Application of MALDI-TOF mass spectrum probe in carboxylesterase detection

Abstract

The invention relates to application of an MALDI-TOF mass spectrum probe in carboxylesterase detection, is used for quantitative analysis of carboxylesterase and screening of related inhibitor drugs, and belongs to the technical field of mass spectrum analysis and testing. Aiming at the problem that carboxylesterase is difficult to detect by using a mass spectrum method, the invention provides the MALDI-TOF mass spectrum probe for realizing rapid, accurate and specific detection of carboxylesterase, and the MALDI-TOF mass spectrum probe is used for detecting a complex system. Compared with the prior art, the method has the advantages that the problem of complex synthesis of carboxylesterase molecular probes is solved, interference of phenomena such as substrate optical signals, light scattering and photobleaching in spectra is avoided, rapid and accurate analysis of carboxylesterase in hepatoma carcinoma cells and detection of inhibitors are realized, and whether high-flux simultaneous analysis can be performed on samples on the MALDI target plate or not is judged; and the analysis speed and efficiency are greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of mass spectrometry analysis and testing, and particularly relates to an application of a MALDI-TOF mass spectrometry probe in carboxylesterase detection. Background Art

[0002] Carboxylesterase (CE) is one of the most abundant serine hydrolases in the human liver. It plays an important role in the metabolism of xenobiotics in the liver and is expected to be a new biomarker candidate for hepatocellular carcinoma (HCC). A database search study showed that CE can be used as a good serological indicator of hepatocellular damage. A recent study showed that based on an enzyme-linked immunosorbent assay of 208 patients (including 57 HCC patients), the CE level in HCC patients was significantly higher than that in healthy donors and other liver diseases.

[0003] Currently, most carboxylesterase detection methods are based on spectrophotometry, chromatography, and fluorescent probe-based detection techniques. However, the need for fluorescent or chromophore labels, overlapping light signals, and complex chemical labeling processes limit the application of such methods.

[0004] Another alternative method is mass spectrometry. Someone has measured the absolute protein abundance of CE in human liver microsomes and cytoplasm by liquid chromatography-tandem mass spectrometry proteomics. However, no one has yet utilized the method of mass spectrometry to detect the activity of carboxylesterase, which may be due to the difficulty of ionization of the substrate of carboxylesterase under mass spectrometry conditions and is not easily detected. At present, matrix-assisted laser desorption ionization (MALDI) mass spectrometry has the advantages of less sample dosage, fast analysis speed, high salt tolerance and high throughput, and can simplify the separation, desalination and labeling steps of complex samples during analysis. MALDI mass spectrometry has become an important tool for the activity analysis of various enzymes and the screening of inhibitor drugs. Therefore, a low-cost, highly sensitive and highly specific MALDI mass spectrometry probe for carboxylesterase activity analysis and drug screening is urgently needed. Summary of the invention

[0005] The purpose of the present invention is to provide a MALDI-TOF mass spectrometry probe for carboxylesterase (CE) detection, to achieve quantitative analysis of CE and screening of related inhibitor drugs. Compared with the prior art, the present invention solves the problem of complex synthesis of carboxylesterase molecular probes, avoids the interference of light signal overlap, achieves rapid and accurate analysis of carboxylesterase in cells and analysis of inhibitor drugs, and can achieve simultaneous analysis of hundreds of samples on the same target plate, greatly improving the analysis speed and efficiency.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A MALDI-TOF mass spectrometry probe is used in the detection of carboxylesterase. The MALDI-TOF mass spectrometry probe molecule has a structure shown in formula (I), and the product after the reaction with carboxylesterase has a structure shown in formula (II):

[0008]

[0009] Furthermore, the present invention provides a method for using a MALDI-TOF mass spectrometry probe for detecting carboxylesterase content, comprising the following steps:

[0010] Step 1: preparing PBS solutions of carboxylesterase standards of different concentrations, and adding mass spectrometry molecular probes of formula (I) to the above solutions for enzyme cleavage reaction to obtain products of formula (II);

[0011] Step 2: Take 1 μL of sample and 1 μL of matrix, mix them evenly, and then spot them on the MALDI target plate. After drying, inject and analyze;

[0012] Step 3: Draw a standard curve with the different activity values ​​of the carboxylesterase in step 1 as the horizontal axis and the ratio of the MALDI signal intensity of formula (ⅠⅠ) in step 2 to the total signal intensity of the formula (Ⅰ) and formula (ⅠⅠ) as the vertical axis;

[0013] Step 4: Prepare a PBS solution of the sample to be tested, add the mass spectrometry probe of formula (I), add acetonitrile after enzyme cleavage reaction and centrifuge, take 1 μL of supernatant and 1 μL of matrix solution, mix them evenly and then spot them on the MALDI target plate, dry them and perform MALDI mass spectrometry analysis, and measure I 式Ⅱ / (I 式Ⅱ +I 式Ⅰ ) value into the standard curve to obtain the activity of carboxylesterase in the sample to be tested.

[0014] As a more optimal technical solution of the present invention: the pH value of the PBS solution in step 1 is preferably 7-9, and more preferably 7.4; the volume of the solution is preferably 100-1000 μL, and more preferably 400 μL.

[0015] As a more optimal technical solution of the present invention: the enzyme cleavage time in step 1 is preferably 0.1 to 1.2 hours, and most preferably 50 minutes.

[0016] As a more optimal technical solution of the present invention: the sample to be tested in step 4 is a sample containing a large amount of protein.

[0017] As a more optimal technical solution of the present invention: the matrix described in step 2 is preferably α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and α-cyano-4-hydroxycinnamic acid (CHCA) is most preferred.

[0018] As a more optimal technical solution of the present invention: the concentration of the formula (I) in step 1 is preferably 5-10 μM, most preferably 8 μM; the volume is preferably 10-100 μL, most preferably 10 μL.

[0019] The present invention provides a method for preparing a MALDI-TOF mass spectrometry probe, the steps of which are as follows:

[0020] (1) 2-Aminoquinoline (4 mmol, 576 mg) was dissolved in DCM, and then 4-dimethylaminopyridine (5 mmol, 610 mg) and sodium carbonate (12.5 mmol, 1325 mg) were added to the organic phase. The mixture was stirred in an ice bath under the protection of nitrogen. Then, furoyl chloride (6 mmol, 780 mg) was slowly added. The reaction was allowed to react for 4 h until complete conversion, and the reaction progress was monitored by TLC.

[0021] (2) After 4 hours, the crude product was extracted with a column and then purified by chromatography filtration (petroleum ether / ethyl acetate = 4:1) to obtain a white-grey product of formula (I).

[0022] The present invention has the following advantages:

[0023] 1. The MALDI-TOF mass spectrometry probe provided by the present invention has the characteristics of simple synthesis, stability under mass spectrometry conditions, high sensitivity, high selectivity and high ionization efficiency.

[0024] 2. The product obtained by hydrolysis of the mass spectrometry probe provided by the present invention is easily detected, avoiding the use of isotope labels or internal standard molecules, and achieving accurate quantification of carboxylesterase activity.

[0025] 3. The mass spectrometry probe provided by the present invention undergoes specific cleavage only under the action of carboxylesterase, while other common inorganic salts, amino acids, proteins, hydrolases, etc. do not cause interference.

[0026] 5. The present invention has good sensitivity, reproducibility and selectivity, and is suitable for rapid screening of inhibitor drugs and quantitative analysis of carboxylesterase in cells.

[0027] 6. The mass spectrometry probe and method provided by the present invention complement each other with MALDI mass spectrometry, can realize high-throughput analysis of actual samples, and are easy to promote and apply in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art; obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 The synthetic route and enzyme cleavage reaction equation of the mass spectrometry probe formula (I) of the present invention;

[0030] Figure 2 is the MALDI signal intensity ratio of 8 μM of the molecule of formula (I) and 8 μM of the molecule of formula (II) of the present invention;

[0031] Figure 3 The MALDI mass spectra of the mass spectrometry probe formula (I) of the present invention before and after the reaction;

[0032] Figure 4 The response MALDI spectra of different concentrations of carboxylesterase (0.1-1.2 U / mL) of the present invention;

[0033] Figure 5 is the quantitative analysis curve of carboxylesterase at different concentrations of the present invention;

[0034] Figure 6 The mass spectrometry probe (I) of the present invention is used to evaluate the response of different inorganic salts, amino acids, proteins, hydrolases and carboxylesterases;

[0035] Figure 7 The mass spectrometry probe formula (I) method of the present invention is used to evaluate the IC50 of the inhibitor drug 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF);

[0036] Figure 8 This is the MALDI measurement spectrum of liver cells and liver cancer cells using the mass spectrometry probe method of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical effect and technical solution of the embodiment of the present invention clearer, the technical solution in the embodiment of the present invention is clearly and completely described below in conjunction with the drawings in the embodiment of the present invention; it is obvious that the described embodiment is a part of the embodiment of the present invention. Based on the embodiment disclosed in the present invention, other embodiments obtained by ordinary technicians in this field without making creative work should all fall within the scope of protection of the present invention.

[0038] The model of the matrix-assisted laser desorption ionization time-of-flight mass spectrometer used in the following examples is Autoflexspeed TOF / TOF, and the laser is a Nd:YAG laser with a wavelength of 355nm. The mass spectrometry test parameters are: acceleration voltage: 20.000kv; delayed extraction voltage: 18.000kv; delayed extraction time: 150ns; reflector voltage: 20.000kv; lens voltage: 6.000kv; frequency: 500Hz; laser energy: 0-100%; and the test mode is the reflection positive ion mode.

[0039] Example 1: Preparation of the mass spectrometry probe of formula (I) of the present invention

[0040] 2-Aminoquinoline (4mmol, 576mg) was dissolved in DCM, and then 4-dimethylaminopyridine (5mmol, 610mg) and sodium carbonate (12.5mmol, 1325mg) were added to the organic phase. The mixture was stirred in an ice bath under the protection of nitrogen. Then, furoyl chloride (6mmol, 780mg) was slowly added. After 4h, the crude product was extracted with a column and purified by column chromatography (petroleum ether / ethyl acetate = 4:1) to obtain a white-grey product of formula (I).

[0041] Figure 1 The above is a synthetic route diagram of mass spectrometry probe formula (Ⅰ).

[0042] Example 2: MALDI mass spectrometry analysis of mass spectrometry probes (I) and (II)

[0043] 8 μM mass spectrometry probe solutions of formula (I) and formula (II) were prepared, 1 μL of sample solution and an equal volume of CHCA matrix (10 mg / mL, dissolved in 50% ACN / 0.1% TFA (v / v)) were mixed and spotted on a MALDI target plate, dried at room temperature and then subjected to MALDI mass spectrometry analysis.

[0044] like Figure 2 The MALDI test intensity ratio diagram of 8μM formula (I) and 8μM formula (II) is shown. In this positive ion mode, the same concentration of formula (I) and formula (II) were mixed and 10 parallel experiments were performed. The RSD of the intensity ratio of the detected substrate formula (I) and product formula (II) was 4.85%, indicating that the substrate and product have similar ionization efficiency and are suitable for quantitative analysis of carboxylesterase activity.

[0045] Figure 3 For the reaction (I) in MALDI mass spectrometry Figure 1 The following is the chemical equation of the enzyme cleavage reaction) before and after the MALDI mass spectrometry. Figure 3 It can be seen that the ionization capabilities of the two are similar, which meets the quantitative requirements.

[0046] Example 3: Quantitative analysis of carboxylesterase at different concentrations and drawing of standard curves

[0047] Prepare 320 μM MALDI molecular probe stock solution of formula (I).

[0048] Prepare 390 μL of carboxylesterase standard PBS buffer (pH = 7.4) with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 U / mL, add 10 μL of mass spectrometry probe stock solution, and react in a 37°C water bath for 50 min. After the reaction, take 1 μL of the supernatant and mix it evenly with 1 μL of CHCA matrix solution, then spot it on the MALDI target plate, and perform MALDI mass spectrometry analysis after drying. The different concentration values ​​of carboxylesterase are used as the horizontal axis, and the ratio of the MALDI signal intensity of the product formula (Ⅱ) to the total intensity of the product and the molecular probe is I 式Ⅱ / (I 式Ⅱ +I 式Ⅰ ) as the vertical axis and draw the standard curve.

[0049] from Figure 4 and Figure 5 It can be seen that the mass spectrometry probe formula (I) and the product formula (II) have clear protonation spectra, which show a good linear relationship with the activity of carboxylesterase, demonstrating good accuracy and reproducibility for the determination of carboxylesterase.

[0050] Example 4: Evaluation of the selectivity of the mass spectrometry probe formula (I) of the present invention for carboxylesterase

[0051] PBS buffer solutions (pH = 7.4) containing different additives were prepared, including blank control, 1 mM H2O2, 1 mM MgCl2, 1 mM NaCl, 200 μM arginine, 200 μM tyrosine, 200 μM L-glutamic acid, 200 μM serine, 200 μM cysteine, 200 μM homocysteine, 200 μM glutamic acid, 200 μM ascorbic acid, 50 μg mL -1 Albumin, 50 μg mL -1 Lysozyme, 50 μg mL -1 Trypsin, 10 U mL -1 Butyrylcholinesterase, 10U mL -1 Acetylcholinesterase, 10 U mL -1 Alkaline phosphatase, 1.5 U mL -1 Carboxylesterase. 10 μL of mass spectrometry probe stock solution was added to the prepared PBS buffer to a total volume of 400 μL. The enzyme digestion reaction was carried out in a 37°C water bath for 60 min. After the reaction, 1 μL of the supernatant was mixed with 1 μL of CHCA matrix solution and spotted on the MALDI target plate. After drying, MALDI mass spectrometry analysis was performed to calculate the I 式Ⅱ / (I 式Ⅱ +I 式Ⅰ )value.

[0052] Figure 6As shown, for different inorganic salts, amino acids, proteins and other hydrolases, the mass spectrometry probe formula (I) only exhibits high selectivity for carboxylesterase, indicating that the mass spectrometry probe formula (I) has good selectivity.

[0053] Example 5: Determination of the IC50 value of the carboxylesterase inhibitor drug 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) using the mass spectrometry probe formula (I) of the present invention

[0054] Prepare PBS buffer solutions (pH = 7.4) containing different concentrations of AEBSF, the concentrations of which are 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, and 2.5 mM. Mix the buffer with 1.5 U mL -1 The carboxylesterase solution was mixed for 5 minutes. After the enzyme was inactivated, 10 μL of mass spectrometry probe stock solution was added. The total volume of the solution was 400 μL. The enzyme digestion reaction was carried out in a 37°C water bath for 50 minutes. After the reaction, 1 μL of the supernatant was mixed with 1 μL of CHCA matrix solution and spotted on the MALDI target plate. After drying, MALDI mass spectrometry analysis was performed. The inhibition efficiency can be expressed by the formula [1-I Ⅱ (I Ⅱ0 +I Ⅰ0 ) / I Ⅱ0 (I Ⅱ +I Ⅰ )] × 100%, where I and I0 correspond to the signal intensity of the probe in the presence and absence of the inhibitor, respectively.

[0055] Figure 7 The inhibitor inhibition curve is shown, and it can be seen that the IC50 value of AEBSF is 1.11957mM.

[0056] Example 6: Detection of Carboxylesterase Activity in Normal Hepatocytes (LO2) and Hepatoma Cells (HepG2)

[0057] After the two cells were cut with trypsin, they were centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the cells were washed twice with PBS. The density was then adjusted to 1×10 7 cell / mL, and the cells were ultrasonically destroyed in an ice bath environment (ultrasound for 3 seconds, interval of 10 seconds, repeated 30 times). Then 20μL of mass spectrometry probe stock solution was added, and the total volume of the solution was 400μL. Reacted in a water bath at 37°C for 50min. After the reaction was completed, 400μL ACN was added for protein precipitation and probe extraction. After precipitation, centrifugation was performed at 12000rpm for 5min. Take 1μL of the supernatant and 1μL of CHCA matrix solution, mix them evenly, and then spot them on the MALDI target plate. After drying, MALDI mass spectrometry analysis was performed to calculate the I 式Ⅱ / (I式Ⅱ +I 式Ⅰ )value.

[0058] Figure 8 The MALDI comparison spectra of the MALDI probe method of the present invention used for two types of hepatocyte determination are shown. Figure 8 The difference in the content of carboxylesterase in the two types of liver cells can be seen in the figure, which confirms that carboxylesterase is highly expressed in liver cancer cells. This shows that the mass spectrometry probe has the ability to detect carboxylesterase activity in cells.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, a person skilled in the art can still modify or make equivalent substitutions to the specific implementations of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are within the scope of protection of the claims of the present invention to be approved.

Claims

1. Application of a MALDI-TOF mass spectrometry probe in carboxylesterase detection, characterized in that: The MALDI-TOF mass spectrometry probe molecule has a structure shown in formula (I), and the product after the reaction with carboxylesterase has a structure shown in formula (II):

2. The use of a MALDI-TOF mass spectrometry probe in carboxylesterase detection according to claim 1, characterized in that: The following steps are involved: Step 1: preparing PBS solutions of carboxylesterase standards of different concentrations, and adding mass spectrometry molecular probes of formula (I) to the above solutions for enzyme cleavage reaction to obtain products of formula (II); Step 2: Take 1 μL of sample and 1 μL of matrix, mix them evenly, and then spot them on the MALDI target plate. After drying, inject and analyze; Step 3: Draw a standard curve with the different activity values ​​of the carboxylesterase in step 1 as the horizontal axis and the ratio of the MALDI signal intensity of formula (ⅠⅠ) in step 2 to the total signal intensity of the formula (Ⅰ) and formula (ⅠⅠ) as the vertical axis; Step 4: Prepare a PBS solution of the sample to be tested, add the mass spectrometry probe of formula (I), add acetonitrile after enzyme cleavage reaction and centrifuge, take 1 μL of supernatant and 1 μL of matrix solution, mix them evenly and then spot them on the MALDI target plate, dry them and perform MALDI mass spectrometry analysis, and measure I 式ⅠⅠ / (I 式Ⅰ +I 式ⅠⅠ ) value into the standard curve to obtain the activity of carboxylesterase in the sample to be tested.

3. The use of a MALDI-TOF mass spectrometry probe in carboxylesterase detection according to claim 2, characterized in that: The pH value of the PBS solution in step 1 is 7-9.

4. The use of a MALDI-TOF mass spectrometry probe in carboxylesterase detection according to claim 2, characterized in that: The enzyme digestion reaction time in step 1 is 0.1 to 1.2 hours.

5. The use of a MALDI-TOF mass spectrometry probe in carboxylesterase detection according to claim 2, characterized in that: The sample to be tested in step 4 is a sample containing a large amount of protein.

6. Use of a MALDI-TOF mass spectrometry probe in carboxylesterase detection according to claim 2, characterized in that: The matrix described in step 1 and step 4 is one of α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.

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