High-throughput detection method for determining fructose-6-phosphoric acid in cell matrix

Abstract

The invention discloses a high-throughput detection method for determining fructose-6-phosphoric acid in a cell matrix, a sample is introduced into a mass spectrum through sound wave ejection based on a sound wave excitation mass spectrum technology for target compound detection and analysis, a carrier liquid of the sample is an alkaline carrier liquid, the sample ejection amount of sound wave ejection is 10-15 nL, and the sample ejection amount of the sound wave ejection is 10-15 nL; the ejection frequency of sound wave ejection is that the interval between every two samples is 1000-4000 milliseconds. According to the detection method provided by the invention, the analysis time can be shortened, the manpower and consumable cost can be saved, the operation is simple and convenient, and the high-throughput detection requirement can be met.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical analysis, and in particular to a high-throughput detection method for measuring fructose-6-phosphate in a cell matrix. Background Art

[0002] Studies have found that tumor cells consume a large amount of glucose during proliferation. Even in the presence of oxygen, glucose will ferment to produce lactic acid, which is called aerobic glycolysis, also known as the "Warburg effect." The Warburg effect provides a biological advantage for cancer cells, allowing them to survive and proliferate in low-oxygen or hypoxic environments. At the same time, the metabolic end product lactic acid will accumulate in the environment around cancer cells, thereby forming an immunosuppressive microenvironment that is conducive to tumor growth and metastasis, which has also become an important indicator of malignant tumor transformation. Therefore, finding drug molecules that can affect the Warburg effect has gradually become one of the hot spots in tumor research.

[0003] Phosphorylation of fructose-6-phosphate (F6P) by phosphofructokinase-1 (PFK1) to produce fructose-1,6-bisphosphate (F-1,6-P) is one of the three necessary steps for the Warburg effect in tumor cells. Detecting F6P to evaluate the effect of drugs on the Warburg effect and screen out suitable drugs to inhibit tumor growth has become one of the strategies for tumor drug development.

[0004] Mass spectrometry has been widely used in the detection of F6P due to its advantages such as high specificity and high reproducibility. Since F6P contains multiple polar functional groups, the compound has a high polarity. In the currently commonly used reversed-phase chromatography analysis technology, this type of compound is difficult to retain. With the development of chromatography technology, scientists have reported a number of analytical methods based on the reversed-phase chromatography retention principle, ion pair reagents or derivatization. However, these analytical methods usually have the following problems:

[0005] 1. The mobile phase composition of reversed-phase chromatography is complex: the mobile phase usually needs to be supplemented with formic acid, ammonium acetate, etc. in different proportions, and the proportion of addition needs to be precisely controlled to avoid the analysis method being irreproducible due to different proportions of the mobile phase, which increases the cost of manpower and reagents, and has the problem of poor reproducibility;

[0006] 2. Long analysis time of reversed-phase chromatography: Reverse-phase chromatography usually requires sufficient equilibrium time to ensure the stability and reproducibility of the analysis method, so it usually takes a long analysis time, which leads to the problem of reduced sample detection throughput;

[0007] 3. Derivatization analysis method: Derivatization usually involves introducing a specific derivatization reagent to bind to F6P, reducing the polarity of F6P, thereby allowing the compound to be retained and detected in a reversed-phase system. This method adds additional steps and time to sample pretreatment. In addition, screening for a suitable derivatization reagent also requires additional time and economic costs;

[0008] 4. Loss of organic solvents and chromatographic columns: Long analysis time, complex mobile phase composition, and large amounts of organic solvents and waste liquids that may be generated during sample processing increase the cost of waste solvent processing. In addition, in daily sample analysis, chromatographic columns are consumables that are expensive and easy to lose. When faced with high-throughput compound screening, more chromatographic columns are needed, which also increases the cost of purchasing consumables.

[0009] In summary, there is an urgent need in the art for a high-throughput detection method for determining fructose-6-phosphate in cell matrix, which can shorten analysis time, save manpower and consumables costs, is easy to operate, and can meet high-throughput detection needs. Summary of the invention

[0010] To solve the above technical problems, the present invention provides a high-throughput detection method for determining fructose-6-phosphate in a cell matrix. The detection method is based on acoustic wave excitation mass spectrometry technology, and the sample is introduced into the mass spectrometer by acoustic wave ejection for target compound detection and analysis. The carrier liquid of the sample is an alkaline carrier liquid, the sample ejection volume of the acoustic wave ejection is 10-15nL, and the ejection frequency of the acoustic wave ejection is 1000-4000 milliseconds per sample interval.

[0011] Specifically, the detection parameters of the mass spectrometer are:

[0012] Flow rate: 600 μL / min; ion pair: 265.0→172.0; declustering voltage: -25 V; collision energy: -16 V; heated nebulizer gas: 90 psi; heated auxiliary gas: 45 psi; curtain gas: 24 psi; ion source temperature: 550°C; ion source voltage: 4500 V.

[0013] Specifically, the method comprises the following steps:

[0014] S1. Prepare the detection solution: the detection solution includes a standard working solution, a cell lysis solution, a reconstitution solution and a sample carrier solution;

[0015] S2. Prepare standard curve and quality control samples: add the standard working solution to blank HepG2 cells, then add the cell lysis solution, mix and centrifuge to prepare a mixed calibration standard series solution and a quality control sample series solution;

[0016] S3. Sample injection detection: Add cell lysis solution to the cell matrix to be tested, mix and centrifuge, take the supernatant, blow dry with nitrogen, add re-solution for re-dissolution, then put it into the microporous ultrasonic injection plate for centrifugation and shaking, and then perform ultrasonic excitation mass spectrometry detection.

[0017] Specifically, the standard working solution is composed of 13 The standard working solution is prepared from C6-fructose-6-phosphate and methanol aqueous solution, and the concentration of the standard working solution is 800-400000 nM.

[0018] Specifically, the cell lysis solution is prepared from tolbutamide, DMSO and methanol aqueous solution, and the concentration of the cell lysis solution is 50 ng / mL.

[0019] Specifically, the concentration gradient range of the mixed calibration standard series solution is 20-10000nM, the mixed calibration standard series solution contains 6-10 concentration values, the concentration gradient range of the quality control sample series solution is 80-8000nM, the quality control sample series solution contains 3-5 concentration values, and the quality control sample series solution is prepared in parallel in 6 groups.

[0020] Specifically, the reconstitution solution is an aqueous solution containing 10% methanol.

[0021] Specifically, the alkaline carrier liquid is an aqueous solution containing 0.01% ammonia water.

[0022] Specifically, the sample ejection volume of the acoustic wave ejection is 12.5 nL, and the ejection frequency of the acoustic wave ejection is 2000 milliseconds per sample interval.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention uses acoustic wave excitation mass spectrometry technology to directly introduce samples into mass spectrometry samples for target compound analysis and detection through acoustic wave ejection. It does not use traditional liquid phase modules, reduces the use of organic solvents and cuts the cost of consumables such as chromatographic columns. At the same time, the analysis speed of the present application can reach 3 seconds per sample, which is 4 times faster than the existing LC-MS method of measuring a substance in 12 seconds. This improves the speed of analysis and detection, shortens the analysis time, and improves the analysis efficiency.

[0025] 2. The detection method provided by the present invention simplifies the operating steps of analyzing the mobile phase ratio and reduces the injection volume to only 1 / 400 of the existing LC-MS method. While saving labor costs and reagent costs, it ensures the reproducibility and stability of the method.

[0026] 3. The detection method provided by the present invention can greatly simplify the operation while ensuring high analytical sensitivity and stability. The quantitative lower limit of F6P in this method is 20nM, which can meet the research of related experiments.

[0027] 4. The detection method provided by the present invention overcomes the defects of the prior art, is easy to operate, and can also meet the needs of high-throughput detection. Take the analysis of 1,000 samples as an example: in terms of time cost, the LC-MS method takes about 200 minutes, while the present method only takes about 50 minutes, which is 1 / 4 of the time of the existing method; in terms of organic solvent cost, the LC-MS method consumes 320 milliliters, while the present method only takes 30 milliliters, and the organic solvent cost is less than 1 / 10 of the existing method; in terms of chromatographic column cost, the LC-MS method requires two chromatographic columns, which is estimated to be about 2,000 yuan, while the present method does not require a chromatographic column and costs 0 yuan. Based on the reduction of costs and the simplification and acceleration of the analysis process, it is possible to quickly screen a large number of candidate compounds. For the needs of high-throughput detection, whether from the perspective of detection efficiency or cost, this method will undoubtedly become the first choice. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 yes 13 Acquisition spectra of C6-fructose-6-phosphate in the range of 2.5-50 nL ejection volume;

[0029] Figure 2 When the ejection interval is 1000 to 4000ms 13 C6-fructose-6-phosphate spectrum;

[0030] Figure 3 yes 13 Acquisition spectra of C6-fructose-6-phosphate under different carrier conditions;

[0031] Figure 4 yes 13 The characteristic m / z signal intensity of C6-fructose-6-phosphate changes with the collision energy;

[0032] Figure 5 yes 13 Standard curve of C6-fructose-6-phosphate and typical collection diagram of quality control samples;

[0033] Figure 6 yes 13 Standard curve of C6-fructose-6-phosphate in the concentration range of 20-10000 nM. DETAILED DESCRIPTION

[0034] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0035] In the following examples, methanol, acetonitrile, and formic acid were of chromatographic grade, and the rest were of analytical grade; deionized water (18 MΩ) was produced in-house using a Millipore Milli-Q water purification system; 13 C6-fructose-6-phosphate standard was purchased from MCE; tolbutamide was purchased from TCI Chemical; ammonium bicarbonate was purchased from Shanghai Laboratory Reagent; ammonia water and ammonium formate were purchased from Yonghua Chemical; and 384 microwell sonic plate was purchased from LABCYTE.

[0036] Instruments and equipment used: Echo MS acoustic excitation coupled mass spectrometry system (acoustic droplet injection platform equipped with SCIEX TripleQuad 6500+ mass spectrometer), purchased from SCIEX.

[0037] Embodiment 1

[0038] In a specific embodiment, the fructose-6-phosphate in the cell matrix is ​​determined using an Echo MS acoustic excitation coupled mass spectrometry system (acoustic droplet jetting platform equipped with a SCIEXTriple Quad 6500+ mass spectrometer), and the specific operation is as follows:

[0039] 1. Solution preparation

[0040] 1. Standard substance stock solution: weigh an appropriate amount of 13 The C6-fructose-6-phosphate standard powder is accurately weighed and prepared into a 10 mM standard stock solution with a 50% methanol aqueous solution and stored in a refrigerator at -40°C for later use.

[0041] 2. Standard working solution: Take an appropriate amount of 13 The C6-fructose-6-phosphate standard stock solution was diluted with an appropriate amount of 80% methanol aqueous solution to obtain a 800-400000 nM standard working solution, which was stored in a refrigerator for later use.

[0042] 3. Cell lysis solution: Accurately weigh an appropriate amount of tolbutamide standard powder, prepare it into 1 mg internal standard stock solution with DMSO, dilute it with 80% methanol aqueous solution to obtain a tolbutamide internal standard solution with a final concentration of 50 ng / mL, and store it in a refrigerator.

[0043] 4. Blank HepG2 cells: HepG2 cell plates cultured for 24 hours without adding unknown test compounds.

[0044] 2. Preparation of standard curve, quality control samples and test samples

[0045] 1. Use a pipette to manually pipette appropriate amounts of 13 C6-fructose-6-phosphate standard working solution was added to blank HepG2 cells, and an appropriate amount of cell lysis solution (80% methanol aqueous solution containing 50 ng / mL tolbutamide) was added, mixed and centrifuged to prepare a mixed calibration standard series solution and a quality control sample series solution. The theoretical concentrations of the mixed calibration standard series solution are 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10000 nM from low to high. The theoretical concentrations of the quality control sample series solution are 80, 800 and 8000 nM from low to high. Six groups of quality control standards were prepared in parallel.

[0046] 2. Add cell lysis solution to the cell matrix to be tested, mix and centrifuge, take 150 μL of supernatant and blow dry with nitrogen, then re-dissolve with 50 μL of aqueous solution containing 10% methanol, take 35 μL into 384 micropore sonic injection plate, centrifuge for 5 minutes, shake for 5 minutes, and perform Echo MS analysis.

[0047] 3. Testing conditions

[0048] Using an aqueous solution containing 0.01% ammonia as the carrier liquid, the acoustic wave ejects 12.5nL of the sample into the detection system, and the detection is performed according to the analysis conditions in Table 1 below. The ejection interval frequency of each sample is 2000 milliseconds, achieving a rapid test of the sample in a 3-second analysis time. 13 C6-fructose-6-phosphate content.

[0049] Table 1 13 Analysis Conditions of C6-Fructose-6-Phosphate on Echo MS System

[0050]

[0051] *SP:surfactant phase, from SCIEX

[0052] 4. Condition Optimization and Result Analysis

[0053] 1. Sample ejection volume optimization

[0054] In order to ensure sufficient detection sensitivity of this method as much as possible, the optimization of the ejection volume is crucial. If the volume is too small, the method sensitivity cannot be achieved and the target substance signal in the sample cannot be accurately determined. If the ejection volume is too large, it may lead to mass spectrometry signal saturation or matrix effect.

[0055] Under the premise of ensuring that other detection conditions remain unchanged, the sample ejection volume is changed, and multiple groups of samples are set to 2.5nL to 50nL, and the ejection volume difference between each adjacent group is 2.5nL (one drop). For every 2.5nL increase in ejection volume, the change in peak area is as follows Figure 1 As shown in Table 2, it can be seen that as the ejection sample volume increases, the peak area of ​​the target compound also increases. 13 After the ejection volume of C6-fructose-6-phosphate reached 12.5 nL, increasing the ejection volume did not significantly increase the signal and gradually tended to saturation. Therefore, considering the signal response and peak shape, the ejection volume determined by this method was 12.5 nL.

[0056] Table 2 13 Peak area of ​​C6-fructose-6-phosphate at 2.5-50 nL ejection volume

[0057] Ejection volume (nL) Peak area Ejection volume (nL) Peak area 2.5 1.72E+04 27.5 6.67E+04 5 2.74E+04 30 6.05E+04 7.5 3.32E+04 32.5 6.64E+04 10 4.01E+04 35 6.08E+04 12.5 4.77E+04 37.5 7.00E+04 15 5.00E+04 40 7.35E+04 17.5 4.91E+04 42.5 7.55E+04 20 5.62E+04 45 7.15E+04 22.5 5.58E+04 47.5 7.44E+04 25 6.54E+04 50 7.22E+04

[0058] 2. Optimize the ejection interval time

[0059] Under this method, all samples are collected in the same data spectrum. To ensure that each peak can be accurately integrated, the peaks need to be completely baseline separated to ensure sufficient reproducibility and stability. If the ejection time is short, the analysis speed will be faster, but poor peak shape may also be obtained, especially at some high-concentration sample points, which may easily cause tailing, and the signal will not return to the baseline before the next sample is collected. If the ejection interval is long, it can ensure that there is good separation between each peak, but the corresponding analysis speed will be slower. Therefore, it is crucial to optimize the ejection interval time.

[0060] Under the premise of ensuring that other detection conditions remain unchanged, the sample ejection time interval is changed to 1000, 1500, 2000, 2500, 3000, 3500 and 4000 milliseconds respectively. The peak shape collection spectrum of the sample and the analysis time required for each sample are as follows: Figure 2 As shown, it can be seen that the acquisition time of each sample is from 2 seconds to 5 seconds, and the interval time starts from 2000 milliseconds. Each sample peak can be well separated, but in order to ensure the stability, reproducibility and analysis rate of the method, this scheme selects the frequency of 2000 milliseconds.

[0061] 3. Optimization of carrier conditions

[0062] Adding different additives to the carrier liquid can affect the signal response and peak shape of the compound. Therefore, finding suitable carrier liquid conditions is also one of the important research points of this application. Under the premise of ensuring that other detection conditions remain unchanged, change the carrier liquid and combine Figure 3, showing the peak shapes and signal spectra obtained by parallel ejection of three samples under six different carrier liquids. Figure 3 In the figure, Figure (a) is a 30% acetonitrile aqueous solution containing 0.1% formic acid, and the mass spectrum response is 9240cps; Figure (b) is a 30% acetonitrile aqueous solution containing 10mM ammonium formate, and the mass spectrum response is 8000cps; Figure (c) is a 30% acetonitrile aqueous solution containing 10mM ammonium bicarbonate, and the mass spectrum response is 6828cps; Figure (d) is a 30% acetonitrile aqueous solution containing 0.1% ammonia, and the mass spectrum response is 6.0e4 cps; Figure (e) is a 10% acetonitrile aqueous solution containing 0.1% ammonia, and the mass spectrum response is 1.09e5 cps; Figure (f) is an aqueous solution containing 0.1% ammonia, and the mass spectrum response is 1.6e5 cps. It can be seen from the figure that under acidic and neutral carrier liquid systems, 13 C6-fructose-6-phosphate showed tailing and low mass spectrometric response. 13 The peak shape of C6-fructose-6-phosphate is more symmetrical, and the mass spectrometry response is improved by nearly 10 times. Combined with the pH recommended range of the electrode in the hardware, 0.01% ammonia water solution (pH=9.9) was finally determined as the carrier liquid of this method.

[0063] 4. Mass spectrometry parameter optimization

[0064] The rationality of mass spectrometry parameters directly affects the signal response of the test compound. In this application, several key parameters are optimized. Under the premise of ensuring that other detection conditions remain unchanged, the collision energy (CE) is changed, and the optimization range is 0~(-50)V. Figure 4 , showing 13 The signal spectrum of the characteristic m / z signal intensity of C6-fructose-6-phosphate changes with the collision energy. The horizontal axis is the CE value and the vertical axis is the signal intensity. It can be seen that the change of collision energy will greatly affect the signal response of the target. 13 C6-fructose-6-phosphate can obtain a higher signal when CE is -16V.

[0065] 5. Linear range and lower limit of quantitation

[0066] The quantitative detection concentration range and the lowest quantitative limit of this method can be evaluated by evaluating the linear fit between the test concentration values ​​of the standard compound at different concentrations and the designed concentration values. Figure 5 and Figure 6 As shown, 13 C6-fructose-6-phosphate standard curve in the concentration range of 20-10000nM, 13 The linear regression equation of C6-fructose-6-phosphate is y=6.846e -5 x+1.717e -4 , (R 2>0.98). The lowest quantitative concentration (LLOQ) was 20nM, and the concentration points of the standard curve were: the concentrations of the standard curve samples C9-C1 were 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000nM, and the low, medium and high concentrations of the quality control samples were: low concentration quality control sample (LQC) 80nM, middle concentration quality control sample (MQC) 800nM, high concentration quality control sample (HQC) 8000nM. The results showed that 13 C6-fructose-6-phosphate had a good linear relationship within the range of 20-10000nM, the lower limit of quantification was 20nM, and the signal-to-noise ratio was greater than 5, all of which met the methodological requirements.

[0067] 6. Accuracy and precision

[0068] The precision and accuracy of the method provided in this application were evaluated by testing the concentration of the quality control samples, and the results are shown in Table 3 below.

[0069] Table 3 13 Accuracy and precision of C6-fructose-6-phosphate in cell matrices

[0070]

[0071]

[0072] It can be seen that in terms of detection accuracy, the detection values ​​of all standard curve samples are within 94.88% to 113.86% of the theoretical concentration. Three groups of quality control samples with different concentrations were used (low concentration quality control sample LQC: 80nM, medium concentration quality control sample MQC: 800nM, high concentration quality control sample HQC: 8000nM), all of which were between 99.73% and 104.65% of the theoretical concentration. All samples were analyzed by repeated ejection 6 times for three consecutive days. In terms of detection precision, the CV% for all standard curve samples within the day was 5.04% to 13.70%, and the deviation (CV%) for quality control samples was between 7.88% and 9.25%. In terms of detection reproducibility, for daytime samples, the CV% of standard curve samples was between 0.91% and 8.13%, and the CV% of quality control samples was between 1.4% and 1.9%. This shows that the method provided in this application is accurate in determining the 13 The precision and accuracy of C6-fructose-6-phosphate concentrations were good.

[0073] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-throughput detection method for determining fructose-6-phosphate in a cell matrix, characterized in that: The detection method is based on acoustic wave excitation mass spectrometry technology, and the sample is introduced into the mass spectrometer through acoustic wave ejection to perform target compound detection and analysis. The carrier liquid of the sample is an alkaline carrier liquid, the sample ejection volume of the acoustic wave ejection is 10-15nL, and the ejection frequency of the acoustic wave ejection is 1000-4000 milliseconds per sample interval.

2. The high-throughput detection method for determining fructose-6-phosphate in a cell matrix according to claim 1, characterized in that: The detection parameters of the mass spectrometer are: Flow rate: 600 μL / min; ion pair: 265.0→172.0; declustering voltage: -25 V; collision energy: -16 V; heated nebulizer gas: 90 psi; heated auxiliary gas: 45 psi; curtain gas: 24 psi; ion source temperature: 550°C; ion source voltage: 4500 V.

3. The high-throughput detection method for determining fructose-6-phosphate in a cell matrix according to claim 1, characterized in that: The method comprises the following steps: S1. Prepare the detection solution: the detection solution includes a standard working solution, a cell lysis solution, a reconstitution solution and a sample carrier solution; S2. Prepare standard curve and quality control samples: add the standard working solution to blank HepG2 cells, then add the cell lysis solution, mix and centrifuge to prepare a mixed calibration standard series solution and a quality control sample series solution; S3. Sample injection detection: Add cell lysis solution to the cell matrix to be tested, mix and centrifuge, take the supernatant, blow dry with nitrogen, add re-solution for re-dissolution, then put it into the microporous ultrasonic injection plate for centrifugation and shaking, and then perform ultrasonic excitation mass spectrometry detection.

4. The high-throughput detection method for determining fructose-6-phosphate in cell matrix according to claim 3, characterized in that: The standard working solution is 13 The standard working solution is prepared from C6-fructose-6-phosphate and methanol aqueous solution, and the concentration of the standard working solution is 800-400000 nM.

5. The high-throughput detection method for determining fructose-6-phosphate in cell matrix according to claim 3, characterized in that: The cell lysis solution is prepared from tolbutamide, DMSO and methanol aqueous solution, and the concentration of the cell lysis solution is 50 ng / mL.

6. The high-throughput detection method for determining fructose-6-phosphate in cell matrix according to claim 3, characterized in that: The concentration gradient range of the mixed calibration standard series solution is 20-10000nM, the mixed calibration standard series solution contains 6-10 concentration values, the concentration gradient range of the quality control sample series solution is 80-8000nM, the quality control sample series solution contains 3-5 concentration values, and the quality control sample series solution is prepared in parallel in 6 groups.

7. The high-throughput detection method for determining fructose-6-phosphate in cell matrix according to claim 3, characterized in that: The reconstitution solution is an aqueous solution containing 10% methanol.

8. The high-throughput detection method for determining fructose-6-phosphate in a cell matrix according to claim 1, characterized in that: The alkaline carrier liquid is an aqueous solution containing 0.01% ammonia water.

9. The high-throughput detection method for determining fructose-6-phosphate in a cell matrix according to claim 1, characterized in that: The sample ejection volume of the acoustic wave ejection is 12.5 nL, and the ejection frequency of the acoustic wave ejection is 2000 milliseconds per sample interval.

Images