PRODUCTION OF STABLE ISOTOPE LABELED HUMAN DNA REPAIR PROTEIN STANDARDS 15N-hPARP1 AND 15N-hOGG1

Abstract

The invention relates to the production of all nitrogen atom-labeled internal standard products of Poly [ADP-ribose] polymerase 1 (PARP1) and 8-oxoguanine-DNA glycosylase (OGG1), which are DNA repair proteins.

Description

[0001] PRODUCTION OF STABLE ISOTOPE LABELED HUMAN DNA REPAIR PROTEIN STANDARDS15N-hPARPl AND15N-hOGGl

[0002] Technical Field

[0003] The present invention relates to the production of internal standard products to be used for the measurement of the absolute amount of Poly [ADP-ribose] polymerase 1 (PARP1) and 8- oxoguanine-DNA glycosylase (OGGI), which are DNA repair proteins, by mass spectrometric methods.

[0004] State of the Art (Prior Art)

[0005] Quantitative analyses of proteins, which are the basic building blocks of living things, are the most commonly applied measurements in basic and clinical sciences. It is very important to measure proteins accurately and specifically in tissues, organs or cells due to the vital tasks of proteins.

[0006] Single or multiple measurements of certain proteins can be performed with different immunological techniques, but the success of these approaches depends on the production and validation of high affinity and specific antibodies. Due to limited specificity and high measurement variations, immunological methods are inadequate, especially in single or multiple measurements of “low amounts of proteins” that have the potential to be clinical biomarkers.

[0007] In the mass spectrometry (MS) based quantitation of proteins found in biological samples, the “bottom-up” proteomics workflow, in which the protein (s) of interest are analyzed by means of “proteotypic (surrogate) peptides” that best represent that protein after being cut with proteolytic enzymes (for example, trypsin), is the main approach used. For the absolute quantitation of proteins, it is important to add the15N and / or13C isotope labeled peptide internal standard (s) of the analyte to be measured, which is called the “internal standard” before the analysis with MS, to the sample, thus standardizing the variability arising from the matrix effect, chromatographic losses and mass spectrometric ionization activity in the sample. The internal standard has the same chromatographic properties as the sample to be analyzed and differs from the endogenous analyte only by mass difference. It is possible to perform absolute quantitation by comparing the peak areas obtained by MS of the internal standards whose concentration is known beforehand with the surrogate peptides of the endogenous analyte.

[0008] At least four / five peptides need to be identified and labeled simultaneously for the detection and quantitation of a protein. On the other hand, considering the wide dynamic range of proteins in biological samples and body fluids, a pre-extraction process is required for the sensitive and accurate analysis of proteins in low amounts in the mass spectrometer, primarily for the removal of other highly available proteins. In this case, it is of great importance that the ideal internal standard for absolute quantitation goes through the same processes as the protein to be examined not only in the mass spectrometric analysis step but also in all preanalytical sample processing steps.

[0009] The13C and15N labeled PARP1 internal standard of “Origene” is sold commercially but has a high cost. For example, in comparative studies of healthy and patient groups, the cost increase is very high in cases where the number of samples is high. However, this product is only obtained by marking atoms in the amino acids of lysine and arginine, rather than marking all the nitrogen and carbon atoms of the hPARPl protein. Commercially available isotopically labeled peptide internal standards are usually added to the samples after isolation and cutting with trypsin of the target protein to be measured. For this reason, the protein is subject to errors related to all stages (extraction, hydrolysis with trypsin, et cetera) before measurement. Another disadvantage of these peptide standards is that they have a high cost; this cost increases even more considering that it is necessary to identify at least four to five peptides at the same time for the detection and quantification of a protein.

[0010] Brief Description and Objects of the Invention

[0011] The object of the present invention is to produce a labeled one-to-one analog of all nitrogens of the target protein. During the production of the stable isotope-labeled protein, which is the product to be produced, all nitrogen atoms of the protein are labeled with15N since the recombinant plasmid containing the gene specific to the target protein will be transferred to the bacterial strain and the cells will be grown in the culture medium containing15N-NH4C1. With the production and use of the completely stable isotope-labeled analog of the targeted protein, the labeled analog is hydrolyzed simultaneously with the protein to be measured in the enzyme-cutting step carried out during the pre-analysis phase of the protein. Thus, it is possible to standardize the variables that will arise from all experimental stages of the analysis, including the pre-extraction method. In addition, peptides can be obtained from the stable isotope-labeled protein internal standard to be produced as much as the number of peptides to be obtained after slaughter with enzymes such as trypsin et cetera specific to the protein to be measured. After MS measurement, internal standard peptide spectra of each of the obtained peptide spectra of the endogenous protein are also obtained. In this way, all peptides selected for absolute quantitation can be quantified.

[0012] With the internal standard to be produced, stable isotope-labeled peptide internal standards specific to all peptides to be obtained after cutting with a proteolytic enzyme such as trypsin are also obtained naturally. This allows the peptides that define the protein during the analysis with LC-MS and that can be used in the quantitation to be easily selected because there are peptide standards with the same retention time and to quantify not only a few peptides but all desired peptides at the same time. It is possible to obtain protein internal standards to be produced once at a high efficiency that can be sufficient for thousands of sample studies.

[0013] For this reason, according to the results obtained by using other commercially available isotope-labeled peptides, the accuracy and precision in the quantitation of the protein have increased and the losses caused by the pretreatment have been normalized with the use of stable isotope-labeled analogs within the scope of the invention.

[0014] Description of the Figures Explaining the Invention

[0015] Figure 1. The pPB-C-His plasmid to be used in the production of labeled hOGGl protein Figure 2. Plasmid pET28 to be used in the production of labeled hPARPl protein

[0016] Detailed Description of the Invention

[0017] With the invention, the expression, purification and characterization of the stable isotopelabeled analog of hPARPl and hOGGl, which play an important role in the BER repair pathway of DNA repair proteins, are carried out. During the production of the stable isotope- labeled protein, which is the product to be produced, all nitrogen atoms of the protein are labeled with15N since the recombinant plasmid containing the gene specific to the target protein will be transferred to the bacterial strain and the cells will be grown in the culture medium containing15N-NH4C1.

[0018] Production of hOGGl and hPARPl by recombinant DNA technology

[0019] It is aimed to produce hOGGl and hPARPl, which are DNA repair proteins, by recombinant DNA technology. For this purpose, clone cDNA containing the hOGGl gene region together with the expression plasmid for hOGGl was also commercially purchased In the study, the plasmid to be tried is specified first; the vector plasmid should contain a promoter region for expression, be kanamycin resistant be attached to 6X-Histidine (required for protein purification) and be transformable to E. coli BL21 (DE3) strain (Figure 1). E. coli BL21 (DE3) strain was grown in a separate medium. The vector plasmid containing hOGGl clone cDNA was transformed into E. coli BL21(DE3) strain by electroporation method.

[0020] The histidine-labeled hPARPl / pET28 plasmid in the DH5a strain, which was commercially purchased for hPARPl protein production (https: / 7 vw.addgene.org / 169815 / '), was propagated in a medium with kanamycin. In the study, the plasmid to be tried was specified first; the vector plasmid should contain a promoter region for expression, be kanamycin resistant be attached to 6X-Histidine (required for protein purification) and be transformable to E. coli BL21 (DE3) strain (Figure 2). E. coli BL21 (DE3) strain was grown in a separate medium. Then, several colonies were isolated from the DH5a strain with a plasmid isolation kit the relevant gene was cut by specific restriction enzymes and gel electrophoresis was performed. As a result of electrophoresis, plasmid and hPARPl were seen in two bands, and it was confirmed that the plasmid isolated by this step was hPARPl / pET28. The isolated plasmid was transformed into E. coli BL21 (DE3) strain by electroporation method.

[0021] Since the plasmids used were resistant to the antibiotic kanamycin, only the bacteria that received the plasmid were colonized in the medium where the kanamycin was added. After the plasmids transferred to the bacteria for both genes were kept overnight at 37°C, several colonies were selected from the petri dish and cut with restriction endonuclease enzymes and it was confirmed which colonies were transformed in agarose gel electrophoresis. Marking of hOGGl and hPARPl transformed bacteria with15N

[0022] Colonies found to have undergone transformation were selected and Lysogeny Broth (LB) containing15N-NH4C1, kanamycin and chloramphenicol was incubated in an agar petri dish in a minimal essential medium (MEM). Then one colony was selected from the petri dish and the LB containing kanamycin, chloramphenicol and glucose was transferred to the medium and shaken overnight at 37°C. This process continued until the optical density (OD600) at 600 nm was between 0.4-0.6. Afterwards, 100 mM zinc sulfate (ZnSO4) was added to the medium and overnight shaking at 37°C continued until OD600 was between 0.8 and 1.0. Zinc sulfate (ZnSO4) is used as it is necessary to inhibit bacterial growth to provide instantaneous measurement of optical density. Since cell density is an important factor for the production of the target protein, these steps should be applied and the optical density should be between 0.8 and 1.0 before proceeding to the next steps. Otherwise, protein production is insufficient. When the process was completed, isopropyl P-D-l -thiogalactopyranoside (IPTG) was added to the medium to induce protein production and the culture was left to shake at 16°C overnight. Afterwards, the cells were centrifuged and the supernatant was discarded and the cells were dissolved in the new environment with the help of the vortex. The cells were stored at -20°C until the protein purification step process.

[0023] Purification of15N-hOGGl and15N-hPARPl by ultracentrifugation and column chromatography

[0024] It was homogenized by adding a lysis buffer to the cells. Cell lysates were ultracentrifuged at 40000 g for 1 h. Using an automated chromatographic purification device from the supernatants obtained after ultracentrifugation, the hOGGl protein was passed through a nickel-agarose resin-filled propylene column, and the hPARPl protein was passed through HiTrap® Chelating column NiC12, HiTrap® Heparin HP column and Sephacryl s200 columns, which are size exclusion chromatography columns, respectively. Purity control of eluents was performed by the SDS-PAGE method. Protein concentration was determined by the Lowry method using BSA standard material. Characterization of15N-labeled hOGGl and hPARPl proteins and determination of molecular mass and isotopic purity to measure the success rate of labeling proteins with15N

[0025] Amino acid analysis of the produced proteins, intact protein analysis, and protein-peptide concentration determination (PICAA) analyses including SI traceable value assignment studies were carried out at the TUBITAK National Metrology Institute (UME) Bioanalysis Laboratory.

[0026] Performing the enzymatic activity determination of15N-hOGGl and15N-hPARPl

[0027] The determination of the enzymatic activity of15N-hOGGl was performed by gas chromatography-mass spectrometry (GC-MS). The calf thymus was used as a DNA sample for the measurement of DNA glycosylase activity of15N-hOGGl. A 50 pg “calf thymus” DNA sample irradiated at a dose of 5 Gy was dissolved in 50 pL incubation buffer (50 mM phosphate buffer (pH 7.4), 100 mM KC1, 1 mM EDTA and 0.1 mM dithiothreitol). Stable isotope-labeled internal standards of specific base lesions removed by hOGGl protein were added (FapyGua-13C,15N2 and 8-OH-GUA-15Ns). Free modified bases were released after hydrolysis of the samples with 2 pg labeled or unlabeled hOGGl at 37°C for 1 hour. 150 pL of cold anhydrous ethanol was added to the samples to stop the reaction and allowed to stand for 1 hour at -20°C. Samples taken from the freezer were centrifuged for 30 minutes at +4°C at 14,000 g. The supernatant has been transferred to glass ampoules. Removal of ethanol was achieved in SpeedVac (approximately 45 min). 200 pL of purified water was added to the samples whose ethanol was removed. The bulbs are covered with a paper cloth and fixed with a rubber band. The samples were frozen with the help of liquid nitrogen and lyophilized with Freeze-dry overnight. 30 pL BSTFA (N, O-Bis (trimethyl silyl) trifluoroacetamide) (1%, v / v) and 30 pL pyridine (1 : 1, v / v) were added under the fume cupboard to the lyophilized supernatant fractions. Nitrogen gas was passed over the samples and the lids were sealed and closed. Incubated for 30 minutes at 120°C. After incubation, the cooled samples were transferred to the injection ampoules (insert vial) with the Hamilton injector without opening the caps. These ampoules were also sealed and loaded into the gas chromatography-mass spectrometer (GC-MS) device. 4 pL of the derivatized samples were injected into the GC arm with an autoinjector and FapyGua and 8-OH-Gua levels were measured. DNA glycosylase activity analysis of15N-hOGGl was performed using electron collision ionization (El) by GC-MS method. SIM mode was used to determine the characteristic ions for identification and quantification. At the same time, the main masses of ions that are widely studied in the literature have been taken into consideration for selection. These masses are: FapyGua, m / z 457 (M+*), FapyGua-13C,15N2; m / z 460, 8-OH-Gua, m / z 455 (M+*), 8- OH-Gua-15N5, m / z 460.

[0028] In the analyses, 5% cross-linked phenylmethylsilicone-coated (0.33 pm) high-resolution fused-silica capillary column (12.5 m, 0.2 mm i.d) was used as the stationary phase, and helium gas of extreme purity was used as the carrier phase. The furnace temperature of the gas chromatograph was set from 130°C to 280°C (increasing rapidly at 130°C for the first two minutes, then 8°C / minute). Integration of the fields of the signals given by the ions of these compounds was used to determine the level of the compounds. hPARPl enzymatic activity chemiluminescence kits were used to perform enzymatic activity determinations of15N-hPARPl. The histone mixture accompanying the mass was diluted with PBS and 50pl was added to the 96-well plate and incubated at 4°C overnight. The plate was washed with a PBST buffer and blocked with a blocking buffer. The master mix was prepared using DTT solution, PARP buffer, PARP substrate mixture and activated DNA solution and added to the plate at 25 pl / well. Then, 5 pl of test inhibitor was added to each well. The PARP1 enzyme was diluted to 0.33 ng / pl and added to the plate at 20 pl / well and incubated at room temperature for 1 hour. Streptavidin-HRP solution was diluted in accordance with the kit procedure and added to the wells. Measurement was performed using the plate reader's Luminescence mode.

[0029] Measurement of manufactured and purified15N-hOGGl and15N-hPARPl internal standard by high-resolution LC-MS

[0030] A high-resolution liquid chromatography mass spectrometer (Thermo Scientific Orbitrap LC- MS) was used to identify the hOGGl and hPARPl proteins of the labeled proteins produced and to demonstrate their usability in absolute quantitation. An electrospray ionization source was used as an ionization source. The15N-hOGGl (150 pg) and15N-hPARPl proteins (150 pg) to be produced were incubated in a trypsin-containing buffer at 37°C for 24 h. For the inactivation of trypsin, the samples were kept at 95°C for 10 minutes and then filtered with ultrafiltration membranes with a molecular mass limit of 3 kDa. After the trypsin process, many peptides of the protein were formed. Tryptic peptides of a protein and its15N-labeled analog are analyzed based on fully scanned mass spectra, usually containing protonated molecular ion (MH+ ion) and double protonated molecular ion [(M+2H)2+], Among the peptides obtained, the peptides with the highest density were selected. Theoretical mass calculations for peptide analyses were performed in the “NIST Mass and Fragment Calculator” program. Analyses of the peptides obtained from the measurement and confirmatory analyses were performed to identify the target protein.

Claims

CLAIMS1. A method of producing15N-labeled analogs of all nitrogen atoms of target proteins, characterized in that it comprises the following process steps: i. providing the recombinant plasmid comprising the promoter region, the antibiotic resistance gene, the protein label and the gene region of the target protein, ii. transforming the provided recombinant plasmid into the bacterial strain, iii. incubating the colonies where the transformation takes place in the medium containing15N-NH4C1 after detection and selection, iv. selecting at least one of the colonies incubated in a medium comprising 15N- NH4C1, transferring it to media comprising antibiotics and agitating it until the optical density 600 (OD600) value reaches a value between 0.4 and 0.6, v. adding a bacterial growth inhibitor agent to the medium and agitating until the OD600 value is between 0.8- 1.0, vi. obtaining labeled analogs of all nitrogen atoms of the target protein by performing protein purification processes.

2. A production method according to Claim 1, characterized in that the recombinant plasmid is transformed into E. coli BL21(DE3) in step ii.

3. A production method according to Claim 1, characterized in that plasmid containing the promoter region, kanamycin resistance gene, 6X-Histidine label and 8-oxoguanine-DNA glycosylase (hOGGl) gene region is provided in step i.

4. A production method according to Claim 1, characterized in that the plasmid comprising the promoter region, the kanamycin resistance gene, the 6X-Histidine label and the Poly [ADP-ribose] polymerase 1 (hPARPl) gene region is provided in step i.

5. A production method according to Claim 1, characterized in that zinc sulfate (ZnSO4) is added as a bacterial growth inhibiting agent in step v.6.15N-hOGGl produced by the production method according to Claims 1, 2, 3 and 5.7.15N-hPARPl produced by the production method according to Claims 1, 2, 4 and 5.

8. The use of15N-hOGGl according to Claim 6 as an internal standard.

9. The use of15N-hPARPl according to Claim 7 as an internal standard.

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