Method for extracting and detecting phosphate group small molecule compound

Abstract

The invention discloses an extraction and detection method of a phosphate group small molecule compound. Titanium dioxide nanoparticles are adopted to enrich and extract phosphate group small molecular compounds in organisms, and then detection is carried out. The titanium dioxide nanoparticles can be combined with phosphate groups through interaction of coordinate bonds, Van der Waals force and the like, target molecules containing the phosphate groups are directly captured in the sample extraction process, then centrifugation is performed to obtain precipitates, soluble sugar, salt, small peptides and the like in supernate are removed, and target compounds in the precipitates are eluted and then detected on a machine. The extraction method provided by the invention can avoid the influence of soluble substances such as sugar on the detection result, and is simple to operate, high in accuracy of the detection result and good in reproducibility.

Description

Technical Field

[0001] The invention belongs to the field of biological analysis, and in particular relates to a method for extracting and detecting a phosphate-based small molecule compound. Background Art

[0002] Phosphate compounds in living organisms (such as adenosine triphosphate, acetyl-CoA, cyclic adenosine monophosphate, etc.) are mainly present in the tricarboxylic acid cycle, glycolysis and oxidative phosphorylation pathways, and are key metabolites involved in energy metabolism. Energy metabolism maintains the most basic life activities of living organisms, while plant adversities and animal diseases are usually accompanied by severe metabolic disorders, and most of them are attributed to energy metabolism disorders. Energy metabolism also affects the function and fate of immune cells, and is related to disease resistance and immune response. At the same time, the modifying groups of protein phosphorylation come from adenosine triphosphate, so abnormal energy metabolism can further lead to changes in upstream protein phosphorylation pathways, seriously affecting the physiological and pathological functions of organisms.

[0003] Therefore, the detection and analysis of phosphate compounds related to energy metabolism in living organisms is of great reference significance for understanding the various physiological, pathological indicators and functions of organisms.

[0004] Phosphate compounds are usually highly polar and easily soluble in polar solvents such as water. However, during the sample extraction process, soluble substances such as sugars, salts, and small peptides are easily introduced, which often lead to ion suppression and discrimination effects during mass spectrometry detection, seriously interfering with the detection and analysis of target compounds. Therefore, how to remove the interference of sugars, salts, small peptides, etc. is an important issue in solving the detection of phosphate compounds. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting and detecting phosphate-based small molecule compounds in an organism. The method can be applied to the concentration, enrichment and extraction of compounds containing phosphate groups, and in particular can greatly reduce the influence of sugars, salts and small peptide compounds introduced by conventional extraction methods on mass spectrometry instruments, thereby improving the stability, reproducibility and reliability of the test results. The method can be used for the extraction and enrichment of compounds containing phosphate groups and strong hydrophilicity, such as cyclic adenosine monophosphate, glucose phosphate, and acetyl coenzyme A.

[0006] The present invention provides a method for extracting and detecting phosphate-based small molecule compounds in an organism. The phosphate-based small molecule compounds are small molecule compounds containing a phosphate group and having strong hydrophilicity, and specifically may be cyclic adenosine monophosphate, glucose phosphate, acetyl coenzyme A, etc. The method uses titanium dioxide nanoparticles to enrich and extract the phosphate-based small molecule compounds in the organism, and then detect them.

[0007] The method comprises the following steps:

[0008] 1) Taking a biological sample, breaking it, adding an extraction solvent to extract, centrifuging to obtain a supernatant, adding chloroform to remove protein, and centrifuging to obtain a supernatant;

[0009] 2) adding titanium dioxide nanoparticles to the obtained supernatant, mixing, incubating, mixing, and centrifuging to obtain a precipitate;

[0010] 3) adding ammonia water to the obtained precipitate, mixing, incubating, centrifuging, and collecting the supernatant;

[0011] 4) Dry the obtained supernatant, re-dissolve it and then detect it.

[0012] In step 1) of the above method, the biological sample includes plant samples, animal samples and microbial samples.

[0013] In one embodiment of the present invention, the biological sample is a whole plant of Arabidopsis thaliana, rice seeds, seedlings and leaves, wheat seeds, seedlings and leaves, soybean seeds, seedlings and leaves, etc.;

[0014] The crushing is carried out under a liquid nitrogen environment;

[0015] The extraction solvent may be pre-cooled water, and may be pre-cooled to 4°C.

[0016] The ratio of the biological sample to the extraction solvent and chloroform can be 100 mg: 300-1000 μL: 300-1000 μL respectively;

[0017] In step 2) of the above method, the particle size of the titanium dioxide nanoparticles may be 5-10 nm;

[0018] The weight ratio of the titanium dioxide nanoparticles to the biological sample may be: 20-60 mg: 100 mg;

[0019] The incubation was performed on ice;

[0020] The incubation time may be 5 min-1 h;

[0021] The centrifugal condition may be centrifugation at 12000 rpm for 10 min;

[0022] In step 3) of the above method, the ammonia water may be specifically 15% ammonia water (mass concentration);

[0023] The ratio of ammonia water to biological sample can be 300-1000 μL: 100 mg;

[0024] The incubation was performed on ice;

[0025] The incubation time may be 5 min-1 h;

[0026] The centrifugal condition may be centrifugation at 12000 rpm for 10 min;

[0027] In step 4) of the above method, the detection method may specifically be ultra-performance liquid chromatography coupled to electrospray quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS);

[0028] The chromatographic columns used were: Acquity UPLC HSST3 chromatographic column (1.8 μm, 2.1 × 100 mm);

[0029] Mobile phase A was 10 mM ammonium formate, and B was methanol;

[0030] The elution gradient is: 0-1min, 10-20% B; 1-2min, 20% B; 2-10min 20-100% B; 10-15min, 100% B; 15-16min, 100%-10% B; 16-20min, 10% B;

[0031] The column oven temperature was 30°C, the sample chamber temperature was 10°C, the injection volume was 5 μL, and the mobile phase flow rate was 0.2 mL / min;

[0032] Mass spectrometry detection was performed in positive ion mode, and MassLynxV4.2 software was used for data acquisition, processing and analysis;

[0033] The desolvation temperature was 325 °C, the ion source temperature was 120 °C, the capillary voltage was 2.5 kV, and the cone voltage was 50 V;

[0034] The data were collected in full scan mode with an acquisition time of 20 min and a scan mass range of m / z 50–1200 Da;

[0035] Nitrogen was used as desolvation and flow gas with flow rates of 900 and 50 L·H -1 ;

[0036] Sodium formate was used for mass calibration;

[0037] In MS / MS, high-purity argon was used as the collision gas and the collision energy varied between 20 and 45 eV.

[0038] The present invention uses titanium dioxide nanoparticles to enrich and extract phosphate-based small molecule compounds in organisms. Titanium dioxide nanoparticles can bind to phosphate groups through coordination bonds, van der Waals forces and other interactions, directly capture target molecules containing phosphate groups during sample extraction, and then centrifuge to obtain precipitates, thereby removing soluble sugars, salts, small peptides, etc. in the supernatant. The target compounds in the precipitate can be eluted and then tested on a machine. Existing extraction methods easily introduce soluble compounds such as sugars, which often lead to ion suppression of target molecules during mass spectrometry detection, produce a discrimination effect, and seriously interfere with the detection and analysis of target molecules. The extraction method of the present invention can remove soluble compounds such as sugars in samples, avoid detection suppression of target molecules, and the method simultaneously enriches and purifies target molecules, is simple to operate, and has high accuracy and good reproducibility in detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The present invention is a flow chart of using titanium dioxide nanoparticles to extract phosphate-based small molecule compounds.

[0040] Figure 2 The results of different extraction methods for detecting 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) in rice seedlings were compared. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0042] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0043] Example reference Figure 1 The flow chart shown uses titanium dioxide nanoparticles to extract phosphate-based small molecule compounds.

[0044] The specific detection process is as follows: take 100 mg of fresh sample (taking the aboveground part of rice seedlings in the three-leaf stage as an example), grind it in a ball mill under liquid nitrogen environment, add 600 μL of water precooled to 4°C for extraction, centrifuge to obtain the supernatant, add 600 μL of chloroform to remove protein, centrifuge to obtain the supernatant, add 50 mg of titanium dioxide nanoparticles (5-10 nm) to the supernatant, shake well to mix, incubate on ice for 10 minutes, then shake again to mix, centrifuge at 12000 rpm for 10 minutes, discard the supernatant, add 600 μL of 15% by mass ammonia water to the precipitate, shake well to mix, incubate on ice for 10 minutes, centrifuge at 12000 rpm to obtain the supernatant, dry and re-dissolve with 50 μL of pure water.

[0045] Sample detection was completed on Waters ACQUITY UPLCI-ClassPLUS / XevoG2-XSQTOF MS, the chromatographic column was Acquity UPLCH SST3 column (1.8μm, 2.1×100mm), the mobile phase A was 10mM ammonium formate, B was methanol, and the elution gradient was: 0-1min, 10-20% B; 1-2min, 20% B; 2-10min 20-100% B; 10-15min, 100% B; 15-16min, 100%-10% B; 16-20min, 10% B. The column oven temperature was 30℃, the sample chamber temperature was 10℃, the injection volume was 5μL, and the mobile phase flow rate was 0.2mL / min. Mass spectrometry detection was carried out in positive ion mode, and MassLynx V4.2 software was used for data acquisition and processing analysis. The desolvation temperature was 325 °C, the source temperature was 120 °C, the capillary voltage was 2.5 kV, and the cone voltage was 50 V. The data were acquired in full scan mode with an acquisition time of 20 min and a scan mass range of m / z 50–1200 Da. Nitrogen was used as the desolvation and sulphurization gas with flow rates of 900 and 50 L·H-1. Sodium formate was used for mass calibration. In the MS / MS experiments, high purity argon was used as the collision gas and the collision energy was varied between 20 and 45 eV.

[0046] Figure 2 Taking the phosphate-based small molecule compound 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) as an example, the test results after extracting 2',3'-cAMP from the aerial part of rice seedlings at the three-leaf stage according to the operation in the above embodiment are compared with the test results of the conventional extraction method. Among them, A is the 2',3'-cAMP standard product, B is the test result of the eluate component after the rice seedling extract is enriched with titanium dioxide, C is the conventional test result of the rice seedling extract (i.e., the supernatant obtained by centrifugation after adding chloroform to remove protein), and D is the test result of the supernatant component after the rice seedling extract is enriched with titanium dioxide. It can be seen from the figure that the cAMP content detected after titanium dioxide enrichment ( Figure 2B, chromatographic peak area 6536) compared with the results of conventional direct detection ( Figure 2 C, chromatographic peak area 1885) increased by nearly 4 times, and the supernatant ( Figure 2 D) Almost undetectable.

[0047] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for extracting and detecting phosphate-based small molecule compounds in an organism, which uses titanium dioxide nanoparticles to enrich and extract phosphate-based small molecule compounds in an organism, and then detects them.

2. The method according to claim 1, characterized in that: The phosphate-based small molecule compounds are all small molecule compounds containing a phosphate group and having strong hydrophilicity.

3. The method according to claim 1 or 2, characterized in that: The phosphate-based small molecule compounds include cyclic adenosine monophosphate, glucose phosphate, and acetyl coenzyme A.

4. The method according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: 1) Taking a biological sample, breaking it, adding an extraction solvent to extract, centrifuging to obtain a supernatant, adding chloroform to remove protein, and centrifuging to obtain a supernatant; 2) adding titanium dioxide nanoparticles to the obtained supernatant, mixing, incubating, mixing, and centrifuging to obtain a precipitate; 3) adding ammonia water to the obtained precipitate, mixing, incubating, centrifuging, and collecting the supernatant; 4) Dry the obtained supernatant, re-dissolve it and then detect it.

5. The method according to claim 4, characterized in that: In step 1), the biological sample includes plant samples, animal samples, and microbial samples; The crushing is carried out under a liquid nitrogen environment; The extraction solvent is pre-cooled water; The ratio of the biological sample to the extraction solvent and chloroform is 100 mg: 300-1000 μL: 300-1000 μL, respectively.

6. The method according to claim 4 or 5, characterized in that: In step 2), the particle size of the titanium dioxide nanoparticles is 5-10 nm; The weight ratio of the titanium dioxide nanoparticles to the biological sample is: 20-60 mg: 100 mg; The incubation was performed on ice; The incubation time is 5min-1h; The centrifugal condition is 12000 rpm for 10 min.

7. The method according to claim 4 or 5, characterized in that: In step 3), the ammonia water is (mass concentration) 15% ammonia water; The ratio of ammonia water to biological sample is 300-1000 μL: 100 mg; The incubation was performed on ice; The incubation time is 5min-1h; The centrifugal condition is 12000 rpm for 10 min.

8. The method according to claim 4 or 5, characterized in that: In step 4), the detection method is ultra-high performance liquid chromatography coupled with electrospray quadrupole time-of-flight tandem mass spectrometry.

9. The method according to claim 8, characterized in that: The chromatographic columns used were: Acquity UPLC HSS T3 column, 1.8 μm, 2.1 × 100 mm; Mobile phase A was 10 mM ammonium formate, and B was methanol; The elution gradient was: 0-1min, 10-20% B; 1-2min, 20% B; 2-10min 20-100% B; 10-15min, 100% B; 15-16min, 100%-10% B; 16-20min, 10% B; The column oven temperature was 30°C, the sample chamber temperature was 10°C, the injection volume was 5 μL, and the mobile phase flow rate was 0.2 mL / min.

10. The method according to claim 8, characterized in that: Mass spectrometry detection was performed in positive ion mode. desolvation temperature, 325 °C; source temperature, 120 °C; capillary voltage, 2.5 kV; cone voltage, 50 V; The data were collected in full scan mode with an acquisition time of 20 min and a scan mass range of m / z 50–1200 Da; Nitrogen was used as desolvation and flow gas with flow rates of 900 and 50 L·H -1 ; Sodium formate was used for mass calibration; In MS / MS, argon was used as the collision gas and the collision energy was varied between 20 and 45 eV.

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