Method for purifying and chromatography-mass spectrometry identifying deoxy-monomethyl guanine nucleotide or / 3-hydroxybenzoic acid and application

Abstract

The application discloses a method for purifying and chromatographing and mass spectrometrically identifying deoxyguanosine or 3-hydroxybenzoic acid and application. After metabolites are extracted by a methanol method, UPLC-MS / MS-ultra high performance liquid chromatography and ABSCIEX QTRAP 5500 triple quadrupole tandem linear ion trap mass spectrometry are used to distinguish the metabolites by using liquid chromatography and mass spectrometry. The MS technology provides the ability to detect and quantify a large number of metabolites. The method optimizes the purification and mass spectrometric identification of the progeria markers in blood, identifies the structures of the differential markers deoxyguanosine and 3-hydroxybenzoic acid in blood by using the mass spectrometric method, and distinguishes the difficult-to-distinguish differential markers. The method for purifying and mass spectrometrically identifying the progeria differential markers in blood is simple and easy to operate, has small experimental dosage, good stability, is fast and accurate, and can make up for the deficiency in searching for the blood differential markers.

Description

Technical Field

[0001] This invention belongs to the field of blood biomarker research, specifically involving a purification and chromatographic mass spectrometry identification method and application of deoxyguanine nucleotide or / 3-hydroxybenzoic acid. It is a stable and reproducible application of this mass spectrometry method for biomarker identification. Background Technology

[0002] Disease biomarkers are biochemical indicators that can objectively measure and evaluate changes in the structure or function of systems, organs, tissues, and cells during a disease state. They are crucial for disease diagnosis, prognosis, and drug efficacy evaluation. Progeria syndrome (HGPS) is a rare and fatal genetic disorder characterized by accelerated aging that begins in childhood, manifesting as low body weight, lipid metabolism disorders, and abnormalities in the skin, musculoskeletal system, and cardiovascular system. Death usually results from myocardial infarction or stroke. (Gordon LB, Rothman FG, López-Otín C, Mistelli T. Progeria: a paradigm for translational medicine[J]. Cell. 2014 Jan 30;156(3):400-7.)(Merideth MA, et al. Phenotype and course of Hutchinson-Gilford progeria syndrome[J]. N Engl J Med. 2008 Feb 7;358(6):592-604.)(Ullrich NJ, Gordon LB. Hutchinson-Gilford progeria syndrome[J]. Handb Clin Neurol. 2015;132:249-64.) Based on the number of confirmed HGPS cases in the United States, the PRF estimates the prevalence of HGPS to be between 1 / 20 million and 1 / 18 million, with an incidence rate of approximately 1 in 4 million to 8 million newborns. In China, the median prevalence of HGPS is 1 / 55 million. HGPS does not show a sex or race predisposition. Currently, the detection of key biomarkers for progeria, such as proteins like Progerin and Lamin A / C, in blood or other biological samples is challenging. Quantification is primarily achieved through molecular techniques such as real-time quantitative PCR, immunofluorescence, or mass spectrometry. These techniques are not only complex but also costly, limiting their widespread application. Therefore, developing detection technologies targeting specific metabolites in the blood is crucial. This non-invasive detection method can provide continuous monitoring of disease activity and real-time assessment of treatment effectiveness, while minimizing disruption to patients. With advancements in technology, improvements in the sensitivity and specificity of detection techniques will make blood metabolite detection a powerful tool for the early diagnosis and treatment monitoring of progeria.

[0003] Deoxyguanine mononucleotide (dGMP, C 10 H 14N5O7P (CAS 902-04-5) is a colorless or white crystalline powder with a molecular weight of 347.22, a melting point of 245℃, and a density of 2.32. It is readily soluble in water. It can be used as a molecular marker for the detection and research of DNA or RNA. Deoxyguanine nucleotide is a nucleotide composed of deoxyguanosine and monophosphate. While there are relatively few studies on its safety and toxicity, it is generally considered a relatively safe compound. 3-Hydroxybenzoic acid (C7H6O3, CAS 99-06-9) is a white crystalline powder with a molecular weight of 138.028, a melting point of 201℃, and a density of 1.4850. It is readily soluble in hot water, soluble in alcohols and ethers, slightly soluble in cold water, and insoluble in benzene. It is flammable when exposed to open flames or high heat. It decomposes under high heat, releasing irritating fumes. It is stable and can react with hydroxyl and carbonyl groups. 3-Hydroxybenzoic acid can be used as a food preservative. (Xie M, Ye H, Hamri GC, Fussenegger M. Antagonistic control of adual-input mammalian gene switch by food additives[J]. Nucleic Acids Res. 2014 Aug;42(14):e116.) 3-Hydroxybenzoic acid, as a biosynthetic intermediate, participates in the biosynthesis of xanthomonin and has a signal transduction function. (Cao XQ, Ouyang XY, Chen B, Song K, Zhou L, Jiang BL, Tang JL, Ji G, Poplawsky AR, HeYW. Genetic Interference Analysis Reveals that Both 3-Hydroxybenzoic Acid and 4-Hydroxybenzoic Acid Are Involved in Xanthomonadin Biosynthesis in the Phytopathogen Xanthomonas campestris pv. Campestris[J]. Phytopathology. 2020 Feb;110(2):278-286.) 3-Hydroxybenzoic acid has anti-biofilm activity and mainly targets the Agr and Sar systems of Staphylococcus aureus.(Ganesh PS, Veena K, Senthil R, Iswamy K, Ponmalar EM, Mariappan V, GirijaASS, Vadivelu J, Nagarajan S, Challabathula D, Shankar EM. Biofilm-Associated Agrand Sar Quorum Sensing Systems of Staphylococcus aureus Are Inhibited by 3-Hydroxybenzoic Acid Derived from Illicium verum[J]. ACS Omega. 2022Apr 20; 7(17):14653-14665.)

[0004] With the completion of the human genome sequencing project, the study of gene function has gradually become a hot topic, leading to a series of "omics" researches, including transcriptomics (studying transcription processes), proteomics (studying all proteins and their functions in a biological system), and metabolomics (studying changes in metabolites and metabolic pathways). Unlike other omics, metabolomics studies the changes in all metabolites produced by a biological system (cell, tissue, or organism) in response to external stimuli. Its research focuses on small molecule compounds with a relative molecular weight <1500 (including sugars, amino acids, organic acids, nucleic acids, lipids, etc.), and identifies and quantifies these small molecule compounds. It employs modern high-throughput, high-sensitivity detection methods to systematically analyze metabolites in biological samples. The core concept is that the metabolic state of an organism reflects its overall condition and is also influenced by factors such as genome coding, diet, and environment (Beger RD, Dunn W, Schmidt MA, Gross SS, Kirwan JA, Cascante M, Brennan L, Wishart DS, Oresic M, Hankemeier T, Broadhurst DI, Lane AN, Suhre K, Kastenmüller G, Sumner SJ, Thiele I, Fiehn O, Kaddurah-Daouk R; for “Precision Medicine and Pharmaceuticals Task Group” - Metabolics Society Initiative. Metabolics can provide precision medicine: "A White Paper, Community" Perspective. Metabolomics. 2016; 12(10):149. doi:10.1007 / s11306-016-1094-6. Epub 2016Sep2. PMID:27642271;PMCID:PMC5009152.). To date, progeria is still diagnosed clinically using genetic testing. However, due to its rarity, primary care physicians often lack experience and are prone to misdiagnosis and missed diagnosis. Furthermore, genetic testing cannot serve as an indicator for monitoring the development and progression of the disease. Therefore, it is crucial to identify biomarkers for the diagnosis of progeria and monitor them in a timely manner.

[0005] Mass spectrometry was first widely used in the analysis of biomolecules (such as proteins, enzymes, nucleic acids and carbohydrates) after the emergence of soft ionization techniques such as MALDI and ESI in the 1980s. The high sensitivity and high throughput of mass spectrometry have made it a powerful tool for the detection of biomarkers (Domon B, Aebersold R. Mass spectrometry and protein analysis[J]. Science (New York, NY), 2006, 312(5771):212-7.). Mass spectrometry is characterized by high precision, high specificity, and high sensitivity. It is cost-effective and has high throughput, enabling the detection of multiple indicators in a single experiment. It has significant advantages and irreplaceable roles in certain fields (newborn screening, low-concentration hormone detection, drug concentration detection, trace element detection, etc.) (Buchberger AR, Delaney K, Johnson J, et al. Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights[J]. Analytical Chemistry, 2018, 90(1):240-65.). Currently, there are no relevant literature or patent reports on the use of mass spectrometry to analyze differential markers of progeria in blood. This method is rapid, simple, and stable, and has broad application prospects.

[0006] This invention verifies that the biomarkers described are closely related to the occurrence and development of progeria, and that their application in early diagnosis has good diagnostic efficacy, with high accuracy, sensitivity, and specificity, providing valuable biological information for the early diagnosis and monitoring of the development of progeria.

[0007] This invention first used UHPLC-QTOF-MS to detect non-targeted metabolomics in the serum of progeria patients (10 control group, 10 progeria patients). The results showed significant differences between the serum of progeria patients and the healthy group. Differential pathway enrichment analysis indicated that serum metabolites of progeria patients showed multi-organ dysfunction. Further significance analysis (fold change 2, p-value < 0.05) revealed that 55 molecules were upregulated and 75 molecules were downregulated in progeria. Using triple quadrupole targeted mass spectrometry, the detection method for two differentially expressed metabolites was identified and validated in a validation set (12 control group, 12 progeria patients). The results showed that these two molecules (dGMP and 3-Hydroxybenzoic acid) showed consistent trends with non-targeted metabolomics screening, both being negatively correlated with progeria. Summary of the Invention

[0008] The purpose of this invention is to provide a method for the purification and chromatographic-mass spectrometric identification of deoxyguanine nucleotides or 3-hydroxybenzoic acid.

[0009] A method for the purification and chromatographic-mass spectrometric identification of deoxyguanine nucleotides or / 3-hydroxybenzoic acid, comprising the following steps:

[0010] (1) Add acetonitrile / methanol to the plasma sample, vortex, put it into a centrifuge, centrifuge, take the supernatant and evaporate to dryness, add acetonitrile aqueous solution, vortex, sonicate, put it into a centrifuge, centrifuge, take the supernatant into a container for UPLC-MS / MS detection, and use the aqueous solution as a substitute matrix to prepare a standard curve.

[0011] (2) The analysis was performed using an LC-MS system, with the mobile phase set as A: acetonitrile containing formic acid and B: water containing ammonium acetate and formic acid, so that deoxyguanine nucleotides and 3-hydroxybenzoic acid could be distinguished.

[0012] (3) Electrospray ionization source was selected for detection. Deoxyguanine nucleotides were detected using positive ion multiple reaction monitoring mode, and 3-hydroxybenzoic acid was detected using positive ion multiple reaction monitoring mode.

[0013] In step (1), the volume ratio of acetonitrile to methanol is 1:0.5 to 1.5. In step (1), the centrifuge conditions are 2 to 6°C and 12,000 to 14,000 rpm.

[0014] In step (2), the LC-MS system used was a Shimadzu Nexera X2 LC-30AD ultra-high performance liquid chromatography system and a SCIEXQTRAP5500 liquid chromatography-mass spectrometry system. In step (2), the chromatographic column used in the LC-MS system was a Waters ACQUITY BEH Amide column, with a column temperature of 38–42℃ and an autosampler temperature of 7–9℃.

[0015] In step (2), mobile phase A is an acetonitrile solution of formic acid with a mass percentage of 0.09%–0.1%; in mobile phase B, the concentration of ammonium acetate is 9–11 mM, the mass percentage of formic acid is 0.09%–0.1%, and the remainder is water. In step (2), deoxyguanine nucleotides are analyzed using positive ion multiple reaction monitoring (MRM). The ion source temperature is 490–510 °C; the ion spray voltage is 5400–5600 V; the curtain gas is 28–32 psi; the nebulizer gas is 53–57 psi; and the auxiliary heating gas is 58–62 psi. The mass spectrometry parameters of deoxyguanine nucleotides are as follows: the mother ion (Q1) m / z is 384.1, the daughter ion (Q3) m / z is 152.1, the declustering voltage (DP) is 80, and the collision energy (CE) is 24. In step (2), hydroxybenzoic acid was analyzed using positive ion multiple reaction monitoring (MRM). The ion source temperature was 490–510 °C; the ion spray voltage was 5400–5600 V; the curtain gas pressure was 28–32 psi; the nebulizer gas pressure was 53–57 psi; and the auxiliary heating gas pressure was 58–62 psi. The mass spectrometry parameters for hydroxybenzoic acid were as follows: the mother ion (Q1) m / z was 139, the daughter ion (Q3) m / z was 79.0, the declustering voltage (DP) was 60, and the collision energy (CE) was 10.

[0016] This invention extracts metabolites using methanol and then distinguishes them using UPLC-MS / MS (equipped with an ABSCIEX Exion LC AD) and an ABSCIEX QTRAP 5500 triple quadrupole tandem linear ion trap mass spectrometer. MS technology provides the ability to detect and quantify large numbers of metabolites. This invention optimizes the purification and mass spectrometric identification of progeria biomarkers in blood, using mass spectrometry to identify the structures of differentially expressed biomarkers deoxyguanine nucleotides (dGMP) and 3-hydroxybenzoic acid, thus enabling the differentiation of difficult-to-distinguish biomarkers. The method for purification and mass spectrometric identification of progeria biomarkers in blood used in this invention is simple, requires small experimental quantities, has good stability, and is rapid and accurate, thus addressing the current shortcomings in the search for differentially expressed biomarkers in blood.

[0017] Application of deoxyguanine nucleotide or 3-hydroxybenzoic acid as a biomarker for progeria in the preparation of progeria reagents or kits.

[0018] A method for purifying and identifying progeria markers in blood by mass spectrometry, which is achieved through the following steps:

[0019] (1) Quantitatively aspirate 50 μL of plasma sample, add 200 μL of acetonitrile / methanol (1:1, v / v), vortex for 3 minutes, place in a centrifuge at 4℃ and centrifuge at 13000 rpm for 10 minutes, quantitatively aspirate the supernatant and evaporate to dryness, quantitatively add 50 μL of 50% acetonitrile aqueous solution to the evaporated EP tube, vortex for 2 minutes, sonicate for 10 minutes, place in a centrifuge at 4℃ and centrifuge at 13000 rpm for 10 minutes, quantitatively aspirate the supernatant and place it in a clean sample bottle for LC-MS / MS detection, and at the same time use the aqueous solution as a substitute matrix to prepare a standard curve;

[0020] (2) Based on the properties of the substances, the UPLC-MS / MS (equipped with ABSCIEX Exion LC AD) ultra-high performance liquid chromatography system and the ABSCIEX QTRAP 5500 triple quadrupole tandem linear ion trap mass spectrometry system of Shimadzu Corporation of Japan were selected for analysis. The chromatographic column was a Waters ACQUITY BEH Amide column (2.1×100mm, 1.7μm), the column temperature was 40℃, and the autosampler temperature was 8℃.

[0021] (3) The mobile phase was set as follows: A: acetonitrile with 0.1% formic acid, B: water with 10mM ammonium acetate and 0.1% formic acid, with a flow rate of 0.2mL / min and an injection volume of 5μL, so that deoxyguanine nucleotide (dGMP) and 3-hydroxybenzoic acid could be distinguished.

[0022] (4) Liquid chromatography elution conditions: The mobile phase was set to consist of acetonitrile (A) with 0.1% formic acid and water (B) with 10mM ammonium acetate and 0.1% formic acid, so that dGMP and 3-Hydroxybenzoic acid could be distinguished.

[0023] (5) Based on the chromatographic results and physicochemical properties, a suitable electrospray ionization (ESI) source was selected for detection. The mode was positive ion, multiple reaction monitoring (MRM), ion source temperature was 500℃, curtain gas was 30 psi, CAD was 9, ion voltage was 5500 V, spray gas was 55 psi, and auxiliary heating gas was 60 psi. The mass spectrometry parameters were as follows: for deoxyguanine nucleotides, the precursor ion (Q1) m / z was 384.1, the daughter ion (Q3) m / z was 152.1, the declustering voltage (DP) was 80, and the collision energy (CE) was 24. For 3-hydroxybenzoic acid, the precursor ion (Q1) m / z was 139, the daughter ion (Q3) m / z was 79.0, the declustering voltage (DP) was 60, and the collision energy (CE) was 10. Deoxyguanine nucleotide and 3-hydroxybenzoic acid were added at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, 5, 10, 20, 50, 100, 200, 500, and 1000 ng / mL.

[0024] In step (1), the serum sample is dried using a vacuum rotary dryer or dried with N2 at room temperature without heating.

[0025] Step (1) No more than 48 hours before the determination, redissolve the product using a reconstitution system. The reconstitution system is: methanol: acetonitrile: H2O = 2:2:1.

[0026] In steps (2)-(5), the liquid phase separation system is an ABSCIEX Exion LC AD ultra-high performance liquid chromatography system and an ABSCIEX QTRAP 5500 LC-MS system, and a Waters ACQUITY BEH Amide column, 2.1×100mm, 1.7μm column. The mobile phase consists of A: acetonitrile with 0.1% formic acid and B: water with 10mM ammonium acetate and 0.1% formic acid. Gradient elution is performed: 0-3min, 10% B-30% B; 3-6min, 30% B-55% B; 6-8min, 55% B; 8-8.1min, 55% B-10% B; 8.1-13min, 10% B. Under these chromatographic and mass spectrometric conditions, the elution times of each marker are as follows: dGMP (Retention Time: 6.01 min, mass-to-charge ratio m / z: 152.1), 3-Hydroxybenzoic acid (Retention Time: 1.90 min, mass-to-charge ratio m / z: 79.0).

[0027] Currently, the quantification of target metabolites is performed using an ABSCIEX Exion LC AD ultra-high performance liquid chromatography system and an ABSCIEX QTRAP 5500 UPLC-MS / MS system. Due to the different biophysical and chemical characteristics of differential markers, the structural spectra for mass spectrometry identification also differ. This invention optimizes the purification and mass spectrometry identification of progeria markers in blood, using mass spectrometry to identify the structures of deoxyguanine nucleotides and 3-hydroxybenzoic acid, enabling the differentiation of difficult-to-distinguish differential markers and facilitating rapid and accurate clinical application.

[0028] This invention provides a method for purifying and identifying differentially expressed aging markers in blood using mass spectrometry. The method extracts deoxyguanine nucleotides and 3-hydroxybenzoic acid from blood using methanol and analyzes them using an ABSCIEX Exion LC AD ultra-high performance liquid chromatography system and an ABSCIEX QTRAP 5500 UPLC-MS / MS system, distinguishing them using liquid chromatography and mass spectrometry. The method for purifying and identifying differentially expressed progeria markers in blood using this invention is simple, requires small experimental quantities, and has good stability, thus addressing the current shortcomings in progeria-related blood markers.

[0029] The innovative points of this invention are summarized as follows: (1) Compared with other previous studies, this invention proposes a mass spectrometry detection method for progeria biomarkers for the first time. By changing the liquid chromatography conditions and mass spectrometry analysis method, it is possible to simultaneously identify two biomarkers after one purification. The confirmed differential metabolites have high accuracy, sensitivity and specificity, providing an important basis for the early diagnosis and monitoring of the development of hormone-sensitive nephrotic syndrome in children, filling a gap at home and abroad. (2) Compared with other previous studies, this invention proposes a method for identifying deoxyguanine nucleotides and 3-hydroxybenzoic acid for the first time, which can be applied to early diagnostic kits for progeria. Attached Figure Description

[0030] Figure 1 PCA and OPLS-DA analyses of non-targeted metabolites in serum; where: (A) Principal component analysis (PCA) plots of non-targeted metabolite profiles in serum samples in positive ion and (B) negative ion modes were obtained by UHPLC-QTOF-MS. The PCA plots show clear metabolite separation between the healthy individuals and the progeria group. (C) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots of metabolites in serum samples in positive ion and (D) negative ion modes.

[0031] Figure 2 Volcano maps of metabolites detected by non-targeted analysis in positive ion mode, including: (A) Volcano maps of metabolites detected by non-targeted analysis in negative ion mode; (B) Disease pathways enriched by differentially metabolites in positive ion mode; (C) Disease pathways enriched by differentially metabolites in negative ion mode; (D) Disease pathways enriched by differentially metabolites in positive ion mode.

[0032] Figure 3 The deoxyguanine nucleotide (dGMP) chromatographic peak was obtained after methanol extraction of the metabolite from blood and analyzed using an ABSCIEX Exion LC-AD system and a SCIEX QTRAP 5500 LC-MS system. The column used was a Waters ACQUITYBEH Amide column (2.1 × 100 mm, 1.7 μm), and the dGMP signal was detected in positive ion mode.

[0033] Figure 4 The chromatographic peak of 3-hydroxybenzoic acid was obtained by extracting the metabolite from blood with methanol and then analyzing it using an ABSCIEX Exion LC-AD system and a SCIEX QTRAP 5500 LC-MS system. The column used was a Waters ACQUITY BEH Amide column (2.1 × 100 mm, 1.7 μm), and the 3-hydroxybenzoic acid signal was detected in positive ion mode.

[0034] Figure 5 : Deoxyguanine monoguanine (dGMP) mass spectrum peak, signal of deoxyguanine monoguanine mass spectrum peak.

[0035] Figure 6 : 3-Hydroxybenzoic acid mass spectrum peak, 3-hydroxybenzoic acid mass spectrum peak signal.

[0036] Figure 7 Targeted quantitative detection of two metabolites in the progeria group. Detailed Implementation

[0037] The present invention will be further described in conjunction with the accompanying drawings and embodiments. Unless otherwise specified, all percentages appearing in the embodiments are mass percentages.

[0038] Reagents and instruments used in the embodiments of this invention:

[0039]

[0040] Example 1: Biomarker Screening

[0041] Step 1. Study population and sample collection, sample preprocessing

[0042] This study was approved by the ethics committee (ethics number: 2023-IRB0186-P-01) and conducted in accordance with the Declaration of Helsinki. The study included participants registered at the hospital for 1–180 months since 2020. Serum samples were collected from 10 patients with progeria and 10 healthy children as controls. Selection criteria for progeria: genetically confirmed LMNA. G608G Blood samples were collected from participants in a non-fasting state and centrifuged at 3000 rpm for 20 minutes at 4°C. Serum was then stored at -80°C for further analysis. Blood samples were collected aseptically from 1 mL of fasting venous blood in the morning into an EDTA anticoagulant tube. Immediately after collection, the sample was centrifuged at 3000 rpm for 5 minutes at room temperature, and the supernatant was collected and stored at -80°C.

[0043] The median ages in the control group and the patient group were 89.1 (95% confidence interval [CI]: 50.2–128.0) and 41.7 (95% CI, 5.7–77.8) months, respectively. The male-to-female ratio in serum and urine samples was approximately 1:1 in the control group and approximately 1:1 in the progeria group. There were no statistically significant differences in age or sex between the two groups (p > 0.05).

[0044] Step 2. Sample preparation for metabolite analysis and detection and analysis of non-targeted metabolites using UHPLC-QTOF-MS.

[0045] Serum samples (200 μL) were extracted using 800 μL of HPLC-grade methanol (methanol:H₂O = 4:1, v / v) in 1.5 mL / 2 mL Eppendorf tubes. The mixture was vortexed at 4–8 °C for 1 min (≤1 min), followed by incubation at -80 °C for 2 h or overnight. The supernatant was centrifuged at 14000 g (or maximum speed) for 20 min at 4 °C using a refrigerated centrifuge, and an equal volume of supernatant was transferred to a new Eppendorf tube. The extracted metabolites were dissolved in 100 μL of 80% methanol and analyzed by LC-MS.

[0046] Analysis was performed using an Agilent 1290 Infinity II UHPLC system and an Agilent 6545 QTOF / MS (Agilent) LC-MS system. Chromatographic separations were achieved on an ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of 15 mM ammonium acetate, 0.3% NH3·H2O aqueous solution and 15 mM ammonium acetate, 0.3% NH3·H2O in a 9:1 acetonitrile / water mixture (B), with a flow rate of 0.3 mL / min. The B phase was eluted at 95% for 1 min, then slowly decreased to 50% over 1 to 8 min, maintained at 50% for 3 min, and subsequently gradiented to 95% over 0.5 min, held for 1.5 min. The injection volume was 5 μL.

[0047] Mass spectrometry data were obtained using electrospray ionization in positive and negative ion modes at 200–1200 m / z. Other mass spectrometry settings included: sheath gas temperature 300 °C, sheath gas flow rate 11 L / min, VCap 3500 V, capillary 0.09 mA, nozzle voltage 0 V, gas temperature 275 °C, fragmenter 150 V, and separator 65 V. Raw data were processed using Profinder 10.0 (Agilent) for peak detection, calibration, and integration. All metabolite variables were obtained from UHPLC-QTOF-MS datasets. T-tests, Wilcoxon tests, and ANOVA were performed between two or more groups, and chi-square tests were used to compare categorical variables. Principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA) were performed using MetaboAnalyst version 4.0 (http: / / www.metaboanalyst.ca). Data were scaled to unit variance prior to PCA. Metabolite enrichment was log-transformed prior to pathway analysis. All statistical tests were two-tailed, with significance set at P < 0.05.

[0048] The results are as follows Figure 1 As shown, in the UHPLC-QTOF-MS dataset, after data annotation, we obtained 160 metabolite features in positive ion mode and 157 metabolic features in negative ion mode. We applied principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to determine whether the metabolic characteristics of progeria patients differed from those of the normal group. In the PCA model, serum samples from both groups were analyzed in positive ion mode (…). Figure 1 A) and negative ion mode ( Figure 1 C) Both groups showed a clear separation trend, and the OPLS-DA scoring plot showed a clear separation trend between the two groups of serum samples. Figure 1 (B, D). We found 55 molecules upregulated and 75 molecules downregulated in progeria. Using triple quadrupole targeted mass spectrometry, we identified two differentially expressed metabolites and validated them on a validation set (12 controls and 12 progeria patients). The results showed that these two molecules (dGMP and 3-Hydroxybenzoic acid) showed a consistent trend with those obtained from non-targeted metabolomics screening, both being negatively correlated with progeria.

[0049] To explore metabolic pathways associated with progeria, such as Figure 2 As shown, 55 significantly upregulated and 75 significantly downregulated metabolites were identified in the progeria group under both positive and negative ion modes. Among these altered metabolites, both positive and negative ion modes validated one different upregulated metabolite and eight different downregulated metabolites. Further enrichment of the significantly different metabolites into disease pathways was performed. Figure 2 A / B). For example... Figure 2 As shown in the C / D diagram, multiple organ dysfunction was predicted in both positive and negative ion modes.

[0050] Step 3. Absolute quantification using triple quadrupole MS technology for the screened biomarkers provides the capability to detect and quantify a large number of metabolites. Currently, the quantification of target metabolites is performed using UPLC-MS / MS (equipped with ABSCIEX Exion LC AD) – ultra-high performance liquid chromatography and ABSCIEX QTRAP 5500 triple quadrupole tandem linear ion trap mass spectrometry, distinguishing them using liquid chromatography and mass spectrometry. Mass Hunter Workstation (Ver B.06.00, Agilent Technologies) software is used for data acquisition and analysis. Two different methods are selected for detection based on the biophysical and chemical differences of the differentially expressed metabolites.

[0051] 2.1 After extracting metabolites using the methanol method, dGMP (deoxyguanine nucleotide, 347.22 g / mol, C) was detected in negative ion mode.10 H 14 N5O7P (CAS 902-04-5) and 3-Hydroxybenzoic acid (138.028 g / mol, C7H6O3, CAS 99-06-9).

[0052]

[0053] Example:

[0054] A quantitative sample of 50 μL of plasma was taken and 200 μL of acetonitrile / methanol (1:1, v / v) was added. The mixture was vortexed for 3 minutes and centrifuged at 13000 rpm for 10 minutes at 4°C. The supernatant was then quantitatively aspirated and evaporated to dryness. 50 μL of 50% acetonitrile aqueous solution was added quantitatively to the evaporated EP tube. The mixture was vortexed for 2 minutes, sonicated for 10 minutes, and centrifuged at 13000 rpm for 10 minutes at 4°C. The supernatant was then quantitatively aspirated and transferred to a clean sample vial for LC-MS / MS detection. A standard curve was prepared using the aqueous solution as a substitute matrix.

[0055] Analysis was performed using a Shimadzu Nexera X2 LC-30AD ultra-high performance liquid chromatography (UHPLC) system and a SCIEX QTRAP5500 LC-MS system. The HPLC conditions were as follows: the column was replaced with a Waters ACQUITY BEH Amide (2.1 × 100 mm, 2.7 μm); the mobile phase consisted of acetonitrile (A) with 0.1% formic acid and water (B) with 10 mM ammonium acetate and 0.1% formic acid; the flow rate was 0.2 mL / min with gradient elution: 0–3 min, 10% B–30% B; 3–6 min, 30% B–55% B; 6–8 min, 55% B; 8–8.1 min, 55% B–10% B; 8.1–13 min, 10% B. The column oven temperature was 40 °C, the injection volume was 5 μL, and the autosampler temperature was 8 °C.

[0056] The mass spectrometry conditions are as follows:

[0057] (1) Deoxyguanine nucleotides were tested in positive ion multiple reaction monitoring (MRM) mode. The ion source (ESI) temperature was 500℃; the ion spray voltage was 5500V; the curtain gas was 30psi; the nebulizing gas was 55psi; and the auxiliary heating gas was 60psi. The MRM conditions for deoxyguanine nucleotides were: mass-to-charge ratio 384.1→152.1; declustering voltage was 80V; and the collision energy was 24V.

[0058] (2) 3-Hydroxybenzoic acid was analyzed using positive ion multiple reaction monitoring (MRM) mode. The ion source (ESI) temperature was 500℃; ion spray voltage was 5500V; curtain gas pressure was 30psi; nebulizer gas pressure was 55psi; and auxiliary heating gas pressure was 60psi. The MRM conditions for 3-hydroxybenzoic acid were: mass-to-charge ratio 139→79; declustering voltage: 60V; collision energy: 10V. Under these conditions, compound data were acquired using Analyst Software 1.7.1 and analyzed using OS 3.1.6 software. Each compound was validated using a separate standard. The two compounds were essentially separated under conventional column chromatography. For detailed results, please refer to [link to relevant documentation]. Figure 3-6 .

[0059] Conclusion: The analytical conditions meet the requirements and can be used to test the sample.

[0060] like Figure 7 As shown, consistent with the non-targeted metabolic profile, the progeria group had lower levels of deoxyguanine nucleotides and 3-hydroxybenzoic acid.

Claims

1. Application of deoxyguanine nucleotide or 3-hydroxybenzoic acid as a biomarker for progeria in the preparation of progeria reagents or kits.

2. The application according to claim 1, characterized in that, The purification and chromatographic-mass spectrometric identification method for deoxyguanine nucleotides or / and 3-hydroxybenzoic acid includes the following steps: (1) Add acetonitrile / methanol to the plasma sample, vortex, put it into a centrifuge, centrifuge, take the supernatant and evaporate to dryness, add acetonitrile aqueous solution, vortex, sonicate, put it into a centrifuge, centrifuge, take the supernatant into a container for UPLC-MS / MS detection, and use the aqueous solution as a substitute matrix to prepare a standard curve. (2) The analysis was performed using an LC-MS system. The mobile phase was set as follows: A: acetonitrile containing formic acid, B: ammonium acetate and water containing formic acid, so that deoxyguanine nucleotides and 3-hydroxybenzoic acid could be distinguished. (3) Select an electrospray ion source for detection. Deoxyguanine nucleotides are detected using positive ion multiple reaction monitoring mode, and 3-hydroxybenzoic acid is detected using positive ion multiple reaction monitoring mode.

3. The application according to claim 2, characterized in that, In step (1), the volume ratio of acetonitrile / methanol is 1:0.5~1.

5.

4. The application according to claim 2, characterized in that, In step (1), the centrifuge conditions are 2~6℃ and 12000~14000rpm.

5. The application according to claim 2, characterized in that, In step (2), the LC-MS system used was the Shimadzu Nexera X2LC-30AD ultra-high performance liquid chromatography system and the SCIEX QTRAP5500 liquid chromatography-mass spectrometry system.

6. The application according to claim 2, characterized in that, In step (2), the LC-MS system uses a Waters ACQUITY BEH Amide column with a column temperature of 38~42 ℃ and an autosampler temperature of 7~9 ℃.

7. The application according to claim 2, characterized in that, In step (2), the mobile phase A is an acetonitrile solution of formic acid with a mass percentage of 0.09%~0.1%; In mobile phase B, the concentration of ammonium acetate is 9~11 mM, the mass percentage of formic acid is 0.09%~0.1%, and the balance is water.

8. The application according to claim 2, characterized in that, In step (2), deoxyguanine nucleotides were processed using positive ion multiple reaction monitoring mode. The ion source temperature was 490~510 ℃; the ion spray voltage was 5400~5600 V; and the curtain gas pressure was 28~32 psi. Nebulizing gas: 53~57 psi; auxiliary heating gas: 58~62 psi.

9. The application according to claim 2, characterized in that, In step (2), hydroxybenzoic acid is used in positive ion multiple reaction monitoring mode, with ion source temperature of 490~510 ℃; ion spray voltage of 5400~5600 V; and curtain gas of 28~32 psi. Nebulized gas: 53~57 psi; The auxiliary heating gas is 58~62 psi.

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