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CN-122017074-A - Method for detecting phosphate-containing metabolite and application thereof

CN122017074ACN 122017074 ACN122017074 ACN 122017074ACN-122017074-A

Abstract

The invention discloses a method for detecting phosphate-containing metabolites, which introduces a stable and characteristic chlorine isotope distribution through a derivatization strategy of a chloro reagent ) The mass spectrum label of the (2) can be used for carrying out quick and high-specificity primary identification on the derivative product based on primary mass spectrum data. By combining an automatic data mining program specially developed for the method, the potential phosphate-containing metabolites can be screened and locked from massive and complex non-targeted metabonomics original data with high flux and accuracy, and the data analysis efficiency and reliability are greatly improved. The method is successfully applied to analysis of actual biological samples such as cholestasis disease models, not only realizes efficient identification and accurate quantification of various key metabolites such as nucleotide, sugar phosphate and the like, but also reveals disturbance of related metabolic pathways, and provides a powerful innovative analysis tool for research of disease mechanisms and biomarker discovery.

Inventors

  • LI NA
  • WU JIANLIN
  • CHEN XIAOLIN
  • Ao Lanjia
  • LI YUJIE

Assignees

  • 澳门科技大学

Dates

Publication Date
20260512
Application Date
20260226

Claims (10)

  1. 1. A method for detecting a phosphate-containing metabolite, comprising the steps of: Performing derivatization treatment of a chloro reagent on phosphate-containing metabolites in a sample to be detected; Carrying out liquid chromatography-mass spectrometry analysis on the sample after the derivatization treatment to obtain mass spectrum data; Identifying a derivatized phosphate-containing metabolite in the sample to be tested based on the characteristic isotope signals derived from chlorine in the chloro reagent in the mass spectrometry data.
  2. 2. The method according to claim 1, characterized in that said identifying based on the characteristic isotope signals, in particular comprises: screening the mass spectrum data for precursor ions having both a mass to charge ratio (M) peak and an isotope (m+2) peak thereof; calculating a peak intensity ratio of the M peak to the (m+2) peak; Precursor ions having the peak intensity ratio within a predetermined range are identified as potential derivatized metabolites.
  3. 3. The method of claim 2, wherein the predetermined range is 2.75 to 3.25.
  4. 4. The method of claim 2, wherein the screening and calculating steps are implemented based on a Python program.
  5. 5. The method of claim 1, wherein the chloro reagent derivatization treatment comprises: Mixing the pretreated sample with a first reaction solution containing carbodiimide and imidazole to perform a first reaction; Then adding a second reaction liquid containing a chloro reagent and methylimidazole to carry out a second reaction; The chloro reagent is [5- (2-chlorophenyl) isoxazol-3-yl ] methylamine.
  6. 6. The method of claim 5, wherein the first reaction is carried out at 40℃for 30 minutes and the second reaction is carried out at 40℃for 2 hours.
  7. 7. The method of claim 1, further comprising a sample pretreatment and enrichment step prior to derivatizing the phosphate-containing metabolite with the chlorinating agent, wherein the enrichment step is performed using a weak anion exchange solid phase extraction column.
  8. 8. The method of claim 7, wherein the sample pretreatment and enrichment step specifically comprises: mixing biological tissue sample with normal saline and precooled methanol, homogenizing, centrifuging, collecting supernatant, mixing, concentrating to obtain residue; redissolving the residue in ammonium acetate buffer solution, and extracting by using an activated weak anion exchange solid phase extraction column; Eluting with ammonium acetate buffer solution and methanol, eluting the target compound with ammonia water methanol solution, and obtaining an enriched sample.
  9. 9. The method of claim 1, wherein in the liquid chromatography-mass spectrometry analysis: The filling material of the chromatographic column is phenyl bonding silica gel; mobile phase A is ammonium bicarbonate aqueous solution, mobile phase B is methanol-aqueous solution of ammonium bicarbonate, wherein the volume concentration of methanol is 90%; gradient elution procedure was used.
  10. 10. Use of a method according to any one of claims 1 to 9 for rapid screening of phosphate-containing metabolites in non-targeted metabonomic analysis or for screening or studying biomarkers associated with cholestatic disease.

Description

Method for detecting phosphate-containing metabolite and application thereof Technical Field The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting phosphate-containing metabolites and application thereof. Background Phosphate-containing metabolites are a vital class of endogenous compounds in organisms that are widely involved in and regulate a number of core life processes such as energy metabolism (e.g., ATP, ADP), cell signaling (e.g., IP 3), genetic information transfer (e.g., nucleotides), and the regulation of the activity of a variety of key enzymes. In metabonomics, exposure histology and disease mechanism research, comprehensive and accurate analysis of phosphate-containing metabolites in biological samples (such as blood, tissues and cells) is realized, and the method has great scientific value and application prospect in revealing physiological functions, finding metabolic disturbance related to diseases and potential biomarkers. Currently, assays for phosphate-containing compounds rely primarily on the use of various chromatographic and detection techniques in combination. The liquid chromatography-mass spectrometry technology has become a mainstream platform for analyzing metabolites in complex systems due to high separation capacity, high sensitivity and strong structural analysis potential. However, the high polarity and strong hydrophilicity of the phosphate-containing groups results in their extremely weak retention on common reverse phase chromatographs, tend to elute near dead time, have poor separation effects, and are susceptible to severe interference from co-eluting components in complex matrices. Hydrophilic chromatography has become a common alternative to improve retention, but it still suffers from long column equilibration times, poor peak shape, limited resolution, and possibly poor batch-to-batch reproducibility, which is challenging when analyzing highly structurally similar isomers (e.g., glucose-1-phosphate and glucose-6-phosphate). Therefore, there is a need in the art to develop a new analysis strategy that can effectively improve the chromatographic separation performance of phosphate-containing metabolites, and finally realize efficient and rapid non-targeted discovery and identification of such key metabolites from complex systems. Disclosure of Invention The present invention aims to solve at least to some extent one of the technical problems existing in the prior art. To this end, the invention provides a method for detecting phosphate-containing metabolites and uses thereof. According to one aspect of the invention, a method for detecting phosphate-containing metabolites is provided, which comprises the following steps of carrying out derivatization treatment on phosphate-containing metabolites in a sample to be detected, carrying out liquid chromatography-mass spectrometry combined analysis on the sample after the derivatization treatment to obtain mass spectrum data, and identifying the derivatized phosphate-containing metabolites in the sample to be detected based on characteristic isotope signals of chlorine elements in the chlorinating agents in the mass spectrum data. Preferably, the identification based on the characteristic isotope signals specifically comprises the steps of screening precursor ions with mass-to-charge ratio (M) peaks and isotope (M+2) peaks thereof existing simultaneously from the mass spectrum data, calculating the peak intensity ratio of the M peaks to the (M+2) peaks, and identifying the precursor ions with the peak intensity ratio in a preset range as potential derivative metabolites. Preferably, the preset range is 2.75 to 3.25. Preferably, the screening and calculating steps are implemented based on a Python program. Preferably, the derivatization treatment of the chloro reagent comprises the steps of mixing a pretreated sample with a first reaction solution containing carbodiimide and imidazole to perform a first reaction, and then adding a second reaction solution containing the chloro reagent and methylimidazole to perform a second reaction, wherein the chloro reagent is [5- (2-chlorophenyl) isoxazol-3-yl ] methylamine. Preferably, the first reaction is carried out at 40℃for 30 minutes and the second reaction is carried out at 40℃for 2 hours. Preferably, the method further comprises a sample pretreatment and enrichment step before the derivatization treatment of the phosphate-containing metabolite by the chlorinating reagent, wherein the enrichment step is carried out by adopting a weak anion exchange solid phase extraction column. Preferably, the sample pretreatment and enrichment step specifically comprises the steps of mixing biological tissue samples with normal saline and precooled methanol, homogenizing, centrifuging, collecting supernatant, combining and concentrating to obtain residues, redissolving the residues in ammonium acetate buffer, extracting the residues by an