Chemical labeling and LC-MS combined method, and applications thereof in nucleotide analysis

A chemical labeling and combination technology, applied in the field of analytical chemistry, can solve the problems of limited sensitivity of nucleotides, and achieve the effects of quantitative analysis, high labeling efficiency, and improved separation

Inactive Publication Date: 2017-07-14
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Chemical labeling and LC-MS combined method, and applications thereof in nucleotide analysis
  • Chemical labeling and LC-MS combined method, and applications thereof in nucleotide analysis
  • Chemical labeling and LC-MS combined method, and applications thereof in nucleotide analysis

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Experimental program
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Effect test

Embodiment 1

[0036] 1. First prepare the working solution: (1) Dissolve the labeling reagent DMPA in chromatographic pure acetonitrile to prepare a DMPA acetonitrile solution with a concentration of 1M; (2) Dilute imidazole with water to prepare a concentration of 2.5mM, pH=6 Imidazole buffer; (3) EDC is dissolved in water to prepare an EDC solution with a concentration of 500 mM; (4) A 3 mL solid phase extraction cartridge is filled with 200 mg of amino silica gel; (5) The volume fraction of ammonia is 0.25%, acetonitrile The volume fraction of solvent S is 80%, and the volume fraction of pure water is 19.75%.

[0037] 2. In addition to protein: take a certain amount of biological sample into a 3mL homogenization tube, and add a methanol aqueous solution pre-cooled to -80°C (methanol: water = 4:1, v / v). Place the homogenization tube in a mixture of ice and water to grind the tissue for 10 minutes, transfer the homogenized mixture to a 5 mL centrifuge tube, and add 1.5 mL of methanol aqueous ...

Embodiment 2

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

[0043] Urine samples were centrifuged twice at 5000×g at 4°C for 10 minutes each time to remove insoluble matter. The supernatant after centrifugation was passed through a nylon filter membrane (13mm×0.22μM, Shanghai Anpu Scientific Instrument Co., Ltd.) to remove protein. After protein removal, the urine sample is enriched with amino silica gel cartridges, then chemically labeled with DMPA, and then liquid-liquid extraction is used to remove excess labeling reagents, and finally Strata X cartridges are used to remove excess activation After adding EDC, it was dried with nitrogen at 37°C, re-dissolved in 100μL of water, injected 70μL, and analyzed by LC-MS.

Embodiment 3

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

[0045] A certain amount of kidney cancer and adjacent tissues were added to a 3 mL homogenization tube, and a methanol aqueous solution (methanol: water = 4:1, v / v) pre-cooled to -80°C was added. Place the homogenization tube in a mixture of ice and water to grind the tissue for 10 minutes, transfer the homogenized mixture to a 5 mL centrifuge tube, and add 1.5 mL of methanol aqueous solution pre-cooled to -80°C (methanol: water = 4 :1, v / v) After cleaning the homogenization tube, it is combined with the extraction solution. The combined extracts were centrifuged at a speed of 14000×g at 4°C for 10 min, the supernatant was taken out, and dried under nitrogen at 37°C.

[0046] After protein removal, the sample is enriched with amino silica gel cartridges, then chemically labeled with DMPA, and then liquid-liquid extraction is used to remove excess labeling reagents, and finally Strat...

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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 specifically relates to a method for combining chemical labels 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 DNA modifications and more than one hundred RNA modifications, methylated nucleotides account for the majority, and among all the modification types, cytosine 5-methylation is currently the most General research, but the quantitative research on methylated cytosine modified nucleotides has not been reported yet. Theoretically, there is 5-methyldeoxycytosine nucleotide (5-Me-dCMP) or 5-methylcytosine nucleotide (5-Me- CMP), and these methylated modified monophosphate nucleotides may be converted into their triphosphate form under the action of phosphokinase, which is reused by DNA or RNA and randomly inserted into...

Claims

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Application Information

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