musculus ) and other mammals such as humans, monkeys, cows, horses, etc., the same protein is expressed, the homology (homology) is 90% or more database (http: //www.ncbi.nlm.nih. gov / blast). For example, PDZ and LIM domain proteins having the amino acid sequence of SEQ ID NO: 1 have 95% homology with human PDZ and LIM domain proteins. Therefore, it is apparent to those skilled in the art that the biomarker can be used for diagnosing myocardial hypertrophy of other mammals including humans.

In the diagnostic method, the detecting step is performed by performing two-dimensional electrophoresis on cardiomyocytes and normal cardiomyocytes suspected of myocardial hypertrophy to confirm the expression level of the biomarker protein, or cardiomyocytes suspected of myocardial hypertrophy. Contacting with a kit comprising an antibody according to one embodiment may comprise confirming the presence of the biomarker protein via an antigen antibody reaction. For example, when using two-dimensional electrophoresis, the proteome of normal cardiomyocytes and cardiomyocytes of a suspected myocardial hypertrophy patient is extracted, and the proteome is profiled by two-dimensional electrophoresis, from which the expression of the biomarker protein is expressed. If this increase or decrease is confirmed, it can be judged as myocardial hypertrophy. On the other hand, immunoassays currently well known as antigen antibody reactions include, for example, enzyme immunoassay (ELISA, Coated tube), a method using magnetic particles bound to antibodies, and a method using latex particles bound to antibodies.

According to one embodiment, a biomarker for diagnosing myocardial hypertrophy, a diagnostic kit including the same, and a method for diagnosing myocardial hypertrophy using the same, may be useful for determining myocardial hypertrophy from biological samples, and as a tool for identifying the pathogenesis of myocardial hypertrophy. Can be used.

Figure 1 shows the intracellular signal transduction pathway of endothelin-1 inducing myocardial hypertrophy.
Figure 2a shows the increase in the nucleus of ANF protein when treated with cells endothelin-1. CON refers to the control group, ET-1 refers to the experimental group treated with endothelin-1.
Figure 2b shows the mRNA transcription level of ANF in cells after treatment with endothelin-1 using RT-PCR.
Figure 3 separates the cell reactants in the case of not treated with endothelin-1 (A) and treated (B) using two-dimensional electrophoresis and displays differently expressed spots.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Identification of Induction of Myocardial Hypertrophy by Endothelin-1 in Myocardial Cell Line

Myocardial cell line HL-1 (Claycomb et al., Proc . Natl . Acad . Sci . USA 1998, 95, 2979-2984) was prepared in 10% FBS (Sigma), 0.1 mM norepinephrin (Sigma), in Claycomb medium (Sigma), 100 units / ml penicillin, 100 µg / ml streptomycin (Gibco / BRL) and 2 mM L-glutamine (Life Technologies) were added and cultured daily at 37 ° C., 5% CO ₂ . Incubate 2 x 10 ⁶ cells in 100 mm Petri dishes coated with 0.02% gelatin / 0.00125% fibronectin (Sigma), and stop cell growth in serum-free DMEM medium for 4 hours later. , 100 nM of endothelin-1 (Calbiochem) was added and left for 48 hours. Cells were then fixed, treated with anti-trial natriuretic factor antibody (Bachem), and then incubated with Alexa 546 labeled secondary mouse antibody (red) (Bachem). The cells were stained with DAPI (Invitrogen), and the images were collected using a confocal microscope (Zeiss).

From the results of FIG. 2A, it was confirmed that when endothelin-1 was treated, ANF protein in the nucleus was increased, and thus a signal inducing myocardial hypertrophy was transmitted by endothelin-1.

The myocardial cell line HL-1 cultured as above was treated with 100 nM endothelin-1 for 24 hours. Then, total RNA was isolated using RNeasy mini kit (Qiagen), and then 1 μg of total RNA was used as a template, and c-DNA synthesis was performed using M-MLV Reverse Transcriptase (Bioneer) to encode ANF protein. In this case, a primer (5'-GGCTCCTTCTCCATCACCCTG-3 ', SEQ ID NO: 125) and a reverse primer (5'-TCTTCGGTACCGGAAGCTGTT-3', SEQ ID NO: 126) were used. PCR was first performed with predenature for 4 minutes at 94 ° C, denature for 1 minute at 94 ° C, annealing for 1 minute at 53 ° C, and elongation for 1 minute at 72 ° C, followed by 10 cycles at 72 ° C. During post-elongation.

Figure 2b shows the results of the synthesis of cDNA encoding the ANF protein. When endothelin-1 was treated, it was found that the transcription of gene encoding ANF, a marker of myocardial hypertrophy, was increased. From this, it was confirmed that endothelin-1 induced myocardial hypertrophy.

Example  2: Identification of Proteins by Two-Dimensional Electrophoresis and Differential Expression of Myocardial Hypertrophy

After washing the endothelin-1 treated myocardial cell line HL-1 three times with cold PBS as in Example 1, lysis buffer (2 M urea, 2 M thiourea, 5% CHAPS, 50 mM DTT, 0.5% IPG buffer) The cells were added to a protease inhibitor cocktail, and then ultrasonically crushed. Thereafter, the mixture was shaken at room temperature for 1 hour, and the supernatant was recovered by centrifugation at 12,000 rpm for 15 minutes.

Rehydrate IPG strips (pH 4-7, 13 cm, linear; GE Healthcare) overnight with rehydration solution (8M urea, 2% CHAPS, 0.5% IPG buffer (pH 4-7), 18 mM DTT, bromophenol blue) rehydration). Then, using the Isoelectric focusing system (GE Healthcare), 100 V (3 hours), 500 V (1 hour), 1000 V (1 hour), 2500 V (1 hour), 8000 V (30 minutes) with a linear gradient, Then it was separated at 8000 V until 100,000 Vh was reached. After isoelectric focusing, the IPG strips were soaked in equilibration buffer (6M urea, 2% SDS, 75 mM Tris / HCl [pH 8.8], 29.3% glycerol, 0.01% DTT, 0.02% bromophenol blue) and then 15 minutes at 25 ° C. Shook for a while. After equilibration, the IPG strips are placed on a 10% polyacrylamide gel (25 x 35 cm), sealed with 0.5% agarose solution, and then electrophoresis buffer (24 mM Tris, 192 mM) in SE 600 Ruby electrophoresis system (GE Healthcare). glycine, 0.1% SDS) was added for 10 minutes at 10 mA, and then electrophoresis was performed under conditions of 30 mA until bromophenol blue was 0.5 cm from the bottom of the gel.

Thereafter, the separated gel was stained with a silver staining kit (GE Healthcare). The silver dyeing process is as follows. After fixing for more than 2 hours to a solution consisting of 50% methanol, 12% acetic acid, 0.05% formaldehyde, washed three times for 20 minutes with 50% ethanol, and then for 1 minute with 0.2% Sodium thiosulfate solution Sensitizing. Thereafter, the mixture was washed with distilled water, and then reacted with 0.1% silver nitrate, 0.075% formaldehyde (37%) solution for 20 minutes, washed again with distilled water, and then pre-cold 6% sodium carbonate, 0.075% formaldehyde (37%). After reacting with the solution for about 7 minutes, the solution was visualized (developing), and then the reaction was stopped by treating with 50% methanol and 12% acetic acid solution. Thereafter, the silver stained gel was scanned with an image scanner (GE Healthcare). Images of endothelin-1 treated gels were analyzed using Progenesis Workstation Software (Nonlinear Dynamics). Compared to the control and endothelin-1 treated gels, the spots that were consistently changed from each of the three gels were selected as differentially expressed proteins.

The results are shown in FIG. 3, and differently expressed spots are indicated by red circles.

Example  3: Identification and analysis of differentially expressed proteins

The spots selected as differentially expressed in Example 2 were cut out from the gel, destained for 5 minutes with a solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate in a 1: 1 mixture, and then distilled water four times for 5 minutes. Washed. The washed gel pieces were incubated in 200 mM ammonium bicarbonate solution at 37 ° C. for 30 minutes and then dehydrated twice with acetonitrile for 10 minutes. The dehydrated gel pieces were rehydrated with 10 ng / ul trypsin dissolved in 25 mM ammonium bicarbonate, which was incubated overnight at 37 ° C. to decompose into peptides. 0.1% trifluoroacetic acid dissolved in 60% acetonitril was incubated three times for 10 minutes to recover the cleaved peptides in a new tube. Finally, gel fragments were dehydrated using acetonitril and then recovered in a new tube. The collected supernatant was dried using a vacuum centrifuge.

After dissolving the dried sample in 0.4% acetic acid, peptides were separated for 40 minutes with a gradient of 5-40% acetonitril using an Agilent Seires 1200LC system (Agilent Technologies) and then for 10 minutes with a 40-80% acetonitril gradient. LTQ electrospray ionization linear single quadrupole ion-trap mass spectrometer (Thermo) was used.

All MS / MS spectra were tested using the International Protein Index mouse protein sequence database (IPI, version 3.24, European Bioinformatic Institute) using the SEQUEST search engine. The peptide mass tolerance was 3.0 Da, the fragment ion mass tolerance was 1.0 Da and the methionine oxidation. The parameters of the modification were set as conditions.

The protein searched by the above method is shown in Table 1 below, and includes the accession number, the molecular weight of the protein, the pI, the number of polypeptides detected, the coverage of the detected polypeptides for the whole protein, and the intensity change in 2D Numerical values) together. In addition, the amino acid sequence and position of the polypeptide fragment identified from the searched protein are shown in Table 2.

Spot No. Protein name Accession No. MW (Da) pI Matched peptide Coverage (%) change in intensity One Isoform C of Lamin-A / C
(SEQ ID NO: 8) P48678_2 65446 6.4 6 14 -5.08 2 Heat shock protein 75 kDa
(SEQ ID NO: 9) Q9CQN1 80209 6.3 30 50 -2.18 3 Heat shock 70 kDa protein 14
(SEQ ID NO: 10) Q99M31 55883 6.4 26 35 -1.03 4 FK506-binding protein 4
(SEQ ID NO: 11) P30416 51441 5.5 4 11 -2.74 5 PDZ and LIM domain protein 1
(SEQ ID NO: 1) O70400 35643 6.4 17 45 2.37 6 ADP-ribosylhydrolase
(SEQ ID NO: 2) Q8BGK2 39885 5.6 16 35 0.42 7 Peptidyl-prolyl cis-trans isomerase (SEQ ID NO: 3) Q9QZH3 33449 5.4 20 41 0.99 8 Eukaryotic translation elongation factor 1 delta isoform b (SEQ ID NO: 4) Q80T06 31292 5.0 6 24 1.34 9 Ran-specific GTPase-activating protein (SEQ ID NO: 5) P34022 23596 5.5 4 28 0.65 10 ATP synthase subunit beta, mitochondrial precursor
(SEQ ID NO: 6) P56480 56300 5.2 18 31 1.77 11 Poly (ADP-ribose) glycohydrolase ARH3
(SEQ ID NO: 12) Q8CG72 39414 4.9 17 36 0.8 12 Ubiquitin thioesterase protein OTUB1 (SEQ ID NO: 13) Q7TQI3 31270 4.9 58 63 -1.18 13 Isoform Short 14-3-3 protein β / γ (SEQ ID NO: 14) Q9CQV8_2 27854 4.8 7 32 -0.28 14 Isoform 1 of Astrocytic phosphoprotein PEA-15
(SEQ ID NO: 7) Q62048_1 15054 4.9 9 37 0.94

Spot
No. Protein Name Position Mass Identified Peptides One

Isoform C of Lamin-A / C

29-41 1628.80 LQEKEDLQELNDR (SEQ ID NO 56) 79-89 1164.54 AAYEAELGDAR (SEQ ID NO 57) 145-155 1118.58 EAALSTALSEK (SEQ ID NO 58) 281-296 1751.86 NSNLVGAAHEELQQSR (SEQ ID NO: 59) 299-311 1429.78 IDSLSAQLSQLQK (SEQ ID NO: 60) 528-541 1490.74 TALINSTGEEVAMR (SEQ ID NO: 61) 2


























Heat shock protein 75 kDa


























61-84 2643.26 STQAAEDKEEESLHSIISNTEAVR (SEQ ID NO 62) 98-105 926.59 KLLDIVAR (SEQ ID NO 63) 117-128 1288.65 ELISNASDALEK (SEQ ID NO: 64) 153-179 2845.49 KGTITIQDTGIGMTQEELVSNLGTIAR (SEQ ID NO: 65) 184-198 1635.81 AFLEALQNQAETSSK (SEQ ID NO: 66) 223-254 3249.56 SAAPESPGYQWLSDGSGVFEIAEASGVRPGTK (SEQ ID NO 67) 279-292 1647.83 YSNFVSFPLYLNGK (SEQ ID NO: 68) 294-306 1559.78 INTLQAIWMMDPK (SEQ ID NO: 69) 307-318 1598.70 DISEFQHEEFYR (SEQ ID NO: 70) 343-359 2031.98 SIFYVPEMKPSMFDVSR (SEQ ID NO 71) 360-370 1180.61 ELGSSVALYSR (SEQ ID NO 72) 391-404 1512.78 GVVDSEDIPLNLSR (SEQ ID NO: 73) 405-414 1170.66 ELLQESALIR (SEQ ID NO 74) 427-433 883.44 FFIDQSK (SEQ ID NO: 75) 442-451 1323.60 FFEDYGLFMR (SEQ ID NO 76) 452-468 1858.95 EGIVTTAEQDIKEDIAK (SEQ ID NO 77) 472-491 2125.03 YESSALPAGQLTSLPDYASR (SEQ ID NO: 78) 508-520 1574.72 HLAEHSPYYEAMK (SEQ ID NO 79) 549-565 2016.04 LISVETDIVVDHYKEEK (SEQ ID NO: 80) 576-589 1737.81 LSEKETEDLMAWMR (SEQ ID NO: 81) 596-604 1062.62 VTNVKVTFR (SEQ ID NO: 82) 605-621 1810.91 LDTHPAMVTVLEMGAAR (SEQ ID NO: 83) 637-649 1491.84 AQLLQPTLEINPR (SEQ ID NO 84) 680-689 1025.55 IAAGLVDDPR (SEQ ID NO 85) 3














Heat shock 70 kDa protein 14













34-44 1204.65 VTPAIVAYSER (SEQ ID NO: 86) 98-109 1452.71 LRYEIDTGEETK (SEQ ID NO 87) 110-118 1011.53 LVNPEDVAR (SEQ ID NO: 88) 153-168 1502.78 SALGEAAGAAGFNVLR (SEQ ID NO 89) 169-190 2287.20 LIHEPSAALLAYGIGQDHPTGK (SEQ ID NO: 90) 191-197 805.47 SNVLVFK (SEQ ID NO: 91) 198-216 1999.97 LGGTSLSLSVMEVNSGMYR (SEQ ID NO: 92) 217-245 3155.52 VLSTNTSDNIGGAHFTDTLAQYLASEFQR (SEQ ID NO: 93) 321-331 1208.57 QTGFTADDINK (SEQ ID NO 94) 403-425 2388.21 GVDESGADRFTVLFPSGTPLPAR (SEQ ID NO 95) 458-469 1402.78 FAQVVLQDLDKK (SEQ ID NO 96) 475-483 1002.58 DILAVLTMK (SEQ ID NO: 97) 4 FK506-binding protein 4 7-27 2063.05 AAENGAQSAPLPLEGVDISPK (SEQ ID NO: 98) 138-151 1531.70 GEDLTEEEDGGIIR (SEQ ID NO: 99) 358-372 1682.85 RGEAHLAVNDFDLAR (SEQ ID NO: 100) 5








PDZ and LIM domain protein 1







17-31 1628.89 LVGGKDFEQPLAISR (SEQ ID NO: 15) 87-98 1413.76 IWSPLVTEEGKR (SEQ ID NO: 16) 103-121 2102.03 MNLASEPQEVLHIGSAHNR (SEQ ID NO: 17) 122-135 1419.68 SAMPFTASPAPSTR (SEQ ID NO: 18) 136-163 3089.46 VITNQYNSPTGLYSSENISNFNNAVESK (SEQ ID NO: 19) 164-195 3396.68 TSASGEEANSRPVVQPHPSGSLIIDKDSEVYK (SEQ ID NO: 20) 209-235 2948.49 QSTSFLVLQEILESDGKGDPNKPSGFR (SEQ ID NO: 21) 6









ADP-ribosylhydrolase









25-38 1572.80 ENSVLGSIQEELQK (SEQ ID NO: 22) 39-51 1270.69 TGGLDSLVLSPGR (SEQ ID NO: 23) 52-61 1198.54 WPVSDNTIMH (SEQ ID NO: 24) 86-101 1857.87 YVETVETLSEHRPDPS (SEQ ID NO: 25) 187-193 854.48 PLVQWGR (SEQ ID NO 26) 212-226 2014.87 HMAEYQEHWFYFEAK (SEQ ID NO 27) 227-234 1169.55 WQFYLEER (SEQ ID NO 28) 236-255 2411.09 IREDAEDKVTFPDNYDAEER (SEQ ID NO 29) 327-335 1115.56 GLYQELEHK (SEQ ID NO: 30) 336-347 1306.71 GRLEDLGAALHR (SEQ ID NO 31) 7








Peptidyl-prolyl cis-trans isomerase E








6-20 1661.86 RVLYVGGLAEEVDDK (SEQ ID NO: 32) 21-45 2844.46 VLHAAFIPFGDITDIQIPLDYETEK (SEQ ID NO: 33) 94-104 1387.68 PVWSDDDWLKK (SEQ ID NO 34) 109-134 2836.33 TLEENKEEEGPEPPKAEAQEGEPTAK (SEQ ID NO: 35) 138-147 1193.58 SNPQVYMDIK (SEQ ID NO: 36) 162-173 1364.64 SDVVPMTAENFR (SEQ ID NO 37) 218-227 1267.64 KFDDENFILK (SEQ ID NO: 38) 262-276 1668.88 HVVFGEVTEGLDVLR (SEQ ID NO 39) 8








Eukaryotic translation elongation factor 1 delta isoform b






6-20 1661.86 RVLYVGGLAEEVDDK (SEQ ID NO: 40) 21-45 2844.46 VLHAAFIPFGDITDIQIPLDYETEK (SEQ ID NO: 41) 94-103 1259.58 PVWSDDDWLK (SEQ ID NO 42) 109-134 2836.33 TLEENKEEEGPEPPKAEAQEGEPTAK (SEQ ID NO: 43) 138-147 1193.58 SNPQVYMDIK (SEQ ID NO: 44) 162-173 1364.64 SDVVPMTAENFR (SEQ ID NO 45) 218-227 1267.64 KFDDENFILK (SEQ ID NO 46) 262-276 1668.88 HVVFGEVTEGLDVLR (SEQ ID NO 47) 9
Ran-specific GTPase-activating protein
6-39 3888.68 DSHEDHDTSTENADESNHDPQFEPIVSLPEQEIK (SEQ ID NO 48) 40-50 1380.63 TLEEDEEELFK (SEQ ID NO 49) 58-68 1334.61 FASENDLPEWK (SEQ ID NO 50) 10 ATP synthase subunit beta, mitochondrial precursor 42-53 1274.64 ELISNASDALDK (SEQ ID NO: 51) 73-82 1193.64 IDIIPNPQER (SEQ ID NO 52) 11










Poly (ADP-ribose) glycohydrolase ARH3










57-75 1946.97 VLSHVESLEPDPGTPGSAR (SEQ ID NO: 101) 76-89 1679.73 TETLYYTDDTAMTR (SEQ ID NO: 102) 90-109 2242.14 ALVQSLLAKEAFDEVDMAHR (SEQ ID NO: 103) 121-131 1110.61 GYGAGVITVFK (SEQ ID NO: 104) 163-178 1663.88 VAGISLAYSSVQDVQK (SEQ ID NO: 105) 240-251 1452.67 ELGMEERPYSSR (SEQ ID NO: 106) 252-266 1697.97 LKKVGELLDQDVVSR (SEQ ID NO: 107) 285-309 2952.41 TAIYCFLRCMEPHPEIPSTFNSLQR (SEQ ID NO: 108) 12

















Ubiquitin thioesterase protein OTUB1
















37-51 1722.93 IQQEIAVQNPLVSER (SEQ ID NO: 109) 52-59 963.56 LELSVLYK (SEQ ID NO: 110) 60-71 1514.65 EYAEDDNIYQQK (SEQ ID NO: 111) 95-113 2175.10 AFGFSHLEALLDDSKELQR (SEQ ID NO: 112) 121-151 3675.73 SKEDLVSQGFTEFTIEDFHNTFMDLIEQVEK (SEQ ID NO: 113) 177-185 1049.59 LLTSGYLQR (SEQ ID NO: 114) 186-198 1581.76 ESKFFEHFIEGGR (SEQ ID NO: 115) 228-238 1325.63 SVSIQVEYMDR (SEQ ID NO: 116) 239-256 1838.84 GEGGTTNPHVFPEGSEPK (SEQ ID NO: 117) 257-271 1912.02 VYLLYRPGHYDILYK (SEQ ID NO: 118) 13




Isoform Short 14-3-3 protein β / γ




42-49 906.52 NLLSVAYK (SEQ ID NO: 119) 104-115 1349.72 YLILNATQAESK (SEQ ID NO: 120) 128-138 1195.57 YLSEVASGENK (SEQ ID NO: 121) 138-157 2286.11 KQTTVSNSQQAYQEAFEISK (SEQ ID NO: 122) 194-212 2158.00 TAFDEAIAELDTLNEESYK (SEQ ID NO: 123) 213-222 1188.65 DSTLIMQLLR (SEQ ID NO: 124) 14

Isoform 1 of Astrocytic phosphoprotein PEA-15
36-54 2169.00 SEEITTGSAWFSFLESHNK (SEQ ID NO 53) 55-71 2091.06 LDKDNLSYIEHIFEISR (SEQ ID NO 54) 72-83 1476.78 RPDLLTMVVDYR (SEQ ID NO: 55)

Attach an electronic file to a sequence list

Claims (7)

delete delete A myocardial hypertrophy diagnostic kit comprising an antibody that specifically binds to an isoform 1 protein or fragment of an astrogenic phosphoprotein PEA-15 having the amino acid sequence of SEQ ID NO: 7. 4. The myocardial hypertrophy diagnostic kit according to claim 3, wherein the antibody is a monoclonal antibody. delete delete The method of claim 3,
PDZ and LIM domain proteins having the amino acid sequence of SEQ ID NO: 1;
ADP-ribosylhydrolase having the amino acid sequence of SEQ ID NO: 2;
Peptidyl-prolyl cis-trans isomerase E having the amino acid sequence of SEQ ID NO: 3;
Translation elongation factor 1 delta isoform b of eukaryotic cells having the amino acid sequence of SEQ ID NO: 4;
Ran-specific GTPase-activating protein having the amino acid sequence of SEQ ID NO: 5;
ATP synthase subunit beta, mitochondrial precursor having the amino acid sequence of SEQ ID NO: 6;
Isoform C protein of Lamin-A / C having the amino acid sequence of SEQ ID NO: 8;
A 75 kDa heat shock protein having the amino acid sequence of SEQ ID NO: 9;
Heat shock 70 kDa protein 14 having the amino acid sequence of SEQ ID NO: 10;
FK506-binding protein 4 having the amino acid sequence of SEQ ID NO: 11;
Poly (ADP-ribose) glycohydrolase ARH3 having the amino acid sequence of SEQ ID NO: 12;
Ubiquitin thioesterase protein OTUB1 having the amino acid sequence of SEQ ID NO: 13; And
The kit further comprises an antibody that specifically binds to at least one protein selected from the group of isoform short 14-3-3 protein β / α or fragments of said protein having the amino acid sequence of SEQ ID NO: 14.

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