KR20200048322A - Composition or kit for diagnosing diabetes mellitus and method of detecting a biomarker for diagnosis of diabetes mellitus using the same - Google Patents

Abstract

Provided are a composition for diagnosing diabetes, a kit for the same, and a method for detecting a protein selected from a group comprising SORT1, CALR, and RAB1A or a fraction thereof for providing information required for diagnosing diabetes using the same. Accordingly, the composition can be used for simply, highly accurately, and specifically diagnosing diabetes, for example type 2 diabetes. Also, the composition is used as a biomarker for predicting progress of the diabetes and a novel treatment target for diabetes treatment. Therefore, a material having an effect on prevention or treatment of the diabetes can be screened.

Description

Composition or kit for diagnosing diabetes mellitus and method of detecting a biomarker for diagnosis of diabetes mellitus using the same}

Diabetes diagnostic composition, kit and a method for detecting biomarkers SORT1, CALR, and RAB1A for diagnosis of diabetes using the same.

Mitochondria are intracellular organs responsible for ATP production from glucose and fatty acid oxidation, and are essential organelles for various cellular activities that require ATP, such as cell proliferation or differentiation. On the other hand, insulin secretion reduction and insulin resistance are known as etiologies of type 2 diabetes. Mitochondrial dysfunction in pancreatic beta cells inhibits insulin secretion, and skeletal muscle mitochondrial dysfunction is associated with reduced fatty acid oxidation, accumulation of lipid metabolites in the cell, and inhibition of the insulin signaling pathway. Accordingly, there is increasing evidence that mitochondrial dysfunction plays an important role in the pathogenesis of type 2 diabetes, because it is involved in both insulin secretion and suppression of reactions in reactive tissues.

Mitochondrial proteome analysis is a method for evaluating mitochondrial function associated with type 2 diabetes by providing comprehensive information on thousands of mitochondrial proteins compared to genome analysis based on a limited number of genes. . Accordingly, a preteome study was conducted in diabetics or animal models, and studies were conducted to discover proteins that increase expression in diabetes.

However, no studies have been conducted on mitochondrial proteomics to confirm the proteome profile of mitochondrial function by comparing diabetic and non-diabetic groups in insulin-responsive tissues.

Therefore, it is necessary to develop a biomarker that can diagnose and diagnose diabetes easily and has high accuracy and specificity of diagnosis.

Provided is a composition for diagnosing diabetes.

Provided is a diabetic diagnostic kit.

It provides a method for detecting biomarkers in a sample derived from muscle tissue for the diagnosis of diabetes.

One aspect is from a sample derived from muscle tissue to Sortilin 1: SORT1, Calreticulin (CALR), and Ras-related protein Rab-1A (RAB1A). It provides a composition for diagnosing diabetes, comprising an agent for measuring the expression level of a protein or fragment thereof selected from the group consisting of.

The sample may be muscle tissue isolated from an individual. The sample may be a whole protein isolated from muscle tissue or a fraction fractionated therefrom. The sample may be a fraction containing mitochondria. The sample may include a polypeptide or nucleic acid isolated from muscle tissue mitochondria.

Sortilin 1: SORT1 is a type I membrane glycoprotein of the vacuolar protein sorting 10 protein (Vps10p) family. SORT1 can also be called Gp95, LDLCQ6, NT3, or NTR3. SORT1 is a protein commonly expressed in many tissues and is present in the central nervous system. At the cellular level, sorbitin functions to transport proteins between the Golgi apparatus, endosomes, lysosomes, and the plasma membrane. In addition, sorbitin is also associated with diseases such as atherosclerosis, coronary artery disease, Alzheimer's disease, and cancer. The sorbitin 1 may include the amino acid sequence of Uniprot No. Q99523 in humans and the amino acid sequence of Uniprot No. Q6PHU5 in mice. The sorbitin 1 is GenBank Accession No. NM_001205228 or NM_002959Q99523, and may include a polypeptide encoded by the nucleotide sequence of GenBank Accession No. It may include a polypeptide encoded by the nucleotide sequence of NM_001271599 or NM_019972.

Calreticulin (CALR) is also known as calregulin, CRP55, CaBP3, calsequestrin-like protein, and endoplasmic reticulum resident protein 60. CALR is a multifunctional protein that binds calcium ions when inactive. CALR has a low affinity to calcium ions, but can bind with high capacity (capacity) to deliver calcium signals. CALR can exist in storage compartments associated with vesicles. CALR can bind to a poorly folded protein and prevent it from being transported from the endoplasmic reticulum to the Golgi apparatus. CALR can also be present in the nucleus and participate in the regulation of transcription. The CALR is known to be associated with systemic lupus, Sjogren's disease, and cancer. The CALR may comprise the amino acid sequence of Uniprot No. P27797 in humans and the amino acid sequence of Uniprot No. P14211 in mice. The CALR is a GenBank Accession No. NM_004343 may include a polypeptide encoded by the nucleotide sequence of GenBank Accession No. It may include a polypeptide encoded by the nucleotide sequence of NM_007591.

Ras-related protein Rab-1A (RAB1A) may be a protein that interacts with CHML, GOLGA2, GOLGA5, MICAL1, RABAC1, and CHM. The small GTPase Rab plays a role in intracellular membrane transport, from the formation of transport vesicles to fusion with the cell membrane. Rab cycles through the inactive GDP-bound and GTP-bound forms, which can attract substances downstream of signaling that are directly responsible for vesicle formation, migration, tethering and fusion. RAB1A regulates vesicular protein transport from endoplasmic reticulum to the Golgi apparatus and to the cell surface and may play a role in IL-8 and growth hormone secretion. It regulates the level of CASR present in the cell membrane. Through its function in protein transport, it can play a role in cell adhesion and cell migration. The RAB1A may comprise the amino acid sequence of Uniprot No. P62820 in humans and the amino acid sequence of Uniprot No. P62821 in mice. The RAB1A is a GenBank Accession No. NM_004161 or NM_015543, and may include a polypeptide encoded by the nucleotide sequence of GenBank Accession No. It may include a polypeptide encoded by the nucleotide sequence of NM_008996.

The composition is selected from the group consisting of cyclooxygenase-2 (COX2) and NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 3: NDUFS3). It may further include an agent for measuring the expression level of the protein or fragment thereof.

Cyclooxygenase (COX), also known as prostaglandin-endoperoxide synthase (PTGS), is an enzyme responsible for the formation of prostanoids, including prostaglandin and thromboxane. to be. Of the cyclooxygenases, cyclooxygenase-2 (COX2) is involved in the conversion of arachidonic acid to prostacyclin H2, an important precursor of prostacyclin. COX2 is underexpressed under normal conditions in most cells, but expression is elevated during the inflammatory response. The COX2 may include the amino acid sequence of Uniprot No. P35354 in humans and the amino acid sequence of Uniprot No. Q05769 in mice. The COX2 is a GenBank Accession No. NM_000963 may include a polypeptide encoded by the nucleotide sequence of GenBank Accession No. It may include a polypeptide encoded by the nucleotide sequence of NM_011198.

NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NDUFS3) is a component of the mitochondrial NADH: ubiquinone oxidoreductase (complex I), a mutation in the gene encoding it causes a mitochondrial complex I abnormality It is associated with Leigh syndrome. The NDUFS3 may include the amino acid sequence of Uniprot No. O75489 in humans and the amino acid sequence of Uniprot No. Q9DCT2 in mice. The NDUFS3 is a GenBank Accession No. NM_004551 may include a polypeptide encoded by the nucleotide sequence of GenBank Accession No. It may include a polypeptide encoded by the nucleotide sequence of NM_026688.

The fragment may be an immunogenic polypeptide as part of the protein.

Diabetes mellitus (DM) is a type of metabolic disease, such as insufficient insulin secretion or normal function, and is characterized by high blood sugar level and high blood sugar level. To release glucose. The diabetes may be type 2 diabetes mellitus (T2DM). Type 2 diabetes is characterized by insulin resistance (the ability of insulin to lower blood sugar, which prevents cells from effectively burning glucose).

The agent may be an antibody or antigen-binding fragment thereof that specifically binds the protein or fragment thereof. It may be an antibody or antigen-binding fragment thereof that specifically binds to the protein or fragment thereof. The antibody can be a polyclonal antibody or a monoclonal antibody. The term "antibody" can be used interchangeably with the term "immunoglobulin". The antibody can be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide comprising an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab ', scFv, diabody, nanobody, minibody, tetrabody, triabodies, V _H domain, or V _L domain. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a surface of a metal chip, plate, or well.

The agent may be a nucleic acid comprising a polynucleotide identical to or complementary to the polynucleotide encoding the protein or fragment thereof. The nucleic acid may be a primer or a probe. The primer or probe may be labeled with a fluorescent material, chemiluminescent or radioactive isotope on its terminal or inside.

Another aspect provides a kit for diagnosing diabetes, comprising an agent that measures the expression level of a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A, from a sample derived from muscle tissue.

The kit may further include a sample necessary for diagnosing diabetes. The kit may include a substrate, a suitable buffer solution, a chromogenic enzyme, a fluorescently labeled secondary antibody, or a chromogenic substrate for immunological detection of a solid support, antibody or antigen-binding fragment. The kit may include a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof for nucleic acid detection. The polymerase is, for example, Taq polymerase.

Another aspect is measuring the expression level of a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A in a sample derived from muscle tissue isolated from a subject suspected of diabetes; And comparing the measured expression level with the protein expression level of a normal control, a method for detecting a protein or a fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A to provide information necessary for diagnosis of diabetes. Gives

The method includes measuring the expression level of a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A in a sample derived from muscle tissue isolated from an individual suspected of having diabetes.

The subject can be a mammal, for example, a human, cow, horse, pig, dog, sheep, goat, or cat. The individual may be an individual suffering from or suspected of having diabetes.

The measuring step may include incubating the sample and an antibody specifically binding to a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A.

The method may further include measuring the expression level of a protein or fragment thereof selected from the group consisting of COX2 and NDUFS3 in the sample. The measuring step may include incubating an antibody specifically binding to a protein or fragment thereof selected from the group consisting of COX2 and NDUFS3.

The measurement steps include muscle biopsy, electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunostaining, protein chip, immunoprecipitation, microarray, Northern blotting, polymer It may be performed by a polymerase chain reaction (PCR), or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, two-dimensional electrophoresis, or a combination thereof. The PCR may be real-time PCR or reverse transcription PCR.

The method includes comparing the measured expression level to the protein expression level of a normal control.

In this method, the measured amount of expression level of SORT1, CALR, or RAB1A can be increased compared to the expression level of a normal control. The normal control group is a group not affected by diabetes, and means a negative control group. For example, if the amount of SORT1, CALR, or RAB1A detected is higher than the amount of a normal control, the subject can be diagnosed as having or is more likely to develop diabetes, eg type 2 diabetes. Treatment of diabetes, for example, insulin injection and metformin administration to an individual diagnosed with type 2 diabetes may be performed.

Other aspects provide SORT1, CALR, RAB1A, COX2, or NDUFS3 biomarkers to predict the progression of diabetes. Another aspect provides a method of screening a substance effective for the prevention or treatment of diabetes by using SORT1, CALR, RAB1A, COX2, or NDUFS3 as a new treatment target for diabetes treatment.

According to a method for detecting a protein or a fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A in order to provide information necessary for diagnosing diabetes, using the composition, kit, and diabetes diagnosis according to one aspect, diabetes, for example It can be used to diagnose type 2 diabetes with ease, high accuracy and specificity.

Figure 1a shows the sequence of the experiment to identify the mitochondrial biomarkers associated with diabetes in skeletal muscle tissue, Figure 1b is for the normalized elution time (NET) in the established database AMT (accurate mass and time tag) database (DB) A graph showing the molecular weight (Da), FIG. 1C is a graph showing the intrinsic mass classification (UMC) identified in each LC-MS / MS dataset as the molecular weight (Da) for the NET, and FIG. 1D NET the designated UMC It is a graph represented by the molecular weight (Da) for.
Figure 2a is the present invention and reference Pagliarini et al. And the mitochondrial protein identified in Lefort et al., FIG. 2B is a graph showing the intracellular location of the identified mitochondrial protein, and FIG. 2C is a location distribution within the mitochondrial of the identified mitochondrial protein Graph, Figure 2d is a graph showing the function of the identified mitochondrial protein, Figure 2e shows the distribution of the identified mitochondrial protein to the mitochondrial complex I to V, Figure 2f is the identified mitochondrial protein by mitochondrial (MT) DNA It is a graph indicating whether it is encoded or encoded by nuclear (Nu) DNA.
Figure 3a is a graph analyzing the protein that is up-regulated or down-regulated compared to the normal control in type 2 diabetes, Figure 3b is a graph showing the intracellular function of the up-regulated protein, Figure 3c is a cell of the down-regulated protein 3D is a schematic diagram of an intracellular network model showing type 2 diabetes-related processes of up- or down-regulated proteins, and FIG. 3E shows membrane and vesicle-related processes of up- or down-regulated proteins. It is a schematic diagram of the intracellular network model shown.
Figure 4a is a graph showing the differential expression level of biomarkers selected from skeletal muscle-derived total proteins of high-fat diet mice (HFD) or normal diet mice (NCD), and FIG. 4b is bio-selected from skeletal muscle-derived mitochondrial proteins of HFD or NCD Graph showing the differential expression level of the marker, Figure 4c is a graph showing the differential expression level of the biomarker selected from the total protein derived from human skeletal muscle, Figure 4d is a differential expression of the biomarker selected from the mitochondrial protein derived from human skeletal muscle It is a graph showing the level (Mann-Whitney test, *: P <0.05, **: P <0.01).

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Identification of mitochondrial biomarkers associated with diabetes in skeletal muscle tissue

1. Identification of intrinsic mass classification of mitochondrial protein derived from skeletal muscle tissue

(1) Sample preparation

To investigate the mitochondrial proteins associated with T2DM, patients with type 2 diabetes mellitus (T2DM) undergoing elective orthopedic surgery at Seoul National University Bundang Hospital (n = 9; 4 men and 5 women) and non-diabetic normal Controls (n = 9; 5 males and 4 females) were selected. T2DM patients diagnosed with diabetes were screened according to American diabetes association (ADA) criteria. The control group was also selected according to the same ADA standard. Written consent was obtained from everyone, and the study was conducted in accordance with the Helsinki Declaration and was approved by the SNUH [SNUBH IRB # B-0710 / 050-009] Ethics Committee. The characteristics of T2DM patients and non-diabetic controls such as age, gender, body mass index, fasting blood glucose, and other experimental values are summarized in Table 1 below.

Normal group (n = 9) Diabetes group (n = 9) p-value age 56.4 ± 14.7 61.0 ± 14.8 0.52 Gender (male: female) 5: 4 4: 5 Duration of diabetes 8.4 ± 11.8 Body mass index (kg / m ² ) 24.7 ± 4.0 27.1 ± 3.7 0.20 Systolic blood pressure (mm / Hg) 129.2 ± 16.8 136.3 ± 15.1 0.36 Diastolic blood pressure (mm / Hg) 75.3 ± 13.0 77.6 ± 11.2 0.70 Fasting blood sugar (mg / dl) 93.9 ± 7.7 140.6 ± 46.3 <0.01 * Hemoglobin A1c (%) 5.5 ± 0.3 7.3 ± 1.3 <0.001 * Total cholesterol (mg / dl) 177.1 ± 16.8 223.1 ± 68.6 0.07 BUN 14.8 ± 5.4 16.2 ± 6.8 0.62 Cr 1.1 ± 0.4 1.0 ± 0.1 0.41

(Data is expressed as mean value ± standard deviation, *: p <0.05)

(2) Separation of mitochondria

After obtaining skeletal muscle tissue from each T2DM patient and normal control, the obtained tissue was homogenized in mitochondrial separation buffer (250 mM sucrose and 1 mM EDTA in 25 mM Tris-HCl pH7.4). Thereafter, the lysate was centrifuged at 1,000 g to separate the nuclear and cytoplasmic fractions, and the cytoplasmic fraction was further centrifuged at 4 ° C. to 10,000 g to obtain crude mitochondrial pellets. The isolated mitochondria were further purified through a 10% to 40% percoll gradient by centrifugation for about 120 minutes at 20000 rpm in a Beckman SW41 rotor. The upper and lower fractions (P1 and P3 fractions, respectively) of the mitochondrial layer during the gradient were carefully discarded. The remaining mitochondrial layer (P2 fraction) was washed twice, and the separated mitochondria were stored at -70 ° C until use.

(3) Preparation of mitochondrial proteins and peptides

To detect membrane proteins closely related to mitochondrial physiology, the mitochondria isolated from skeletal muscle tissue was dissolved in 4% (w / v) SDS lysis buffer in Tris HCl at pH 7.6, and the solution contained 2 mm glass beads. Transferred to a 1.5 ml e-tube. The mitochondrial containing solution was crushed in a repeat stroke for 30 seconds at 1,000 rpm with a Mini-BeadBeater ^™ grinder (Cole-Parmer Instrument, Vernon Hills, IL) and cooled with ice for 1 minute. This process was repeated until no pellets were visible (about 10-15 times). The supernatant was transferred to a new tube and centrifuged at 5,000 rpm for 10 min at 20 ° C. to remove debris. The supernatant was transferred to a new tube, sonicated for 30 seconds using a Q55 sonicator (Qsonica, Newtown, CT) and cooled for 1 minute. The ultrasonic dissolution was repeated 10 times on ice. The lysate was centrifuged at 14,000 g at 20 ° C. for 10 minutes, the supernatant was transferred to a new tube, and the protein concentration was measured using BCA protein assay (Pierce, Rockford, IL) and bovine serum albumin.

Mitochondrial peptide samples were prepared by a modified filter-aided sample preparation (FASP) method. Briefly, 100 μg of protein lysate was reduced in 100 μl of SDT (0.1 M Tris-HCl, SDS 4% (w / v) SDS and 0.1 M DTT in pH 7.6) at 37 ° C. for 45 min. 10 Boil for a minute to denature the protein. After sonication for 10 minutes, samples were centrifuged at 16,000 g for 5 minutes. The reduced protein target was mixed with 200 μl of 8 M urea in Microcon filter YM-30 (Millipore Corporation, Bedford, MA), and the filter was centrifuged at 14,000 g for 60 minutes. All centrifugation steps were performed at 20 ° C. To remove residual SDS, 200 μl of 8 M urea in 0.1 M Tris-HCl (pH 8.5) was added to the filter, and then the filter was centrifuged at 14,000 g. This washing step was repeated twice. Thereafter, 100 μl of 50 mM iodine acetamide of 8 M urea in 0.1 M Tris-HCl was added to the concentrate for alkylation, alkylated at 25 ° C. for 25 minutes in a dark room, and centrifuged at 14,000 g for 30 minutes to generate an alkylation reagent. Was removed. The obtained sample was washed twice with 200 μl of 8 M urea in 0.1 M Tris-HCl, and then twice with 100 μl of 50 mM NH ₄ HCO ₃ . The alkylated protein sample was digested by proteolysis using trypsin (1:50 enzyme to protein ratio (w / w), Promega, Madison, WI) at 600 rpm for 1 minute at 37 ° C., followed by thermo-mixer. Incubation overnight for 12 hours without stirring at comfort (Eppendorf, Hamburg, Germany). The second trypsin digestion (1: 100 enzyme to protein ratio) was performed for 6 hours. The cleaved peptide was eluted by centrifugation at 14,000 g for 20 minutes. The filter was further rinsed with 60 μl of 50 mM NH ₄ HCO ₃ and centrifuged at 14,000 g for 30 minutes. The eluent was mixed with the first eluent. The peptide sample was dried in a vacuum concentrator SPD1010 (Thermo, San Jose, CA) and stored at -80 ° C.

(4) LC-MS / MS analysis

LC-MS / MS experiments were repeated three times on each of the mitochondrial protein samples obtained from nine T2DM patients and nine non-diabetic controls to obtain a 54 (18 × 3) LC-MS / MS dataset.

Specifically, peptides were isolated using a modified version of the nanoACQUITY UPLC system. The analytical column (75 mm ID × 360 mm OD × 70 cm long) was fused with C18 material (3 mm diameter, 300 mm 3 pore size, Jupiter, Phenomenex, Torrance, CA) using acetonitrile slurry (Polymicro Technologies) , Phoenix, AZ) and prepared in-house. Solid phase extraction (SPE) columns are prepared by packing the same C18 material into a 1 cm long linear column (250 mm ID) of an internal reducer (1/16 "to 1/32", VICI, Houston, TX). Did. The column temperature was set to 50 ° C. using a semi-rigid gasoline heater (1/4 ”ID, 60 cm long, WATLOW, Winona, MN.) LC separation gradient was 98% (v / v) solvent A (H ₂ O 5 minutes in 0.1% formic acid), 115% in 2% (v / v) to 50% (v / v) solvent B (0.1% formic acid in 99.9% acetonitrile), 50% to 80% (v / v) ), 10 minutes in solvent B, and 80% in solvent B. The flow rate of the mobile phase was set to 400 nL / min 7-Tesla Fourier transform ion cyclotron resonance mass spectrometer (FTICR, LTQ-FT, Thermo Electron) , San Jose, CA) The peptides eluted from the LC were ionized at an electron injection potential of 2.0 kV Electrospray ionization (ESI) was a fusion-silica capillary emitter. (20 mm ID × 150 mm OD). The temperature of the desolvation capillary was set to 250 ° C. The MS precursor ion scan (m / z 500-2,000) was 1 × 10 ⁶ A GC target values, mass resolution of 1 × 10 ⁵ and maximum ion accumulation time of 500 ms were obtained in full profile mode.The mass spectrometer was operated in a data-dependent tandem MS mode, and the seven most abundant ions detected in the precursor MS scan were By selecting dynamically for MS / MS experiments with dynamic exclusion options (exclusion mass range lower limit = 1.10 Th, exclusion mass range upper limit = 2.10 Th; exclusion list size = 120; and exclusion duration = 30 seconds) of the same peptide. Re-acquisition of the MS / MS spectrum was prevented The collision-induced dissociation of precursor ions was performed in an ion trap (LTQ) with collision energy and separation width set at 35% and 3 Th, respectively Xcalibur software package (v. 2.0 SR2, Thermo Electron). All LC-MS / MS data was deposited with the ProteomeXchange consortium through the PRIDE partner repository with dataset identifiers PXD004312 and 10.6019 / PXD004312 (username: reviewer16736@ebi.ac.uk, password: F64K10U7).

(5) Identification of peptides

To the resulting LC-MS / MS for the data set, assigns the correct precursor (precursor) by mass in tandem mass spectrometry data was applied to PE i-MMR (integrated post experiment monoisotopic mass refinement) analysis method. Briefly, DeconMSn was used to further correct the precursor mass and generate improved MS / MS data (DTA file) via PE-MMR. The obtained mass-improved DTA file was subjected to multivariate mass error correction using DTARefinery. DTA files were converted to mgf files using in-house software. The obtained mgf file is a database using the UniProt human reference database (April 2014, 88,608 items) and MS-GF + (version 9881) against a complex target-decoy database containing forward and reverse protein sequences of 179 common contaminants. Used for search. The MS-GF + search was performed with the following parameters: 10 ppm precursor mass tolerance, semi-trypsin, static modification of carbaamidomethylation to cysteine (57.021460Da), and methionine at the N-terminus and Variable modification of oxidation (15.994920Da) to carbamylation (43.005810Da). Peptide spectrum match (PSM) was obtained using a false discovery rate (FDR) of 1%.

(6) Designation of MS intensity for peptide identification

MS intensity-based, label-free quantitative analysis was applied to the LC-MS / MS dataset to identify the identified peptides according to the unique mass class (UMC).

 Briefly, during PE-MMR analysis, MS characteristics of peptides that appeared during the period of LC elution time in LC-MS / MS experiments were grouped into a unique mass class (UMC). Each UMC included all mass spectral properties of the peptide, such as charge state, abundance, number of scans, and measured monoisotopic mass. The peptide is represented by UMC. For each UMC, the UMC mass was calculated as the intensity weighted average of the monoisotopic masses of all UMC components, and the sum of the abundances (UMC intensities) for all spectral components of the UMC was calculated to determine the abundance of the corresponding peptide. Was marked. During PE-MMR analysis, the precursor mass (or DTA file) of the MS / MS spectrum matched the UMC mass and replaced with UMC mass if a match was identified. In this process, DTA information was linked to the matched UMC. If the linked DTA file became a peptide sequence of FDR≤1% after MS-GF + search and target-decoy analysis, the peptide ID was recorded in UMC and the UMC intensity was assigned to the peptide ID. To minimize the LC elution time variation of the same peptide in different LC / MS runs, normalized elution time (NET) was calculated for the identified UMC.

2. Identification of the intrinsic mass classification of mitochondrial proteins derived from cytoplasmic hybrid cells

Wild-type cytoplasmic (cybrid) cells were described in Chae S et al., Sci. Signal 2013; 6 (264): Prepared as described in rs4. Example 1. Mitochondria were isolated from cytoplasmic hybrid cells, as described in (2) and (3).

The isolated mitochondria were fractionated by mitochondrial peptide by offgel electrophoresis. Specifically, 500 μg trypsin cleavage peptide from the P2 mitochondrial fraction of cytoplasmic hybrid cells was fractionated with 3100 OFFGEL Fractionator (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol, and the offgel fraction was performed in 26 fractions. A tray of 24 wells was used as a 24 cm IPG gel strip of a linear pH gradient ranging from 3 to 10 (Immobiline DryStrip, GE Healthcare Life Sciences, Uppsala, Sweden), and the gel strip was 15 minutes with 25 mL of rehydration solution per well During rehydration. Trypsin cleavage peptide was dissolved in 0.72 ml deionized water and mixed with 2.88 ml offgel stock solution. To each well, 150 ml of the obtained solution was loaded. Focusing was performed at a voltage range of 500 to 8,000 V at 20 ° C. and a maximum current of 50 mA at 50 kVh. After focusing, the peptide fraction was collected from 24 wells, as well as the extra acid (positive end) and extra base (negative end) fractions. To remove glycerol, a spin desalting column was used. The peptide was then dried with a vacuum concentrator.

The obtained off-gel fractions were analyzed by LC-MS / MS according to Examples 1.1 (4) to (6), and after identifying the peptide, the identified peptide was classified according to UMC.

3. Construction of mitochondrial master precision mass and time tag (AMT) database (DB) and alignment of peptides between samples

Master AMT DB with information from UMC with peptide IDs of all 80 LC-MS / MS datasets (LC-MS / MS experiment of 3 replicates of 18 samples and 26 LC-MS / MS experiment of off-gel fraction) Edited in (Oracle Database 10g Enterprise Edition Release 10.2.0.1.0; Figure 1a). Monoisotopic masses and NETs are a collection of AMTs, unique peptides that are determined experimentally. When the peptide was measured several times, the average mass and median of NET were recorded for each AMT. In this process, a master DB containing 42,132 peptides (or AMT) was obtained (each point in FIG. 1B).

Because of the undersampling of the current protein analysis platform, a large portion of UMCs in LC-MS / MS experiments were not identified by MS / MS experiments and protein database searches. For example, from an individual LC-MS / MS dataset, 42,132 UMCs were measured in the MS spectrum, of which 5,602 UMCs were identified by protein database searches (identified as UMCs; FIG. 1C).

Peptide ions detected in MS scan but without the opportunity for MS / MS analysis, or fragmented but poor quality ions are the main cause of this gastronomic UMC. To assign peptide IDs to the categorized UMC (red dot in FIG. 1C), the master AMT DB was matched under mass and NET tolerances (ie, each ± 10 ppm, ± 0.025 NET, FIG. 1D). The process of assigning peptide IDs to unidentified UMCs is 3,363 additional UMCs identified by the master AMT DB information, thereby increasing the total number of UMCs having peptide ID assignments to 8,965 (assigned UMCs).

To align the identified peptides between samples, the master AMT DB is used to assign the distinct UMC to the peptide ID, followed by 3 replicates of 54 LC-MS / MS datasets (i.e., 18 individual mitochondrial peptide samples) The results assigned from the LC-MS / MS dataset) were combined into an alignment table with peptide IDs corresponding to the UMC intensity in each column from each LC-MS / MS experiment. Quantile normalization of sorted data was performed to correct for systemic deviations in peptide abundance between individual LC-MS / MS datasets.

Of the 42,132 peptides in the master AMT DB, 7,635 peptides detected in more than half of the T2DM (n ≧ 5) and control (n ≧ 5) samples were selected and mapped to 1,711 proteins. Among them, 1,171 proteins (1,150 protein-coding genes) having two or more sibling peptides were selected as mitochondrial proteins with high reliability.

4. Analysis of mitochondrial proteins

In order to identify the differentially expressed protein between the T2DM patient and the non-diabetic control group, first, 1) a peptide set in which the protein has two or more non-overlapping peptides was selected. 2) In order to increase statistical power, a set of peptides detected in more than 50% of all individuals in each group was selected. For this selected peptide, a statistical hypothesis test was performed incorporating the Student's t-test and the log2-median ratio test.

Briefly, for the selected peptides, 1) a student's t-test and a log ₂ -median ratio test were applied to calculate the T-value and log ₂ -median ratio, and 2) 2 estimated from random permutation experiments previously described. Corrected T-values and log ₂ -median ratios for each peptide are calculated using the empirical null distribution for the eggplant assay, and 3) P corrected from the two assays using the Stouffer method for each peptide. -Integrate the values, 4) After calculating the FDR for the integrated P-value using the Storey method, select differentially expressed peptides satisfying FDR ≤0.1 and expression ratio ≥ 1.5 times, and then differentially expressed Protein groups containing peptides (differentially expressed DEP) were selected. In addition, to understand the cellular function associated with DEP, the intracellular mechanism (GOBP) represented by DEP was identified using DAVID software, an intracellular function enrichment analysis tool. GOBP showing a change by DEP was selected for mechanisms with P≤0.05.

5. Characteristics of skeletal muscle-derived proteins

To assess the inclusiveness of bone tissue-derived mitochondrial proteins, 1,150 proteins were compared to the most comprehensive mouse and human mitochondrial proteins reported in previous papers. Pagliarini et al., Cell 2008; 134 (1): 112-23 are derived from 1) LC-MS / MS profile of mitochondria isolated from 14 mouse tissues, 2) mitochondrial proteins reported in previous literature, and 3) integrated analysis of GFP tag microscopic data Provided an overview of the mitochondria of 1,098 protein-coding genes. 1,098 genes mapped 1,012 human heterologous homology based on human-mouse orthoology information from the Mouse Genome Informatics database. Lefort et al., J Proteomics 2009; 72 (6): 1046-60 identified 823 proteins after profiling mitochondrial proteins isolated from human skeletal muscle and mapped them to 803 protein-coding genes. As a result of the comparison, 1,150 mitochondrial protein-coding genes were found in 14 mouse tissues and human skeletal muscle tissue, respectively, 397 (1,012 39.2%) and 481 (803 60.0%) protein-coding genes (total 558) ) (FIG. 2A). The remaining 592 of the 1,150 protein-coding genes were not reported in two previous mitochondrial proteins.

To understand the characteristics of mitochondrial proteins, the intracellular location of 1,150 proteins was investigated based on Gene Ontology Cellular Components (GOCCs). The largest number of proteins (455 out of 1,150; 39.6%) was located in the mitochondria, followed by the plasma membrane and cytoplasm / cytoskeleton (FIG. 2B). The comparison of mitochondrial proteins detected by LC-MS / MS was based on GOCC, where a fraction of mitochondrial annotated protein (39.6%) was previously described in Paglianini et al. And Lefort et al .. In addition, the location distribution of the 455 mitochondrial proteins is located in the entire spectrum of the sub-mitochondrial compartment, ranging from the mitochondrial outer membrane and inner membrane to the inner membrane space and the mitochondrial matrix. It was found to include (Fig. 2c). Next, enrichment analysis of Gene Ontology Biological Processe (GOBP) was performed using DAVID software to investigate intracellular function represented by 1,150 proteins. Cell processes that are significantly (P <0.05) marked by mitochondrial proteins include oxidative phosphorylation (OXPHOS), glycolysis tricarboxylic acid (TCA) cycles, mitochondrial organization / transportation, and fatty acid beta- Processes related to T2DM-associated mitochondrial function in skeletal muscle, such as oxidation, response to oxidative stress, and translation (FIG. 2D). For the OXPHOS complex, the protein provided comprehensive coverage (48-100%) of the protein in all 5 complexes, regardless of mitochondrial or nuclear-encoded OXPHOS protein (Figure 2f) (Figure 2e). These data confirm that, in addition to comprehensiveness, mitochondrial proteins provide a wide range of mitochondrial proteams involved in various pathological processes occurring in the sub-mitochondrial compartment.

6. Mitochondrial protein of skeletal muscle changed in T2DM

In skeletal muscle, T2DM is known to be associated with mitochondrial abnormalities with changes in the presence of mitochondrial proteins. In the 54 LC-MS / MS dataset (27 for 9 T2DM patients and 27 for 9 non-diabetic controls), to identify mitochondrial proteins with altered presence in T2DM compared to non-diabetic controls After sorting the measured peptides, 523 peptides differentially expressed between T2DM and non-diabetic controls were identified as described in Example 1.3.

335 proteins containing 523 peptides were selected as differentially expressed protein (DEP) between T2DM and a non-diabetic control. Of the 335 DEPs, 135 proteins were up-regulated in T2DM compared to the non-diabetic control, and 200 proteins were down-regulated (FIG. 3A). In order to understand the functional linkage between DEP and T2DM, DAVID software was used to identify the intracellular mechanisms represented by up- and down-regulated proteins.

As a result of this analysis, 135 up-regulated proteins mainly consist of molecular transport (calcium ion transport, protein transport, and calcium ion homeostasis) and cytoskeletal tissue (muscle contraction, actin filament-based processes, and actin cytoskeletal tissue) (Figure 3B). , It has been shown to be primarily involved in the processes involved in molecular transport and cytoskeletal tissue, which is consistent with previous findings that molecular transport and cytoskeleton organization are involved in T2DM etiology. On the other hand, 200 down-regulated proteins were mainly involved in metabolic processes, such as glucose metabolism (corresponding process), fatty acid metabolism process (fatty acid beta-oxidation) and TCA cycle, which can greatly contribute to T2DM etiology (Fig. 3c). . In particular, both up-regulated and down-regulated proteins (6 and 16 proteins, respectively) showed significant OXPHOS. The pattern of opposing changes in the OXPHOS protein was consistent with the pattern found for the OXPHOS gene in previous studies.

To explore the overall function of the T2DM-related process, a network model analysis was performed to describe the interaction between DEPs involved in GOBP related to molecular transport, metabolism, and cytoskeletal tissue. Specifically, for network model analysis for DEP, some of the DEPs belonging to the T2DM-related GOBPs represented by DEPs were selected, and then proteins for DEPs selected from four protein-protein interaction databases -Protein interaction information was collected: Biomolecular Interaction Network Database (BIND), Human Protein Reference Database (HPRD), Biological General Repository for Interaction Datasets (BIGRID), and Molecular INTeraction Database (MINT). The initial network model was constructed with the selected DEP and interaction information using the visualization tool Cytoscape. After that, the nodes were arranged according to the GOBP associated with each protein and the intracellular signal path, so that nodes with similar functions were placed close together. Groups of nodes related to the same GOBP were labeled with the corresponding GOBP. The reconstructed network model is shown in FIGS. 3D and 3E. The network model in FIG. 3D includes glycolysis pathways (HK1, GPI, PGM1, ENO3, and PKM), TCA cycles (ACO2, IDH3A, OGDH, DLST, SUCLA2, SDHA, FH, and MDH1), and fatty acid β oxidation (ACADM) , DECR1, ECH1, HADH, and HADHB) related proteins were significantly down-regulated in T2DM compared to non-diabetic controls. Down-regulation of these pathways as well as transport of glucose and aspartate (SLC25A12), pyruvate (MCP1 / 2), malate and α-ketoglutarate (SLC25A11), and fatty acids (ACSL1 and CPT1B / 2) It also shows that it was down-regulated in T2DM. In addition, the proteins of the five OXPHOS complexes are significantly downregulated in T2DM, while several proteins (ATP5O and NDUFA6 / A7 / A12 / AB1 / S6) are inversely upregulated, suggesting a complex pattern of changes in the OXPHOS system in T2DM. . In addition, mitochondrial protein transport (TIMM44) was down-regulated in T2DM, along with molecular chaperones (HSPA1A / D1). In addition, the network model also showed down regulation of calcium (MICU1 and VDAC1 / 3) and ATP transport (SLC25A4 and VDAC1 / 3). Collectively, down-regulation of these major mitochondrial metabolic processes in the network model indicates that the overall activity of mitochondrial metabolism in T2DM is reduced compared to non-diabetic controls.

On the other hand, the network model shown in FIG. 3E showed up-regulation of several processes originally reported to be related to vesicles (ER): ER calcium transport (RYR1 and ATP2A1 / A3) (above FIG. 3E) and ER unfolded. Protein reaction (PDIA3 / 4 and ERP29) and vesicle-mediated protein transport (SORT1, RAB1A / 10, STX7, and COPB1) (middle of Figure 3e). This ER cell process, represented by the mitochondrial protein up-regulated in T2DM, indicates the potential association of mitochondrial function with ER in T2DM.

The network model of FIG. 3e showed up-regulation of cytoskeletal constituent pathways (ITGB1, RHOA, MYLPF, DOCK2, PDLIM3, ACTN4, DES, DYNC1H1 and TPM1) in T2DM compared to non-diabetic controls, while multiple molecules of the pathway (FLNA, ACTA1 / C1, ACTN2, and TNNC1) were inversely downregulated.

Recently, a subset of mitochondria has a close relationship with ER, forms an interface called a mitochondria-associated ER membrane (MAM), and increases the function of MAM when mitochondrial function is reduced under pathological conditions. It was reported to do. Of the MAM proteins previously reported in several studies, a significant portion of them (40.1%; 15 of the 37 previous MAM proteins) were detected in mitochondrial proteins. These data show that the mitochondrial sample prepared in Example 1.1 contains MAM, and the up-regulated MAM protein (FIG. 3E) can also be used as a mitochondrial profile indicating the function of T2DM-associated mitochondria.

7. Mitochondrial protein profile indicating T2DM pathophysiology

In human and rodent skeletal muscles with T2DM, mitochondrial dysfunction was observed in insulin resistance. Dysfunction is associated with impaired OXPHOS, glycolysis, and TCA cycles, and is consistent with down-regulation of this process in T2DM compared to non-diabetic controls (FIGS. 3C-3D). Based on this data, as mitochondrial protein profiles showing T2DM-related mitochondrial function, the following representative proteins were initially selected for downregulated OXPHOS: 1-2) Downregulated NDUFS3 (Complex I) and COX2 (Complex IV) ). Unlike this down-regulated process, the mechanism of the up-regulated cellular process was unclear (Fig. 3e).

On the other hand, mitochondrial dysfunction has been linked to impaired calcium transport, after which it has been shown to alter calcium homeostasis in MAM. Similarly, the network model in FIG. 3e showed upregulated calcium transport in MAM.

In addition, mitochondrial dysfunction has been reported to result in a decrease in protein transport to mitochondria, which in turn has been reported to alter protein transport in MAM. Similarly, the network model in FIG. 3E showed that protein transport is upregulated in MAM. Based on these data, the following representative proteins for calcium and protein transport were further selected as additional mitochondrial protein profiles exhibiting T2DM-associated mitochondrial function: 3) Up-regulated CALR for calcium transport and 4-5) protein transport Up-regulated SORT1 for.

8. Verification of selected mitochondrial protein profiles indicating T2DM

To experimentally test the effectiveness of the selected protein as described in Example 1.7, first, five types were screened using Western blotting analysis in a diabetic mouse model, a high fat diet (HFD) -induced obesity model. Differential expression of proteins was confirmed.

It was confirmed that HFD mice (6 weeks of age) had significantly higher body weight, fat mass, blood sugar level, and blood insulin levels than mice fed the normal diet. For immunoblotting analysis, total lysate and crude mitochondria were prepared from skeletal muscle tissue of HFD mice and skeletal muscle tissue of normal-fed mice as controls. To prepare the total lysate, tissues were treated with 10 mM Na ₄ P ₂ O ₇ , 100 mM NaF, 2 mM Na ₃ VO ₄ , 1% NP-40, and protease inhibitor cocktail (10 μg / μl aprotinin, 10 μg / μl Leupeptin and 1 mM PMSF) was dissolved in 20 mM Tris-HCl buffer (pH 7.4). Lysates were sonicated twice for 15 seconds, and cell debris was removed by centrifugation (13,000 rpm) at 4 ° C. for 30 minutes. To prepare the crude mitochondria, the tissue was homogenized in a mitochondrial separation buffer (250 mM sucrose, 25 mM Tris-HCl pH7.4, 1 mM EDTA) at 4 ° C. Thereafter, the lysate was centrifuged at 1000 g to separate the nucleus and cytoplasmic fractions, and the cytosolic fraction was further centrifuged at 4 ° C. to 10000 g to obtain crude mitochondrial pellets. The pellet was dissolved in protein lysis buffer. About 20-30 μg total lysate and crude mitochondria were isolated on SDS-PAGE. The isolated protein was transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked with 5% skim milk in Tris-buffered saline-Tween20 (TBS-T) for 1-2 h at room temperature, and then probed at 4 ° C. overnight with specific antibodies against selected mitochondrial proteins. The band intensity was quantified, and the results are shown in FIGS. 4A and 4B. 4A and 4B, similar to LC-MS / MS data, SORT1, CALR, and RAB1A were both in skeletal muscle tissue (FIG. 4A) of HFD-mouse and mitochondria isolated from skeletal muscle tissue (FIG. 4B). , Compared to the sample of normal-fed mice, it was significantly upregulated (P <0.05). In addition, it was confirmed that COXII and NDUFS3 were significantly down-regulated in both skeletal muscle tissue of HFD-mouse and mitochondria isolated therefrom (FIG. 4B) (P <0.05).

In addition, to confirm the differential expression of five selected proteins in human skeletal muscle tissue, based on the same criteria as described in Example 1.1 (1), from an independent set of 14 T2DM patients and 12 non-diabetic controls. Skeletal muscle tissue was collected. The characteristics of T2DM patients and non-diabetic controls such as age, gender, body mass index, fasting blood glucose, and other experimental values are summarized in Table 2 below.

Normal group (n = 12) Diabetes group (n = 14) p-value age 56.1 ± 16.5 70.0 ± 12.8 0.03 * Gender (male: female) 6: 6 3:11 Body mass index (kg / m ² ) 26.1 ± 2.4 25.0 ± 3.3 0.36 Systolic blood pressure (mm / Hg) 129.8 ± 9.7 127.2 ± 17.6 0.95 Diastolic blood pressure (mm / Hg) 74.8 ± 8.3 70.6 ± 13.0 0.33 Fasting blood sugar (mg / dl) 100.1 ± 18.3 151.5 ± 65.5 0.01 * Hemoglobin A1c (%) 5.6 ± 0.3 6.7 ± 1.4 0.02 * Total cholesterol (mg / dl) 186.8 ± 43.9 180.1 ± 42.1 0.69 BUN 14.5 ± 5.5 18.7 ± 6.2 0.08 Cr 0.8 ± 0.2 1.1 ± 0.2 0.01 *

(Data is expressed as mean value ± standard deviation, *: p <0.05)

The results of separating mitochondria from the collected tissue and performing immunoblotting are shown in FIGS. 4C and 4D. 4C and 4D, similar to LC-MS / MS data, SORT1, CALR and RAB1A were significantly upward in both skeletal muscle tissue of T2DM patients (FIG. 4C) and mitochondria isolated therefrom (FIG. 4D). The adjusted pattern was shown (P <0.05). Similarly, COX2 and NDUFS3 showed significantly (P <0.05) down-regulated patterns in both skeletal muscle tissue (Fig. 4c) and mitochondria (Fig. 4d) of T2DM patients compared to non-diabetic controls (P <0.05). 0.05).

Therefore, it was confirmed that the selected mitochondrial protein profile may act as a marker of T2DM-associated dysregulation of mitochondrial function, up-regulation of MAM function and down-regulation of OXPHOS.

Claims (12)

From a sample derived from muscle tissue, from the group consisting of Sortilin 1: SORT1, Calreticulin (CALR), and Ras-related protein Rab-1A (RAB1A) A composition for diagnosing diabetes, comprising an agent that measures the expression level of a selected protein or fragment thereof. The composition according to claim 1, wherein the sample is a fraction containing mitochondria. The composition according to claim 1, wherein the diabetes is type 2 diabetes. The method according to claim 1, Cyclooxygenase-2 (Cyclooxygenase-2: COX2) and NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 3: NDUFS3) The composition further comprises an agent for measuring the expression level of a protein or fragment thereof. The method according to claim 1, wherein the formulation
An antibody or antigen-binding fragment thereof that specifically binds the protein or fragment thereof; or
A composition comprising a polynucleotide that is identical to or complementary to a polynucleotide encoding the protein or fragment thereof. The composition according to claim 5, wherein the antibody is a polyclonal antibody or a monoclonal antibody. The composition according to claim 5, wherein the nucleic acid is a primer or a probe. A kit for diagnosing diabetes, comprising an agent for measuring the expression level of a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A from a sample derived from muscle tissue. Measuring the expression level of a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A in a sample derived from muscle tissue isolated from a subject suspected of diabetes; And
Comprising the step of comparing the measured expression level with the protein expression level of the normal control,
A method of detecting a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A to provide information necessary for diagnosing diabetes. The method of claim 9, wherein the measuring step comprises incubating the sample with an antibody specifically binding to a protein or fragment thereof selected from the group consisting of SORT1, CALR, and RAB1A. The method of claim 9, wherein the method further comprises measuring the expression level of a protein or fragment thereof selected from the group consisting of COX2 and NDUFS3 in the sample. The method according to claim 9, wherein the measuring step is muscle biopsy, electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay: ELISA), immunostaining, protein chip, immunoprecipitation, microarray, Northern (Northern) ) Blotting, polymerase chain reaction (PCR), or a combination thereof.

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