Method for detecting 217-phosphorylated tubulin fragment on basis of chemical derivatization method, and use thereof

Abstract

The present invention relates to a method for detecting a 217-phosphorylated tubulin fragment on the basis of a chemical derivatization method, and the use thereof. The detection method comprises the following steps: S1, adding TMPP-Ac-OSu and a triethylamine solution to a p-Tau 217 solution, mixing same and then performing a derivatization reaction, and then adding an aqueous ammonia solution to perform a quenching reaction so as to obtain a sample solution; and S2, performing LC-MS / MS detection on the sample solution. In the present invention, TMPP-Ac-OSu is innovatively utilized to perform a specific chemical derivatization reaction with p-Tau 217, such that the signal intensity of the resulting chemical derivatization product is significantly enhanced, and the detection capability is improved. Analysis can be performed under Vanquish UHPLC liquid phase conditions, and paired with a more cost-effective TSQ AltisTM Triple Quadrupole instrument, thereby greatly reducing the detection costs. Moreover, the method involves short time consumption and thus can be used for rapid extraction of a large number of samples. The detection method of the present invention is of great significance for the early detection of Alzheimer's disease.

Description

Methods and applications for detecting 217-phosphorylated tubulin fragments based on chemical derivatization Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a method and application for detecting 217-phosphorylated tubulin fragments based on chemical derivatization. Background Technology

[0002] In the diagnosis of Alzheimer's disease (AD), accurate detection of plasma levels of 217-phosphorylated tubulin fragment (p-Tau 217), an Alzheimer's disease (AD)-related biomarker, is crucial. However, due to the extremely low concentration of p-Tau 217 in plasma, conventional mass spectrometry detection techniques face challenges in sensitivity. Alzheimer's disease is a neurodegenerative disease, and early diagnosis is essential for its management and treatment. p-Tau 217, as an important biomarker of AD, plays a vital role in the early diagnosis and progression monitoring of the disease. However, current detection methods rely on high-performance but expensive mass spectrometry equipment and require lengthy chromatographic separations, increasing detection costs and complexity.

[0003] Traditional p-Tau 217 detection methods, such as the technique developed by Washington University in St. Louis, employ a nanoAcquity UPLC system for liquid phase separation after sample processing, utilizing high-end Fusion chromatography. TM Lumos TM Tribrid TM Mass spectrometry was used to analyze p-Tau 217; another example is the LC-MS method developed by the University of Gothenburg, where, after sample processing, nano-LC liquid chromatography was performed using a Dionex 3000 system, followed by analysis using Eclipse. TM Tribrid TM p-Tau 217 was analyzed using mass spectrometry. All of the above methods require high-end mass spectrometers for detection, increasing the cost and complexity of the analysis.

[0004] Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing technologies, this invention proposes a method and application for detecting 217-phosphorylated tubulin fragments based on chemical derivatization. This method overcomes the limitations of existing technologies by improving the mass spectrometry detection sensitivity of p-Tau 217 (217-phosphorylated tubulin fragments). Specifically, this invention includes the following steps: First, a p-Tau 217 sample is derivatized with a specific chemical reagent to increase its mass spectrometry detection response signal. Then, the derivatized sample is separated using high-performance liquid chromatography (HPLC). Finally, the separated sample is detected using mass spectrometry. Compared with existing technologies, the chemical derivatization method of this invention significantly improves the detection sensitivity of p-Tau 217, specifically by increasing the mass spectrometry signal of p-Tau 217 (below picograms per milliliter) by 20 times. This technological breakthrough not only reduces dependence on high-end mass spectrometry equipment but also reduces detection costs and time, making early diagnosis of Alzheimer's disease (AD) possible under a wider range of experimental conditions. In this method, the signal of the chemically derivatized products is significantly enhanced, increasing the detection capability. It can be combined with the more cost-effective TSQ Altis under Vanquish UHPLC conditions. TM The analysis using the Triple Quadrupole instrument significantly reduces the cost of detection. Furthermore, the method is time-efficient and can be used for rapid extraction of large numbers of samples.

[0006] The objective of this invention can be achieved through the following methods:

[0007] In a first aspect, the present invention provides a method for detecting 217-phosphorylated tubulin fragments based on a chemical derivatization method, the method comprising the following steps:

[0008] S1. Add TMPP-Ac-OSu and triethylamine solution to p-Tau 217 solution, mix and carry out derivatization reaction, then add ammonia solution to quench reaction to obtain sample solution;

[0009] S2. Take the sample solution and perform LC-MS / MS detection.

[0010] In one embodiment of the present invention, in step S1, the concentration of the p-Tau 217 solution is 0.001-1000 pg / mL.

[0011] In some preferred embodiments, the p-Tau 217 solution is prepared using an aqueous solution of 2 wt.% acetonitrile.

[0012] In one embodiment of the present invention, in step S1, the ratio of p-Tau 217 to TMPP-Ac-OSu is 0.001-1000 pg / mL: 1-300 μg.

[0013] In one embodiment of the present invention, in step S1, the ratio of p-Tau 217, triethylamine solution, and ammonia solution is 0.001-1000 pg / mL: 0.01-10 μL: 1.01-10 μL.

[0014] Furthermore, the concentration of the ammonia solution is 18-23 wt.%; the concentration of the triethylamine solution is 18-22 wt.%.

[0015] Furthermore, the concentration of the p-Tau 217 solution is 0.1-1000 pg / mL.

[0016] As one embodiment of the present invention, in step S1, the mixing method includes agitation and stirring, wherein the agitation and stirring time is 0.5-2 hours and the agitation and stirring frequency is 800-1200 RPM.

[0017] In one embodiment of the present invention, in step S1, p-Tau 217 undergoes a derivatization reaction to generate a p-Tau 217-CHEM derivative.

[0018] In one embodiment of the present invention, in step S1, hydroxylamine solution is used instead of ammonia solution.

[0019] As one embodiment of the present invention, in step S2, the sample solution is transferred into a vial or a 96-well injection plate and then loaded for LC / MS / MS detection.

[0020] In one embodiment of the present invention, the parameters detected by LC / MS / MS in step S2 include:

[0021] Chromatographic conditions:

[0022] Chromatographic column: Ruikang C18 column;

[0023] Mobile phase A: Water + 0.1% ammonia;

[0024] Mobile phase B: Acetonitrile + 0.1% ammonia;

[0025] The mobile phase gradient is as follows:

[0026] The mass spectrometry acquisition parameters are as follows:

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. This invention creatively employs a specific chemical derivatization reaction between TMPP-Ac-OSu and p-Tau 217. By modifying p-Tau 217 with TMPP-Ac-OSu, the mass spectrometry detection sensitivity of p-Tau 217 can be increased by 20 times, effectively solving the detection challenge caused by its extremely low concentration in plasma. At the same time, this derivatization reaction can be carried out at extremely low equivalents (the total amount of p-Tau 217 is less than 0.1 pg, and the reaction is still applicable and effective).

[0029] 2. Existing detection technologies rely on high-performance but expensive mass spectrometry equipment, and the long chromatographic separation time increases the cost and complexity of detection. The method of this invention improves detection efficiency through chemical derivatization, thereby reducing reliance on high-end mass spectrometry equipment. It can be used in conjunction with the more cost-effective TSQ Altis under Vanquish UHPLC conditions. TM The Triple Quadrupole instrument reduces testing costs and time.

[0030] 3. p-Tau 217, as an important biomarker for Alzheimer's disease (AD), plays a significant role in the early diagnosis and progression monitoring of the disease. This invention improves detection sensitivity, making early diagnosis of AD possible under a wider range of experimental conditions, thus promoting early diagnosis of Alzheimer's disease and having important clinical value for disease management and treatment.

[0031] 4. This derivatization reaction has a short reaction time, mild conditions, and is simple to operate and easy to apply. Attached Figure Description

[0032] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0033] Figure 1 shows the calibration curves for Example 1 without derived p-Tau 217;

[0034] Figure 2 shows the calibration curve of the derived p-Tau 217 in Example 1;

[0035] Figure 3 shows the detection spectrum of p-Tau 217 (Cal 4) at the lowest point of the calibrator in Example 1 without the derivation of p-Tau 217 (Cal 4);

[0036] Figure 4 shows the detection spectrum of p-Tau 217-CHEM(Cal 1) derived from the lowest point of the calibrator in Example 1;

[0037] Figure 5 is a schematic diagram of the derivatization reaction in Example 1 and the signal intensity / retention time relationship of p-Tau 217 and p-Tau 217-CHEM. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0039] Prepare reagents:

[0040] p-Tau 217 solution (p-Tau 217 dissolved in LCMS pure water);

[0041] TMPP-Ac-OSu solution (TMPP-Ac-OSu dissolved in anhydrous DMF, concentration 50 mg / mL);

[0042] Ammonia solution (20 wt.% ammonia solution);

[0043] Triethylamine solution (triethylamine dissolved in 10 wt.% acetonitrile solution);

[0044] The reaction apparatus used: Thermo Shaker Incubator (heating) (Model: TM-100H).

[0045] Example 1

[0046] Derivatization reaction and liquid chromatography-mass spectrometry analysis:

[0047] Take out the p-Tau 217 solution and dilute it to the level of 5-1000 pg / mL in an aqueous solution containing 2 wt.% acetonitrile.

[0048] Take 20 μL of the diluted p-Tau 217 solution and transfer it to a reaction centrifuge tube. Add 0.8 μL of LMPP-Ac-OSu solution (50 mg / mL), 5 μL of triethylamine (20 wt.%), and a mixed solvent of acetonitrile and water (2 wt.% acetonitrile) to make the reaction volume 45 μL. Place the tube in a reaction apparatus and shake and stir at room temperature for 1 hour at a shaking frequency of 1000 RPM to carry out the derivatization reaction, forming p-Tau 217-CHEM. The derivatization reaction is shown in the upper part of Figure 5 (the target peptide is p-Tau 217, and the chemically modified peptide is p-Tau 217-CHEM). Figure 5 shows the signal intensity / retention time relationship of p-Tau 217 and p-Tau 217-CHEM. It is evident that the p-Tau 217-CHEM derivative, formed by TMPP-Ac-OSu modification of p-Tau 217, significantly improves the signal intensity, thereby enhancing its detection sensitivity.

[0049] After the reaction was complete, 5 μL of ammonia solution was added to quench the reaction. After the reaction was complete, the solution was gently shaken on a vibrating plate for 60 s to mix. Then, all the solution was transferred to a vial or a 96-well plate, and 20 μL was taken as the experimental group and control group for LC-MS / MS analysis. The control group was maintained identically to the experimental group without the addition of TMPP-Ac-OSu solution. The reaction was performed under Vanquish UHPLC conditions with TSQ Altis... TM The analysis was performed using a Triple Quadrupole instrument.

[0050] The chromatographic conditions for LC-MS / MS detection are as follows:

[0051] Chromatographic column: Ruikang C18 column;

[0052] Mobile phase A: Water + 0.1% ammonia;

[0053] Mobile phase B: Acetonitrile + 0.1% ammonia;

[0054] The mobile phase gradient is shown in Table 1:

[0055] Table 1

[0056] The mass spectrometry acquisition parameters are shown in Table 2:

[0057] Table 2

[0058] The calibration curves obtained by LC-MS / MS detection are shown in Figure 1 (underdered p-Tau 217) and Figure 2 (derderified p-Tau 217-CHEM), and Table 3 shows the relationship between compound concentration (injection volume) and signal, while Table 4 shows the relative deviation between the calculated actual and theoretical injection volumes. These analytical results were all performed according to standard procedures using mass spectrometry software, including the calculation of detection signal and standard deviation. Table 3 shows that when the injection volume is below 800 femt, the underderified p-Tau 217 (control group) cannot be effectively detected. However, the derivatized p-Tau 217-CHEM (experimental group) can be effectively detected even with an injection volume of 40 femt, demonstrating its signal enhancement effect. Table 4 shows that the mass spectrometry signal of the derivatized p-Tau 217-CHEM (experimental group) has a good linear relationship with the actual injection volume; for each concentration sample, the deviation of the linear fitting result is less than 15%.

[0059] Table 3

[0060] Table 4

[0061] Furthermore, when detecting the lowest point of the calibrator, Cal 1, the signal-to-noise ratio of the compound is greater than 10 (Figures 3 and 4), which meets the detection requirements.

[0062] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for detecting 217-phosphorylated tubulin fragments based on chemical derivatization, characterized by, The method comprises the following steps: S1, adding TMPP-Ac-OSu, triethylamine solution to the p-Tau 217 solution, mixing and then performing a derivatization reaction, and then adding an ammonia solution to perform a quenching reaction to obtain a sample solution; S2, taking the sample solution for LC-MS / MS detection.

2. The method of claim 1, wherein, In step S1, the concentration of the p-Tau 217 solution is 0.001-1000 pg / mL.

3. The method of claim 1, wherein, In step S1, the ratio of the amount of p-Tau 217 to TMPP-Ac-OSu is 0.001-1000 pg / mL: 1-300 μg.

4. The method of claim 1, wherein, In step S1, the ratio of the amount of p-Tau 217, triethylamine solution and ammonia solution is 0.001-1000 pg / mL: 0.01-10 μL: 1.01-10 μL.

5. The method of claim 1, wherein, In step S1, the concentration of the ammonia solution is 18-23 wt.%, and the concentration of the triethylamine solution is 18-22 wt.%.

6. The method of claim 1, wherein, In step S1, the mixing method comprises oscillation stirring, the oscillation stirring time is 0.5-2 hours, and the oscillation stirring frequency is 800-1200 RPM.

7. The method of claim 1, wherein, In step S1, the ammonia solution is replaced by a hydroxylamine solution.

8. The method of claim 1, wherein, In step S2, the sample solution is moved into a sample vial or a 96-well sample plate, and then loaded into LC / MS / MS for detection.

9. The method of claim 1, wherein, In step S2, the chromatographic conditions of the LC / MS / MS detection are as follows: Chromatographic column: Rekon C18 chromatographic column; Mobile phase A: water + 0.1% ammonia water; Mobile phase B: acetonitrile + 0.1% ammonia water; The mobile phase gradient was as follows:

10. The method of claim 1, wherein, In step S2, the mass spectrometry acquisition parameters of the LC / MS / MS detection are as follows:

Images