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CN-122017079-A - Method for detecting pegoviril enzyme activity by mass spectrometry

CN122017079ACN 122017079 ACN122017079 ACN 122017079ACN-122017079-A

Abstract

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

Inventors

  • WU HONGLIN
  • LU KAIZHI
  • HE XIUMEI

Assignees

  • 中国人民解放军总医院第五医学中心

Dates

Publication Date
20260512
Application Date
20260319

Claims (10)

  1. 1. A method for detecting pegoverine enzyme activity by mass spectrometry is characterized by comprising 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 the incubation in the step S1; s3, detecting peak areas of trans-cinnamic acid and an internal standard in the sample to be detected obtained in the step S2 by adopting a liquid chromatography-tandem mass spectrometry; s4, calculating the activity of pegoverine according to the peak area of the sample to be detected obtained in the step S3.
  2. 2. The method for detecting pegvavilia enzyme activity by mass spectrometry according to claim 1, wherein the concentration of L-phenylalanine in the step S1 is 0.1-600. Mu. Mol/L.
  3. 3. A method for detecting pegoverine enzyme activity by mass spectrometry according to claim 1, wherein the incubation temperature in step S1 is 25-45 ℃, preferably 37 ℃; The incubation time is 30-90min, preferably 60min; the shaking speed is 500-900rpm, preferably 700rpm.
  4. 4. A method for detecting pegoverine enzyme activity by mass spectrometry according to claim 1, wherein the internal standard in the step S2 is alpha-methyl cinnamic acid, and the internal standard extract is a methanol solution containing 3.5-4.5. Mu. Mol/L alpha-methyl cinnamic acid.
  5. 5. The method for detecting pegoverine enzyme activity by mass spectrometry according to claim 1, wherein in the step S3, the liquid chromatography condition is that a chromatographic column adopts a C18 column, a mobile phase A is pure water, a mobile phase B is 0.1% methanolic formate, and gradient elution is adopted; Preferably, the gradient elution procedure is that the mobile phase A is reduced from 80% to 2%, the mobile phase B is increased from 20% to 98%, the mobile phase A is maintained to be 2%, the mobile phase B is maintained to be 98%, the mobile phase A is increased from 2% to 80%, the mobile phase B is reduced from 98% to 20%, the mobile phase A is maintained to be 80%, and the mobile phase B is maintained to be 20% for 2.10-2.50 min, wherein the gradient elution procedure is 0-1.20 min, the mobile phase A is maintained to be 2% and the mobile phase B is maintained to be 98% for 1.20-2.00 min.
  6. 6. The method for detecting pegvavilia enzyme activity by mass spectrometry according to claim 1, wherein the mass spectrometry conditions in the step S2 are electrospray ion source, negative ion mode and selective reaction monitoring mode.
  7. 7. The method for detecting pegvavilia enzyme activity by mass spectrometry according to claim 1, wherein in the step S3, the liquid chromatography-tandem mass spectrometry is performed, a standard sample, a blank control sample and a sample to be detected are sequentially subjected to sample injection detection, and the peak area ratio of trans-cinnamic acid to an internal standard in each sample is recorded.
  8. 8. The method for detecting pegvavle enzyme activity by mass spectrometry according to claim 1, wherein the step S4 comprises the steps of: s41, preparing trans-cinnamic acid standard solutions with serial concentrations, performing liquid chromatography-tandem mass spectrometry detection after processing according to steps S1-S3, recording the peak area ratio of trans-cinnamic acid and an internal standard in each standard solution, and establishing a standard curve according to the peak area ratio and the trans-cinnamic acid concentration; S42, substituting the peak area ratio of the sample to be detected obtained in the step S3 into the standard curve established in the step S41, and calculating the generation amount of trans-cinnamic acid; S43, calculating to obtain the activity of pegvavlase according to the generation amount of trans-cinnamic acid; specific calculation formula, enzyme activity (U/L) =c/t×f; Wherein, C is the concentration (mu mol/L) of trans-cinnamic acid calculated by a standard curve, t is the incubation time (min), and F is the mass spectrum detection reaction coefficient.
  9. 9. The method of claim 1, wherein the sample to be tested in step S1 comprises one of pure enzyme standard, pharmaceutical preparation of pegvaprex, serum, plasma or tissue fluid.
  10. 10. The method for detecting pegvavilia enzyme activity by mass spectrometry according to claim 1, wherein the linear detection range of the sample to be detected in step S1 is 2-700U/L.

Description

Method for detecting pegoviril enzyme activity by mass spectrometry Technical Field The invention belongs to the technical field of detection of pegvavlase activity, and particularly relates to a method for detecting pegvavlase activity by using a mass spectrometry. Background Phenylketonuria (Phenylketonuria, PKU) is an autosomal recessive inherited congenital metabolic disorder, mainly caused by mutations in the gene encoding phenylalanine hydroxylase (PHENYLALANINE HYDROXYLASE, PAH), resulting in a significant decrease or complete loss of the enzyme activity. PAH is a key enzyme in the liver that converts dietary-derived phenylalanine (Phe) to tyrosine, and its functional deficiency causes abnormal accumulation of phenylalanine in blood and brain tissues to neurotoxic levels. Untreated PKU patients may exhibit severe clinical manifestations of neurocognitive dysfunction, developmental delay, seizures, behavioral abnormalities, hypopigmentation, and the like. Although neonatal screening and early low phenylalanine dietary intervention can effectively prevent mental retardation, long term strict dietary restriction compliance is poor, and even with traditional management, adult patients have difficulty maintaining guidelines recommended blood phenylalanine concentrations (typically <600 μmol/L) and are at constant neuropsychiatric risk. To overcome the limitations of dietetic therapy, enzyme replacement therapy has become an important breakthrough in PKU therapy. Pegovitamin Li Mei (PEGVALIASE) is a recombinant phenylalanine ammonia lyase (PHENYLALANINE AMMONIA-lyase, PAL) modified by polyethylene glycol (PEG), and is derived from blue algae Anabaena variabilis, and is formed by genetic engineering and covalent connection of 20 kDa linear PEG chains. Unlike PAH, which relies on tetrahydrobiopterin (BH 4) cofactor in mammals, PAL is a non-mammalian source enzyme that directly catalyzes phenylalanine deamination to produce non-toxic trans-cinnamic acid (trans-CINNAMIC ACID) and ammonia, thereby bypassing PAH-deficient pathways and effecting metabolic clearance of phenylalanine by non-phenylalanine hydroxylase pathways. This mechanism not only provides a completely new metabolic bypass, but also makes the patient hopeful to get rid of strict dietary restrictions. Along with the introduction of PEGylated enzyme drugs such as pegovitamin Li Mei into clinical application, accurate evaluation of the catalytic activity of the drugs is not only an auxiliary task of pharmacological research, but also a key decision basis throughout the whole treatment process. The efficacy of such drugs depends directly on their ability to convert phenylalanine to trans-cinnamic acid, however, PEG modification brings pharmacokinetic advantages but introduces multiple analytical challenges. In particular, steric hindrance of the PEG chain may mask the active site of the enzyme, interfere with recognition of substrate transformations by traditional uv spectrophotometry, fluorescence methods, etc., leading to underestimation of activity and even false negative results. Currently, pegoverine enzyme activity is mainly detected by an ultraviolet spectrophotometry, and the enzyme activity is calculated by monitoring absorbance change based on characteristic ultraviolet absorption of trans-cinnamic acid product at 290 nm. The method has the obvious technical defects that firstly, the specificity is poor, other impurities with ultraviolet absorption in a sample can interfere with a detection result, the method is particularly suitable for detecting biological samples (such as blood plasma and tissue fluid), the background interference problem is outstanding, secondly, the sensitivity is low, the detection accuracy of a low-activity enzyme sample or a trace sample is insufficient, the accurate quantitative requirement of low-concentration enzyme activity cannot be met, thirdly, the dynamic range is narrow, and when the concentration of a substrate or a product exceeds a certain range, the linear relation between absorbance and concentration deviates, so that the detection error is increased. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a high-sensitivity and high-specificity technology in enzyme activity measurement, and is particularly suitable for accurate analysis of enzymatic reaction kinetics in complex biological matrixes. The basic principle is that the consumption of a substrate or the generation of a product in an enzyme reaction system is quantitatively monitored, so that the reaction rate is calculated, and the enzyme activity level is reflected. Compared with the traditional colorimetric method or fluorescent method, the LC-MS/MS can directly identify and quantify the chemical structure of the target molecule, avoids false positive or false negative results caused by cross reaction or background interference, and is particularly suitable for samples containing a large amount of endogenous metabolites, prote