WO2022186803A1 - Biochemical medical diagnostic kit - Google Patents
Biochemical medical diagnostic kit Download PDFInfo
- Publication number
- WO2022186803A1 WO2022186803A1 PCT/TR2022/050179 TR2022050179W WO2022186803A1 WO 2022186803 A1 WO2022186803 A1 WO 2022186803A1 TR 2022050179 W TR2022050179 W TR 2022050179W WO 2022186803 A1 WO2022186803 A1 WO 2022186803A1
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- WO
- WIPO (PCT)
- Prior art keywords
- gly
- boc
- oet
- stock solution
- asn
- Prior art date
Links
- 238000009007 Diagnostic Kit Methods 0.000 title description 2
- 230000006240 deamidation Effects 0.000 claims abstract description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- 238000005259 measurement Methods 0.000 claims abstract description 23
- 150000001875 compounds Chemical class 0.000 claims abstract description 21
- 239000011550 stock solution Substances 0.000 claims abstract description 21
- 239000008367 deionised water Substances 0.000 claims abstract description 19
- 239000012153 distilled water Substances 0.000 claims abstract description 19
- BPUCEFIHQRKEGS-QMMMGPOBSA-N ethyl 2-[[(2s)-4-amino-2-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxobutanoyl]amino]acetate Chemical compound CCOC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)OC(C)(C)C BPUCEFIHQRKEGS-QMMMGPOBSA-N 0.000 claims abstract description 16
- -1 Boc-Asn-Gly-OEt compound Chemical class 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 239000012472 biological sample Substances 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 9
- 238000013207 serial dilution Methods 0.000 claims description 8
- 230000002269 spontaneous effect Effects 0.000 claims description 7
- 238000011088 calibration curve Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000011166 aliquoting Methods 0.000 claims description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 2
- 238000000691 measurement method Methods 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 21
- 102000004169 proteins and genes Human genes 0.000 description 21
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 4
- 108010016626 Dipeptides Proteins 0.000 description 4
- 230000032683 aging Effects 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 229960001230 asparagine Drugs 0.000 description 4
- 235000009582 asparagine Nutrition 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 125000003368 amide group Chemical group 0.000 description 2
- 229940009098 aspartate Drugs 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000006338 isoaspartate formation Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 125000000979 2-amino-2-oxoethyl group Chemical group [H]C([*])([H])C(=O)N([H])[H] 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 201000011240 Frontotemporal dementia Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000000538 Protein D-Aspartate-L-Isoaspartate Methyltransferase Human genes 0.000 description 1
- 108010002119 Protein D-Aspartate-L-Isoaspartate Methyltransferase Proteins 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000003164 beta-aspartyl group Chemical group 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 108091006028 deamidated proteins Proteins 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000011935 selective methylation Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N2440/16—(de-)amidation
Definitions
- the invention relates to a kit suitable for biological samples in the field of in vitro diagnostics (biochemical medical diagnosis, etc.) or for use in other fields (pharmacy, chemistry, etc.).
- the invention relates to a kit suitable for use to measure the effect of solutions on the deamidation rate of the proteins they contain.
- Proteins are one of the basic structural and functional molecules that make up living organisms. Proteins are large organic compounds that are formed as a result of the binding of amino acids to each other in chains. Peptides are short polymers formed by linking a- amino acids in a defined order. The bond between one amino acid residue and another is known as an "amide bond" or peptide bond. Proteins are polypeptide molecules (or contain multiple polypeptide subunits). The main difference is that peptides are short while polypeptides/proteins are long.
- Deamidation is an important factor not only in aging, but also in the stability of protein- containing pharmaceutical drugs (such as vaccines). It is necessary to measure the effect of the solutions in which these drugs are dissolved on the protein deamidation rate, and to make interventions to slow the deamidation rate when added to the solutions. In the state of the art, there are studies on this subject.
- the invention relates to methods and tools for the quantitative determination of the isoaspartyl content of polypeptides by selective methylation of their fragments catalyzed by a protein L-isoaspartyl methyltransferase enzyme. Since the deamidation of asparagine side chains at certain protein sites and the resulting isoaspartate formation appear to be an important contributor to protein degradation under mild conditions, the invention also relates to a method for quantifying protein degradation associated with isoaspartate formation,
- the present invention relates to a deamidation rate measurement kit that meets the above- mentioned requirements, eliminates all disadvantages and brings some additional advantages.
- the primary aim of the invention is to develop a kit that measures the effect on the deamidation rate when added to solutions containing protein pharmaceuticals.
- One aim of the invention is to develop a measurement kit that enables to determine the factors affecting the deamidation rate, to examine the correlation of individual differences in deamidation rate with aging and age-related diseases, and to detect diseases characterized by an increase in deamidation rate.
- the invention is a deamidation rate measurement kit containing a stock solution in deionized/distilled water containing the compound Boc-Asn- Gly-OEt and a stock solution in deionized/distilled water containing the compound Boc-Asp- Gly-OEt.
- the deamidation rate measurement kit which is the subject of the invention, in its most basic form; contains stock solution in deionized/distilled water containing the compound Boc-Asn- Gly-OEt (preferably in the range of 0.000001 - 1000 mM, more preferably at the concentration of 0.25 mM) and stock solution in deionized/distilled water containing the compound Boc-Asp-Gly-OEt (preferably 0.000001 - 1000 mM) mM, more preferably at a concentration of 0.25 mM).
- Boc-Asn-Gly-OEt compound a sample view of which is given in Figure 1 , is used both as a calibrator and analytical standard in measurements, and as a deamidated element by being added to biological samples.
- Boc- (1) and -Oet (4) are for blocking Asparagine (Asn) (2) and glycine (Gly) (3) dipeptides respectively, as details shown in the figure.
- the “NH2” structure is removed by deamidation, while the “OH” structure is attached instead.
- the Boc-Asn-Gly-OEt compound turns into the Boc-Asp-Gly-OEt compound and its molecular weight increases by about 1 (0.98) g/mol.
- the Asn (2) and Gly (3) dipeptides are one of the structures with the fastest spontaneous deamidation known. For this reason, it is used in measurements as as the calibrator/analytical standard or the compound subject to deamidation after adding to biological samples. Since Boc-Asn-Gly-OEt is converted into Boc-Asp-Gly-OEt compound as a result of spontaneous deamidation, Boc-Asp-Gly-OEt compound is used as Calibrator and analytical standard in drawing the calibration curve.
- the Boc-Asp-Gly-OEt compound is not commercially available but has been specially synthesized. Deionized/distilled water allows to dissolve Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt compounds and is used as analytical blank.
- the deamidation rate measurement kit developed includes the following steps of process: Preparation of stock solution and serial dilutions of Boc-Asn-Gly-OEt compound in deionized/distilled water; Preparation of stock solution and serial dilutions of Boc-Asp-Gly-OEt compound in deionized/distilled water; Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asn-Gly- OEt stock solution; Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asp-Gly-OEt stock solution; aliquoting the biological sample whose effect on the deamidation rate will be tested in equal volumes in three different tubes; addition - preferably in the range of 1/1000 - 1/2 of the final reaction volume (more preferably in the range of 1/10) - of Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt (as an internal standard
- the present invention is based on the principle of spontaneous deamidation of Boc-Asn-Gly- OEt compound in solution into Boc-Asp-Gly-OEt compound and measuring the resulting Boc- Asp-Gly-OEt compound by an appropriate analytical method.
- Boc-Asn-Gly-OEt solution is added to any sample whose effect on the spontaneous deamidation rate is desired to be measured.
- Spontaneous deamidation of Boc-Asn-Gly-OEt in this mixture which is kept at a temperature between 1-100 °C for 1 minute - 1 month, is ensured.
- This change in mass that is, the resulting Boc-Asp-Gly-OEt and Boc-lsoasp-Gly-OEt can be measured by mass spectrometry, as well as with all kinds of optimized methods based on the different chemical properties of the -NH 2 group in the asparagine of the compound and the -OH group of the newly formed aspartate in the compound.
- the test is provided to be used as a marker to determine the susceptibility to these diseases and for their early diagnosis.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the deamidation rate measurement kit, which includes a stock solution of the compound Boc-Asn-Gly-OEt in deionized/distilled water, and a stock solution of the compound Boc-Asp-Gly-OEt in deionized/distilled water.
Description
BIOCHEMICAL MEDICAL DIAGNOSTIC KIT
Technical Field
The invention relates to a kit suitable for biological samples in the field of in vitro diagnostics (biochemical medical diagnosis, etc.) or for use in other fields (pharmacy, chemistry, etc.).
In particular, the invention relates to a kit suitable for use to measure the effect of solutions on the deamidation rate of the proteins they contain.
State of the Art
Proteins are one of the basic structural and functional molecules that make up living organisms. Proteins are large organic compounds that are formed as a result of the binding of amino acids to each other in chains. Peptides are short polymers formed by linking a- amino acids in a defined order. The bond between one amino acid residue and another is known as an "amide bond" or peptide bond. Proteins are polypeptide molecules (or contain multiple polypeptide subunits). The main difference is that peptides are short while polypeptides/proteins are long.
Asparagine (Asp, N) and glutamine (Gin, Q), two of the 20 different amino acids in the structure of proteins, have amide groups in their side chains. Removal of amide groups in proteins containing these amino acids is called protein deamidation. Protein deamidation can occur enzymatically or spontaneously. As a result of deamidation, changes occur in the structure and charge of proteins. As a result of deamidation, especially in long-lived proteins found in long-lived cells such as neurons, deamidated proteins may accumulate over time, causing structural and functional disorders in these proteins and therefore in the cells in which these proteins are present. A great deal of scientific evidence supporting the role of protein deamidation in Alzheimer's, frontotemporal dementia and many other chronic diseases has been reported in the literature.
For this reason, it is necessary to determine the factors or interventions that affect the deamidation rate, to develop strategies that can prevent/slow down deamidation, and to determine the deamidation rates in individuals. In this way, it is expected that they can make
very critical contributions to the prevention and postponing of aging and age-related diseases.
Deamidation is an important factor not only in aging, but also in the stability of protein- containing pharmaceutical drugs (such as vaccines). It is necessary to measure the effect of the solutions in which these drugs are dissolved on the protein deamidation rate, and to make interventions to slow the deamidation rate when added to the solutions. In the state of the art, there are studies on this subject.
United States patent application number US5273886 The invention relates to methods and tools for the quantitative determination of the isoaspartyl content of polypeptides by selective methylation of their fragments catalyzed by a protein L-isoaspartyl methyltransferase enzyme. Since the deamidation of asparagine side chains at certain protein sites and the resulting isoaspartate formation appear to be an important contributor to protein degradation under mild conditions, the invention also relates to a method for quantifying protein degradation associated with isoaspartate formation,
Although there are studies on this subject in the state of the art, they only allow to indirectly analyze the components found in the interior of pure proteins. Since it can only be used to measure the deamidation of purified proteins, it cannot be used for biological samples with more complex matrices such as blood plasma. There is still no test method that can be used to measure the effect of biological samples (serum, plasma, saliva, cerebrospinal fluid, synovial fluid, urine, cell/tissue/organ extracts, etc.) of humans and other living things on the rate of protein deamidation. For this reason, there is a need to develop a test that has the potential to be used in medical biochemistry laboratories, to measure the effect of biological samples on the protein deamidation rate and to detect interventions that affect the deamidation rate.
As a result, due to the negativities described above and the inadequacy of the existing solutions on the subject; It has been necessary to develop kits that measure the deamidation rate.
Aim of the Invention
The present invention relates to a deamidation rate measurement kit that meets the above- mentioned requirements, eliminates all disadvantages and brings some additional advantages.
The primary aim of the invention is to develop a kit that measures the effect on the deamidation rate when added to solutions containing protein pharmaceuticals.
One aim of the invention is to develop a measurement kit that enables to determine the factors affecting the deamidation rate, to examine the correlation of individual differences in deamidation rate with aging and age-related diseases, and to detect diseases characterized by an increase in deamidation rate.
To fulfill the above-described purposes, the invention is a deamidation rate measurement kit containing a stock solution in deionized/distilled water containing the compound Boc-Asn- Gly-OEt and a stock solution in deionized/distilled water containing the compound Boc-Asp- Gly-OEt.
The structural and characteristic features of the invention and all its advantages will be understood more clearly thanks to the detailed explanation written below, and therefore the evaluation should be made by taking this detailed explanation into consideration.
Figures to Help Explain the Invention
Figure 1 : Structure of the Boc-Asn-Gly-OEt compound
Explanation of References
1 Boc-
2 Asn
3 Gly
4 -Oet
5 Region being deamidated
Detailed Explanation of the Invention
In this detailed explanation, the deamidation rate measurement kit, which is the subject of the invention, is explained only for a better understanding of the subject and without causing any limiting effect.
The deamidation rate measurement kit, which is the subject of the invention, in its most basic form; contains stock solution in deionized/distilled water containing the compound Boc-Asn-
Gly-OEt (preferably in the range of 0.000001 - 1000 mM, more preferably at the concentration of 0.25 mM) and stock solution in deionized/distilled water containing the compound Boc-Asp-Gly-OEt (preferably 0.000001 - 1000 mM) mM, more preferably at a concentration of 0.25 mM).
The Boc-Asn-Gly-OEt compound, a sample view of which is given in Figure 1 , is used both as a calibrator and analytical standard in measurements, and as a deamidated element by being added to biological samples. Boc- (1) and -Oet (4) are for blocking Asparagine (Asn) (2) and glycine (Gly) (3) dipeptides respectively, as details shown in the figure. In the deamidated region (5) shown in the figure, the “NH2” structure is removed by deamidation, while the “OH” structure is attached instead. Thus, the Boc-Asn-Gly-OEt compound turns into the Boc-Asp-Gly-OEt compound and its molecular weight increases by about 1 (0.98) g/mol. Boc- (1) and -OEt (4) structures -in the structure of the Boc-Asn-Gly-OEt compound in solution- allow to block the binding of Asn (2) and Gly (3) dipeptides with unwanted chemical groups during the process. The Asn (2) and Gly (3) dipeptides are one of the structures with the fastest spontaneous deamidation known. For this reason, it is used in measurements as as the calibrator/analytical standard or the compound subject to deamidation after adding to biological samples. Since Boc-Asn-Gly-OEt is converted into Boc-Asp-Gly-OEt compound as a result of spontaneous deamidation, Boc-Asp-Gly-OEt compound is used as Calibrator and analytical standard in drawing the calibration curve. The Boc-Asp-Gly-OEt compound is not commercially available but has been specially synthesized. Deionized/distilled water allows to dissolve Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt compounds and is used as analytical blank.
In a sample application of the invention, the deamidation rate measurement kit developed includes the following steps of process: Preparation of stock solution and serial dilutions of Boc-Asn-Gly-OEt compound in deionized/distilled water; Preparation of stock solution and serial dilutions of Boc-Asp-Gly-OEt compound in deionized/distilled water; Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asn-Gly- OEt stock solution; Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asp-Gly-OEt stock solution; aliquoting the biological sample whose effect on the deamidation rate will be tested in equal volumes in three different tubes; addition - preferably in the range of 1/1000 - 1/2 of the final reaction volume (more preferably in the range of 1/10) - of Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt (as an internal standard) stock solutions to different tubes, and of deionized/distilled water to another tube as blank; at the beginning of the measurement, making measurements from all three tubes, preferably by LC-MS method; after incubation making measurements of all three tubes, preferably by LC-
MS method; subtraction of the measurement results in the tubes with added Boc-Asn-Gly- OEt and Boc-Asp-Gly-OEt from the measurement in the tube with deionized/distilled water; determination of deamidation rate by measuring Boc-Asp-Gly-OEt resulting from spontaneous deamidation in the tube with added Boc-Asn-Gly-OEt.
The present invention is based on the principle of spontaneous deamidation of Boc-Asn-Gly- OEt compound in solution into Boc-Asp-Gly-OEt compound and measuring the resulting Boc- Asp-Gly-OEt compound by an appropriate analytical method. Boc-Asn-Gly-OEt solution is added to any sample whose effect on the spontaneous deamidation rate is desired to be measured. Spontaneous deamidation of Boc-Asn-Gly-OEt in this mixture, which is kept at a temperature between 1-100 °C for 1 minute - 1 month, is ensured. During the incubation period, as a result of the deamidation of the Boc-Asn-Gly-OEt dipeptide in the mixture, the — NH2 group [Figure 1 :(5)] will spontaneously be released from the molecule, while the asparagine in the compound will be converted to isoaspartate or aspartate with the addition of the -OH group, and a molecular weight increase of approximately 1 (0.98) g/mol mass will occur. This change in mass, that is, the resulting Boc-Asp-Gly-OEt and Boc-lsoasp-Gly-OEt can be measured by mass spectrometry, as well as with all kinds of optimized methods based on the different chemical properties of the -NH2 group in the asparagine of the compound and the -OH group of the newly formed aspartate in the compound.
By measuring the effect of the biological samples from individuals/patients on the deamidation rate by using the deamidation rate measurement test, which is the subject of the present invention, and by examining the correlation of individual differences -to be detected and that affect the deamidation rate- with aging and age-related diseases, and if diseases characterized by an increase in deamidation rate are detected, the test is provided to be used as a marker to determine the susceptibility to these diseases and for their early diagnosis.
Claims
1. It is a deamidation rate measurement kit, and its feature is; it contains a stock solution in deionized/distilled water containing Boc-Asn-Gly-OEt compound and a stock solution in deionized/distilled water containing Boc-Asp-Gly-OEt compound.
2. It is a kit according to claim 1 and its feature is; the stock solution in deionized/distilled water containing the Boc-Asn-Gly-OEt compound in the concentration range of 0.000001 - 1000 mM.
3. It is a kit in accordance with claim 2 and its feature is; its concentration is 0.25 mM.
4. It is a kit according to claim 1 and its feature is; it is a stock solution in deionized/distilled water containing Boc-Asp-Gly-OEt compound in the concentration range of 0.000001 - 1000 mM.
5. It is a kit in accordance with claim 4 and its feature is; its concentration is 0.25 mM.
6. It is a rate measurement method made with a deamidation rate measurement kit in accordance with any of the claims 1 to 5, and its feature is;
- Preparation of stock solution and serial dilutions of Boc-Asn-Gly-OEt compound in deionized/distilled water;
- Preparation of stock solution and serial dilutions of Boc-Asp-Gly-OEt compound in deionized/distilled water;
- Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asn-Gly-OEt stock solution;
- Creation of the calibration curve on the instrument to be measured using serial dilutions of the Boc-Asp-Gly-OEt stock solution;
- Aliquoting the biological sample whose effect on the deamidation rate will be tested in equal volumes in three different tubes;
- Adding Boc-Asn-Gly-OEt stock solution to one of the sample tubes, Boc-Asp-Gly-OEt (as an internal standard) stock solution to the other, and deionized/distilled water to the last one as the blank, in the range of 1/1000 - 1/2 of the final reaction volume;
- Making measurements from all three tubes at the beginning of the measurement;
Measurement of all three tubes after incubation;;
- Subtracting the measurement results in the tubes with added Boc-Asn-Gly-OEt and Boc-Asp-Gly-OEt from the measurement in the tube with added deionized/distilled water;
- Determination of the deamidation rate by measuring Boc-Asp-Gly-OEt, which is formed as a result of spontaneous deamidation in the tube with added Boc-Asn-Gly-
OEt,
7. It is a method according to claim 6 and its feature is; the measurements are made by LC-MS method.
8. It is a method according to claim 6 and its feature is; The reaction volume is 1/10.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA3210290A CA3210290A1 (en) | 2021-03-05 | 2022-03-01 | Biochemical medical diagnostic kit |
| JP2023547693A JP2024509716A (en) | 2021-03-05 | 2022-03-01 | biochemical medical diagnostic kit |
| CN202280018246.4A CN116964451A (en) | 2021-03-05 | 2022-03-01 | Biochemical medical diagnosis kit |
| KR1020237031814A KR20230154432A (en) | 2021-03-05 | 2022-03-01 | biochemical medical diagnostic kit |
| US18/264,591 US20240110923A1 (en) | 2021-03-05 | 2022-03-01 | Biochemical medical diagnostic kit |
| AU2022229699A AU2022229699A1 (en) | 2021-03-05 | 2022-03-01 | Biochemical medical diagnostic kit |
| EP22763708.9A EP4302086A1 (en) | 2021-03-05 | 2022-03-01 | Biochemical medical diagnostic kit |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR2021/004329 | 2021-03-05 | ||
| TR202104329 | 2021-03-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022186803A1 true WO2022186803A1 (en) | 2022-09-09 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/TR2022/050179 WO2022186803A1 (en) | 2021-03-05 | 2022-03-01 | Biochemical medical diagnostic kit |
Country Status (2)
| Country | Link |
|---|---|
| EP (1) | EP4302086A1 (en) |
| WO (1) | WO2022186803A1 (en) |
-
2022
- 2022-03-01 WO PCT/TR2022/050179 patent/WO2022186803A1/en active Application Filing
- 2022-03-01 EP EP22763708.9A patent/EP4302086A1/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| KAMEOKA D, UEDA T, IMOTO T.: "A Method for the Detection of Asparagine Deamidation and Aspartate Isomerization of Proteins by MALDI/TOF-Mass Spectrometry Using Endoproteinase Asp-N", JOURNAL OF BIOCHEMISTRY, OXFORD UNIVERSITY PRESS, GB, vol. 134, no. 1, 1 July 2003 (2003-07-01), GB , pages 129 - 135, XP055014099, ISSN: 0021-924X, DOI: 10.1093/jb/mvg120 * |
| TERASHIMA I. ET AL.: "Identification of deamidation and isomerization sites on pharmaceutical recombinant antibody using H2180", ANALYTICAL BIOCHEMISTRY, vol. 368, no. 1, 1 September 2007 (2007-09-01), pages 49 - 60, XP022156831, DOI: 10.1016/j.ab. 2007.05.01 2 * |
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