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A Method of Chemical Labeling Combined with LC-MS and Its Application in Nucleotide Analysis

A chemical labeling and combination technology, applied in the field of analytical chemistry, can solve the problem of limited sensitivity of nucleotides, and achieve the effect of facilitating quantitative analysis, reducing matrix interference, and avoiding contamination of mass spectrometry.

Inactive Publication Date: 2018-05-29
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of direct LC-MS analysis of nucleotides is still limited

Method used

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  • A Method of Chemical Labeling Combined with LC-MS and Its Application in Nucleotide Analysis
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  • A Method of Chemical Labeling Combined with LC-MS and Its Application in Nucleotide Analysis

Examples

Experimental program
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Effect test

Embodiment 1

[0036] 1. First prepare the working solution: (1) dissolve the labeling reagent DMPA in chromatographically pure acetonitrile to prepare a DMPA acetonitrile solution with a concentration of 1M; (2) dilute imidazole with water to prepare a solution with a concentration of 2.5mM and pH=6 Imidazole buffer solution; (3) EDC is dissolved in water, is mixed with the EDC aqueous solution that concentration is 500mM; (4) fills 200mg amino silica gel in 3mL solid-phase extraction small column; (5) the volume fraction of preparation ammonia water is 0.25%, acetonitrile Solvent S with a volume fraction of 80% and a volume fraction of pure water of 19.75%.

[0037]2. Protein removal: Take a certain amount of biological sample and put it into a 3mL homogenization tube, add methanol aqueous solution (methanol:water=4:1, v / v) pre-cooled to -80°C. Place the homogenization tube in the ice-water mixture to grind the tissue for 10 min, transfer the homogenized mixture to a 5 mL centrifuge tube, ...

Embodiment 2

[0042] Example 2: Analysis of Nucleotides in Urine Samples

[0043] Urine samples were centrifuged twice at 4°C at 5000×g for 10 min each time to remove insoluble matter. After centrifugation, the supernatant was passed through a layer of nylon filter membrane (13 mm×0.22 μM, Shanghai Anpu Scientific Instrument Co., Ltd.) to remove proteins. The urine sample after protein removal was enriched with amino silica gel cartridges, then chemically labeled with DMPA, and then the excess labeling reagent was removed by liquid-liquid extraction, and finally the excess activation was removed with Strata X cartridges After EDC, blow dry with nitrogen at 37°C, redissolve in 100 μL water, inject 70 μL, and analyze by LC-MS.

Embodiment 3

[0044] Example 3: Analysis of Nucleotides in Human Renal Cancer Tissue and Paracancerous Tissue

[0045] A certain amount of kidney cancer and para-cancerous tissues were taken and added to a 3 mL homogenization tube, and methanol aqueous solution (methanol:water=4:1, v / v) precooled to -80°C was added. Place the homogenization tube in the ice-water mixture to grind the tissue for 10 min, transfer the homogenized mixture to a 5 mL centrifuge tube, add 1.5 mL of methanol aqueous solution (methanol: water = 4 :1, v / v) combined with the extract after cleaning the homogenate tube. The combined extracts were centrifuged at 14,000×g at 4°C for 10 min, the supernatant was taken out, and dried under nitrogen at 37°C.

[0046] After protein removal, the nucleotides in the sample were enriched with amino silica gel column, then chemically labeled with DMPA, and then the excess labeling reagent was removed by liquid-liquid extraction, and finally the excess activator EDC was removed with...

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Abstract

The invention discloses a chemical labeling and LC-MS combined method, and applications thereof in nucleotide analysis. According to the chemical labeling and LC-MS combined method, N,N-dimethyl-p-phenylenediamine is used for chemical labeling of 10 monophosphate nucleotides containing two methylation modified nucleotides, so that the retention behavior of the modified nucleotides in reversed phase liquid chromatography is improved obviously, and detection sensitivity of the modified nucleotides in mass spectrometric detection is increased. The chemical labeling and LC-MS combined method is high in sensitivity and selectivity; liquid-liquid extraction can be adopted to remove excess labeling reagent DMPA effectively, Strata X solid phase extraction columns can be used for removing excess activator EDC, quantitative determination of extremely-low-abundance methylation modified cytidylate in living bodies can be realized, LC separation sensitivity and ESI-MS detection sensitivity of nucleotides can be improved obviously, and signal responsiveness is increased by 1 to 2 magnitude orders.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method of chemical labeling combined with LC-MS and its application in nucleotide analysis. Background technique [0002] DNA / RNA modification refers to the modification on DNA, RNA bases A\T\C\G\U and ribose. At present, although there are more than a dozen kinds of DNA modifications and more than one hundred kinds of RNA modifications, methylated nucleotides account for the majority, and among all the modification types, methylation at the 5-position of cytosine is currently the most widely studied, but quantitative studies of methylated cytosine-modified nucleotides have not been reported. Theoretically, 5-methyldeoxycytosine nucleotides (5-Me-dCMP) or 5-methylcytosine nucleotides (5-Me- CMP), and these methylated monophosphate nucleotides may be converted into their triphosphate forms under the action of phosphokinases and be reused by DNA or RNA and random...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8813
Inventor 袁必锋曾欢
Owner WUHAN UNIV
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