Method for detecting activity of pegoviril enzyme by mass spectrometry

Abstract

The invention discloses a method for detecting the activity of pegobi enzyme by mass spectrometry, which comprises the following steps: S1, mixing a sample to be detected with L-phenylalanine, and incubating to obtain trans-cinnamic acid; s2, adding an internal standard substance to terminate incubation in the step S1; s3, detecting the peak areas of the trans-cinnamic acid and the internal standard in the to-be-detected sample obtained in the step S2 by adopting a liquid chromatography-tandem mass spectrometry method; and S4, calculating the activity of the pegobi enzyme according to the peak area of the to-be-detected sample obtained in S3. According to the detection method, specificity is optimized, an LC-MS / MS MRM mode is adopted, characteristic ion pair screening is combined, the product trans-cinnamic acid can be specifically recognized, interference of PEG chains, protein impurities and other ultraviolet absorption substances in a sample is effectively eliminated, and the problem that an existing method is poor in specificity is solved.

Description

Technical Field

[0001] This invention belongs to the field of pegovilase activity detection technology, and in particular relates to a method for detecting pegovilase activity using mass spectrometry. Background Technology

[0002] Phenylketonuria (PKU) is an autosomal recessive inherited congenital metabolic disorder primarily caused by mutations in the gene encoding phenylalanine hydroxylase (PAH), leading to a significant reduction or complete loss of this enzyme's activity. PAH is a key enzyme in the liver that converts dietary phenylalanine (L-phenylalanine, Phe) into tyrosine. PKU dysfunction results in the abnormal accumulation of phenylalanine in the blood and brain tissue, reaching neurotoxic levels. Untreated PKU patients may exhibit severe neurocognitive impairment, developmental delay, epilepsy, behavioral abnormalities, and hypopigmentation. Although newborn screening and early low-phenylalanine dietary intervention can effectively prevent intellectual disability, long-term strict dietary restrictions are poorly adhered to, and even with conventional management, adult patients often struggle to maintain guideline-recommended serum phenylalanine concentrations (usually <600 μmol / L), thus facing ongoing neuropsychiatric risks.

[0003] To overcome the limitations of dietary therapy, enzyme replacement therapy has become a significant breakthrough in the treatment of PKU. Pegvaliase, a polyethylene glycol (PEG)-modified recombinant phenylalanine ammonia-lyase (PAL), is derived from the cyanobacterium *Anabaena variabilis* and genetically engineered with a 20 kDa linear PEG chain. Unlike PAHs in mammals, which rely on the tetrahydrobiopterin (BH4) cofactor, PAL is a non-mammalian enzyme that directly catalyzes the deamination of phenylalanine to produce non-toxic trans-cinnamic acid and ammonia, thus bypassing the PAH-deficient pathway and achieving the metabolic clearance of phenylalanine via a non-phenylalanine hydroxylase pathway. This mechanism not only provides a novel metabolic bypass but also offers hope for patients to break free from strict dietary restrictions.

[0004] With the clinical application of PEGylated enzymes such as pegovilase, the accurate assessment of their catalytic activity is no longer merely a supplementary task in pharmacological research, but a crucial decision-making basis throughout the entire treatment process. The efficacy of these drugs directly depends on their ability to convert phenylalanine to trans-cinnamic acid. However, while PEG modification brings pharmacokinetic advantages, it also introduces multiple analytical challenges. Specifically, the steric hindrance of the PEG chain may obscure the enzyme's active site, interfering with the identification of substrate conversion using traditional UV spectrophotometry and fluorescence methods, leading to underestimated activity or even false negative results.

[0005] Currently, pegovic acid enzyme activity detection mainly employs ultraviolet spectrophotometry, based on the characteristic ultraviolet absorption of the product trans-cinnamic acid at 290 nm, calculating enzyme activity by monitoring absorbance changes. However, this method has significant technical drawbacks: First, it has poor specificity; other impurities with ultraviolet absorption in the sample can interfere with the detection results, especially when detecting biological samples (such as plasma and tissue fluid), where background interference is prominent. Second, it has low sensitivity; its accuracy in detecting low-activity enzyme samples or trace samples is insufficient, failing to meet the precise quantitative requirements for low-concentration enzyme activity. Third, it has a narrow dynamic range; when the substrate or product concentration exceeds a certain range, the linear relationship between absorbance and concentration deviates, leading to increased detection errors.

[0006] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become a highly sensitive and specific technique for enzyme activity determination, particularly suitable for the precise analysis of enzyme reaction kinetics in complex biological matrices. Its basic principle lies in quantitatively monitoring the consumption of substrates or the formation of products in the enzyme reaction system to calculate the reaction rate, thereby reflecting the enzyme activity level. Compared to traditional colorimetric or fluorescence methods, LC-MS / MS can directly identify and quantify the chemical structure of target molecules, avoiding false positive or false negative results caused by cross-reactions or background interference. It is especially suitable for samples containing large amounts of endogenous metabolites, proteins, or pharmaceutical excipients, such as plasma, serum, or tissue homogenates. Summary of the Invention

[0007] In view of this, the present invention aims to provide a method for detecting pegovic acid enzyme activity by mass spectrometry, so as to solve at least one technical problem in the background art.

[0008] This invention aims to overcome the shortcomings of existing ultraviolet spectrophotometric methods for detecting pegovilase activity, and to achieve highly specific, highly sensitive, and wide dynamic range quantitative detection of pegovilase activity, applicable to the detection of pegovilase activity in pure enzyme samples, pharmaceutical preparations, and biological samples.

[0009] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A method for detecting pegovic acid enzyme activity using mass spectrometry includes the following steps: S1: Mix the sample to be tested with L-phenylalanine and incubate to obtain trans-cinnamic acid; S2: Add internal standard to terminate the incubation in step S1; S3: The peak areas of trans-cinnamic acid and internal standard in the sample obtained in step S2 were detected by liquid chromatography-tandem mass spectrometry. S4: Calculate the activity of pegolive enzyme based on the peak area of ​​the sample to be tested obtained in S3.

[0010] Furthermore, the concentration of L-phenylalanine in step S1 is 0.1~600 μmol / L.

[0011] Furthermore, the incubation temperature in step S1 is 25-45℃, preferably 37℃; The incubation time is 30-90 minutes, preferably 60 minutes; The oscillation speed is 500-900 rpm, preferably 700 rpm.

[0012] Further, the internal standard in step S2 is α-methylcinnamic acid, and the internal standard extraction solution is a methanol solution containing 3.5-4.5 μmol / L α-methylcinnamic acid.

[0013] Furthermore, in step S3, the liquid chromatography conditions are as follows: a C18 column is used, mobile phase A is pure water, mobile phase B is 0.1% formic acid methanol, and gradient elution is employed. Preferably, the gradient elution program is as follows: 0~1.20 min, mobile phase A decreases from 80% to 2%, and mobile phase B increases from 20% to 98%; 1.20~2.00 min, mobile phase A is maintained at 2%, and mobile phase B at 98%; 2.00~2.10 min, mobile phase A increases from 2% to 80%, and mobile phase B decreases from 98% to 20%; 2.10~2.50 min, mobile phase A is maintained at 80%, and mobile phase B at 20%.

[0014] Furthermore, in step S2, the mass spectrometry conditions are an electrospray ionization source, negative ion mode, and selected reaction monitoring mode for detection.

[0015] Furthermore, in step S3, the liquid chromatography-tandem mass spectrometry detection involves sequentially injecting the standard sample, blank control sample, and test sample for detection, and recording the peak area ratio of trans-cinnamic acid to the internal standard in each sample.

[0016] Further, step S4 includes the following steps: S41: Prepare a series of standard solutions of trans-cinnamic acid, process them according to steps S1-S3, and then perform liquid chromatography-tandem mass spectrometry detection. Record the peak area ratio of trans-cinnamic acid to internal standard in each standard solution, and establish a standard curve based on the peak area ratio and the concentration of trans-cinnamic acid. S42: Substitute the peak area ratio of the sample to be tested obtained in step S3 into the standard curve established in step S41 to calculate the amount of trans-cinnamic acid produced. S43: The activity of pegovic acid enzyme was calculated based on the amount of trans-cinnamic acid produced. The specific calculation formula is: enzyme activity (U / L) = C / t * F.

[0017] Where C is the concentration of trans-cinnamic acid (μmol / L) calculated from the standard curve, t is the incubation time (min), and F is the mass spectrometry detection reaction coefficient.

[0018] Furthermore, the sample to be tested in step S1 includes one of the following: pure enzyme standard, pegovir enzyme pharmaceutical preparation, serum, plasma, or tissue fluid.

[0019] Furthermore, the detection linear range of the sample to be tested in step S1 is 2-700 U / L.

[0020] Compared with existing technologies, the method for detecting pegovic acid enzyme activity using mass spectrometry described in this invention has the following advantages: 1. The detection method of this application has optimized specificity. It adopts the MRM mode of LC-MS / MS and combines characteristic ion pair screening to specifically identify the product trans-cinnamic acid, effectively eliminating interference from PEG chains, protein impurities and other ultraviolet absorbing substances in the sample, thus solving the problem of poor specificity of existing methods.

[0021] 2. The detection method of this application has improved sensitivity. Through internal standard correction, the matrix effect is reduced, and the detection limit can reach 2 U / L, which is more than 10 times more sensitive than the ultraviolet spectrophotometry method, and can realize the accurate detection of low-activity enzyme samples.

[0022] 3. The reaction system adaptability of this application is optimized by taking into account the PEG modification characteristics of pegovic acid enzyme, optimizing the ionic strength of the buffer, substrate concentration and reaction temperature to ensure that the enzyme maintains its natural catalytic activity, while avoiding the inhibitory effect of PEG chain on mass spectrometry detection signal.

[0023] 4. The detection scope of this application is wide and can be used for enzyme activity detection in biological samples such as pure enzyme standards, pegovir enzyme pharmaceutical preparations, serum and tissue fluid, with a dynamic range of 2~700U / L, meeting the detection needs of different scenarios. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The chromatograms of trans-cinnamic acid under different mobile phase formulations proposed in this invention are shown below (a is formulation ①, b is formulation ②, c is formulation ③, and d is formulation ④). Figure 2 This is a graph showing the effect of substrate concentration on the amount of product generated in an enzyme-catalyzed reaction. Figure 3 This is a graph showing the effect of reaction temperature on the amount of enzyme-catalyzed reaction products. Figure 4 This is a typical chromatogram of trans-cinnamic acid and internal standard methylcinnamic acid; Figure 5 This is a standard curve for the mass spectrometry detection of trans-cinnamic acid. Detailed Implementation

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0026] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0027] Reagents: Standards: L-phenylalanine and trans-cinnamic acid standards were purchased from Shanghai Aladdin Company; Internal standard: α-methylcinnamic acid was purchased from Shanghai Aladdin Company; Pegóvelibrine enzyme reagent: 10 mg / 0.5 mL, store at 2-8℃, manufactured by BioMarin Pharmaceutical Inc. Methanol (chromatographic grade, Fisher); Formic acid (mass spectrometry grade, Fisher); Acetic acid-potassium acetate buffer (Shanghai Aladdin Company). Potassium phosphate buffer (Shanghai Aladdin Company). Tris buffer (Shanghai Aladdin Company): Tris(hydroxymethyl)aminomethane buffer (pH=8.0); PBS (Solepro): Phosphate-buffered saline solution; Ultrapure water (Millipore); equipment: YS EXACT 9900MD High Performance Liquid Chromatography-Tandem Mass Spectrometry Detection System (Shandong Yingsheng Biotechnology Co., Ltd.); MCA3.6P-2CCN-M Parts per million electronic balance (Sartorius). Sorvall Legeug Micro 21R high-speed refrigerated centrifuge (Thermo Fisher); IQ7005 Pure Water System (Millipore); Pipettes (Eppendorf); ST07-2 Microplate Thermostatic Oscillator (MIULAB); MS3DS025 Mixer (IKA); ChromCore 120 C18 (30*2.1mm, 3.0 μm) chromatographic column (nanospectral analysis); The mass spectrometer is used with TraceFinder 5.1 software.

[0028] Research indicators: trans-cinnamic acid, with the corresponding homologue internal standard being α-methylcinnamic acid, both purchased from Shanghai Aladdin Company.

[0029] Solution preparation; 1.1.1 Preparation of standard stock solutions; Accurately weigh an appropriate amount of L-phenylalanine (99.4% purity) solid, dilute to volume with ultrapure water, vortex and sonicate until the solid is completely dissolved, prepare a 20 mM substrate stock solution, label it, and store at -20℃. Accurately weigh an appropriate amount of trans-cinnamic acid (99.1% purity) solid, dilute to volume with methanol, vortex until the solid is completely dissolved, prepare a 2 mM product stock solution, label it, and store at -20℃.

[0030] 1.1.2 Preparation of internal standard stock solution; Accurately weigh an appropriate amount of solid methyl cinnamic acid (purity 99.73%), dissolve it in methanol and dilute to volume to prepare an internal standard stock solution with a concentration of 2 mM. Label the solution and store at -20℃.

[0031] 1.1.3 Preparation of internal standard extraction solution; Take 200 μL of 2 mM methyl cinnamic acid internal standard stock solution, add 99.8 mL of methanol, vortex to mix, and prepare 4 μM methyl cinnamic acid internal standard solution.

[0032] 1.2 Preparation of standard solutions; 1.2.1 Preparation of a series of standard working solutions of varying concentrations; Take an appropriate amount of the trans-cinnamic acid substrate stock solution prepared in 1.1.1, dilute it with PBS to prepare a 100 μmol / L standard solution STD6, ​​and dilute STD6 with PBS to obtain a series of standard solutions with different concentrations as shown in Table-1. The concentration unit in Table-1 is μmol / L.

[0033] Table 1 Concentration Standard Solutions 1.2.2 Preparation of quality control solution; Take an appropriate amount of the trans-cinnamic acid substrate stock solution prepared in 1.1.1, dilute it with PBS to prepare a 75 μmol / L standard quality control solution QCH, and dilute QCH with PBS to obtain a series of standard quality control solutions with different concentrations as shown in Table-2. The concentration unit in Table-2 is μmol / L.

[0034] Table 2. Concentration of trans-cinnamic acid 1.2.3 Preparation of incubation solution; Accurately weigh 1 mL of 20 mM L-phenylalanine standard solution, add 99 mL of diluent, vortex for 3 min, and sonicate for 15 min to obtain a 200 μM L-phenylalanine solution, which is the enzyme reaction substrate incubation solution.

[0035] 1.3 Procedure for detecting pegovic acid enzyme activity in samples; 1.3.1 Establishment of the enzyme-catalyzed reaction system; ① Sample addition: Add 20 μL of a negative sample (blank whole blood sample, clinically collected) without pegovilase and a sample containing pegovilase to a polystyrene 96-well plate, respectively; ② Add reaction substrate: Add 700 μL of 200 μM L-phenylalanine solution to the above sample, attach the sealing film, and vortex for 5 seconds, then start timing immediately; ③ Incubation: Place the 96-well polystyrene plate containing the sample in an incubator shaker and incubate at 700 rpm and 37°C for 60 min.

[0036] ④ Termination of reaction: Remove the sealing film, take 30 μL of incubation solution from each well into a well containing 150 μL of internal standard solution, cover with the sealing film, shake to mix, centrifuge at 14000 rpm for 10 min, and then perform LC-MS / MS analysis.

[0037] 1.4 Pretreatment of standard samples; Take 30 μL each of the 6-level standard solution and quality control solution prepared in 1.2.1 and 1.2.2 above, add them to a polystyrene 96-well plate, add 150 μL of internal standard solution to each well, cover with a cover film, shake to mix, and then perform LC-MS / MS analysis.

[0038] 1.5 Mass spectrometry detection; The standard sample, blank control sample, and test sample were injected sequentially for analysis. The peak area ratio of trans-cinnamic acid to the internal standard in each sample was recorded. The product yield was then calculated by substituting the values ​​into the standard curve. (See figure) Figure 4 and Figure 5 .

[0039] 1.6 Enzyme activity calculation; One unit of enzyme activity (U) is defined as the amount of enzyme that, under specified conditions, catalyzes the conversion of 1 micromole (μmol) of L-phenylalanine to trans-cinnamic acid per minute. Enzyme activity is calculated experimentally based on the amount of trans-cinnamic acid produced during the enzymatic reaction. 2. High-performance liquid chromatography-tandem mass spectrometry detection conditions; 2.1 High-performance liquid chromatography conditions; Mobile phase A: pure water; Mobile phase B: 0.1% formic acid in methanol; Chromatographic column: ChromCore 120 C18 (30*2.1mm, 3.0 μm); Gradient elution is shown in Table 3. The column temperature is 40 °C, the flow rate is 0.4 mL / min, and the injection volume is 1 μL.

[0040] Table 3. Mobile phase gradient table; 2.2 Mass spectrometry conditions; In electrospray ionization mode, selected reaction monitoring (SRM, Ingenic Biotech (Thermo Fisher Scientific) mass spectrometer used SRM) was employed for negative ion mode scanning to detect trans-cinnamic acid and its internal standard signal. The ion source parameters are shown in Table 4 below. Table 4 shows the ion source parameters. The ion pair parameters are shown in Table 5 below: Table 5 Ion parameters; Note: "*" represents quantitative ions.

[0041] 3.1. Investigation of the mobile phase system; Trans-cinnamic acid standard solutions were prepared and analyzed using the mobile phase formulations shown in Table 6, according to the mass spectrometry conditions described above. The peak shape and intensity of trans-cinnamic acid under different mobile phase formulations were investigated. The optimal mobile phase system of water-0.1% formic acid-methanol was ultimately determined to be the best. The chromatograms of different mobile phase formulations are shown below. Figure 1 .

[0042] Table 6 Mobile Phase Formulation 3.2 Buffer solution investigation; A substrate solution of a certain concentration was prepared, and PBS buffer, potassium phosphate buffer, acetate-potassium acetate buffer, and Tris buffer were selected as diluents. The incubation solutions prepared with different diluents were investigated, and enzymes of a certain concentration were added to induce enzymatic reactions. The amount of product generated after the enzymatic reaction in the incubation solutions prepared with different diluents was determined, and the system with the largest product production was identified as the optimal buffer solution. Finally, 50 mM Tris buffer was selected as the diluent for the experiment.

[0043] 3.3 Substrate concentration investigation; Medium-concentration enzyme activity samples were prepared, and L-phenylalanine substrate concentration gradients of 50, 100, 200, 400, and 600 μmol / L were set to investigate the optimal substrate concentration that yielded the maximum product yield. Ultimately, 200 μM L-phenylalanine was selected for the experiment. See [link to substrate concentration investigation details] Figure 2 .

[0044] 3.4 Reaction temperature; Medium-concentration enzyme activity samples were prepared, and incubation temperatures of 25, 30, 37, 45, and 60°C were set. The temperature at which the maximum product yield was obtained under different incubation temperatures was determined to be the optimal reaction temperature. Based on laboratory results and the temperature environment of the enzyme in the human body, 37°C was selected as the reaction temperature. The reaction temperature observation graph is shown below. Figure 3 .

[0045] 3.5 Recovery rate experiment; Three concentrations of trans-cinnamic acid standard were added to the incubation solution for spiked recovery experiments. Samples were processed according to the sample pretreatment procedure, and the recoveries were measured to be 96.2%–104.6%, with RSD ≤ 5.0%, indicating good accuracy of the method. The experimental results are shown in Table 7. Table 7 Experimental Results The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting pegovic acid enzyme activity by mass spectrometry, characterized in that: Includes the following steps: S1: Mix the sample to be tested with L-phenylalanine and incubate to obtain trans-cinnamic acid; S2: Add internal standard to terminate the incubation in step S1; S3: The peak areas of trans-cinnamic acid and internal standard in the sample obtained in step S2 were detected by liquid chromatography-tandem mass spectrometry. S4: Calculate the activity of pegolive enzyme based on the peak area of ​​the sample to be tested obtained in S3.

2. The method for detecting pegvorinase activity by mass spectrometry according to claim 1, characterized in that: In step S1, the concentration of L-phenylalanine is 0.1~600 μmol / L.

3. The method for detecting pegovic acid enzyme activity by mass spectrometry according to claim 1, characterized in that: The incubation temperature in step S1 is 25-45℃, preferably 37℃; The incubation time is 30-90 minutes, preferably 60 minutes; The oscillation speed is 500-900 rpm, preferably 700 rpm.

4. The method for detecting pegovic acid enzyme activity by mass spectrometry according to claim 1, characterized in that: The internal standard in step S2 is α-methylcinnamic acid, and the internal standard extraction solution is a methanol solution containing 3.5-4.5 μmol / L α-methylcinnamic acid.

5. The method for detecting pegvorinase activity by mass spectrometry according to claim 1, characterized in that: In step S3, the liquid chromatography conditions are as follows: a C18 column is used, mobile phase A is pure water, mobile phase B is 0.1% formic acid methanol, and gradient elution is used. Preferably, the gradient elution program is as follows: 0~1.20 min, mobile phase A decreases from 80% to 2%, and mobile phase B increases from 20% to 98%; 1.20~2.00 min, mobile phase A is maintained at 2%, and mobile phase B at 98%; 2.00~2.10 min, mobile phase A increases from 2% to 80%, and mobile phase B decreases from 98% to 20%; 2.10~2.50 min, mobile phase A is maintained at 80%, and mobile phase B at 20%.

6. The method for detecting pegvorinase activity by mass spectrometry according to claim 1, characterized in that: In step S2, the mass spectrometry conditions are electrospray ionization source, negative ion mode, and selected reaction monitoring mode for detection.

7. The method for detecting pegovic acid enzyme activity by mass spectrometry according to claim 1, characterized in that: In step S3, the liquid chromatography-tandem mass spectrometry detection involves sequentially injecting the standard sample, blank control sample, and test sample for detection, and recording the peak area ratio of trans-cinnamic acid to the internal standard in each sample.

8. The method for detecting pegovic acid enzyme activity by mass spectrometry according to claim 1, characterized in that: Step S4 includes the following steps: S41: Prepare a series of standard solutions of trans-cinnamic acid, process them according to steps S1-S3, and then perform liquid chromatography-tandem mass spectrometry detection. Record the peak area ratio of trans-cinnamic acid to internal standard in each standard solution, and establish a standard curve based on the peak area ratio and the concentration of trans-cinnamic acid. S42: Substitute the peak area ratio of the sample to be tested obtained in step S3 into the standard curve established in step S41 to calculate the amount of trans-cinnamic acid produced. S43: The activity of pegovic acid enzyme was calculated based on the amount of trans-cinnamic acid produced. The specific calculation formula is: Enzyme activity (U / L) = C / t * F; Where C is the concentration of trans-cinnamic acid (μmol / L) calculated from the standard curve, t is the incubation time (min), and F is the mass spectrometry detection reaction coefficient.

9. The method for detecting pegovic acid enzyme activity by mass spectrometry according to claim 1, characterized in that: The sample to be tested in step S1 includes one of the following: pure enzyme standard, pegovir enzyme pharmaceutical preparation, serum, plasma, or tissue fluid.

10. The method for detecting pegovic acid enzyme activity by mass spectrometry according to claim 1, characterized in that: The detection linear range of the sample to be tested in step S1 is 2-700 U / L.

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