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Method for quickly detecting proteins in bombyx batryticatus according to biological mass spectrometry technology

A biological mass spectrometry and protein technology, applied in the field of traditional Chinese medicine, can solve the problems of inaccurate determination of protein molecular weight, cumbersome steps, time and reagent consumption, etc., and achieve the effect of rich mass-to-charge ratio signals, fast measurement speed, and shortened sample preparation time

Inactive Publication Date: 2015-12-30
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has inherent defects such as cumbersome steps, high consumption of time and reagents, and SDS-PAGE cannot accurately determine the molecular weight of proteins.

Method used

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  • Method for quickly detecting proteins in bombyx batryticatus according to biological mass spectrometry technology

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Crush 20 mg of silkworms to obtain fine powder, add 200 μL of water, ultrasonically extract for 30 minutes, and centrifuge at 12,000 r / min for 15 minutes; slowly add the supernatant to 6 mL of saturated ammonium sulfate solution, vortex for a few seconds, and stand overnight at 4°C; The mixed solution was centrifuged again at 12000r / min for 15 minutes, the supernatant was discarded, and the precipitate was collected; the above precipitate was dissolved with about 100 μL of water to obtain a solution; the protein in the sample solution was adsorbed by reversed-phase bonded solid-phase extraction MilliporeC4Ziptip at 70 % Acetonitrile 10 μL eluted to obtain the test solution; draw 1 μL of the test solution, apply the sample, then take 1 μL of the saturated 3,5-dimethoxy-4-hydroxycinnamic acid matrix solution, mix and evaporate to dryness; the sample Plates were loaded into a MALDI-TOF mass spectrometer for analysis.

Embodiment 2

[0040] Crush 20 mg of silkworms to obtain fine powder, add 1000 μL of water, ultrasonically extract for 30 minutes, and centrifuge at 12000 r / min for 15 minutes; slowly add the supernatant to 50 mL of saturated ammonium sulfate solution, vortex for a few seconds, and stand overnight at 4 °C; The mixed solution was centrifuged again at 12000r / min for 15 minutes, the supernatant was discarded, and the precipitate was collected; the above precipitate was dissolved in about 100 μL of water to obtain a solution; the protein in the sample solution was adsorbed by reverse-phase bonded solid-phase extraction MilliporeC4Ziptip, and the protein in the sample solution was extracted at 80 % Acetonitrile 5 μL eluted to obtain the test solution; draw 0.5 μL of the test solution, apply the sample, then take 0.5 μL of the saturated 3,5-dimethoxy-4-hydroxycinnamic acid matrix solution, mix, and evaporate to dryness; Load the sample plate into a MALDI-TOF mass spectrometer for analysis.

Embodiment 3

[0042] Crush 20 mg of silkworms to obtain a fine powder, add 1200 μL of water, ultrasonically extract for 30 minutes, and centrifuge at 12,000 r / min for 15 minutes; slowly add the supernatant to 60 mL of saturated ammonium sulfate solution, vortex for a few seconds, and stand overnight at 4°C; The mixed solution was centrifuged again at 12000r / min for 15 minutes, the supernatant was discarded, and the precipitate was collected; the above precipitate was dissolved in about 100 μL of water to obtain a solution; the protein in the sample solution was adsorbed by reversed-phase bonded solid-phase extraction MilliporeC4Ziptip at 90 % Acetonitrile 5 μL eluted to obtain the test solution; draw 0.5 μL of the test solution, apply the sample, then take 0.5 μL of the saturated 3,5-dimethoxy-4-hydroxycinnamic acid matrix solution, mix, and evaporate to dryness; Load the sample plate into a MALDI-TOF mass spectrometer for analysis.

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Abstract

The invention relates to a method for quickly detecting proteins in bombyx batryticatus according to the biological mass spectrometry technology, and belongs to the technical field of traditional Chinese medicine. The method comprises the following steps: crushing the bombyx batryticatus, adding water for ultrasonic extraction, carrying out centrifugation to obtain a supernatant, adding the supernatant into a saturated ammonium sulfate aqueous solution for a vortex effect, and the carrying out still standing over night; carrying out centrifugation again to collect precipitates; dissolving the collected precipitates in water to obtain a sample solution; according to a reversed phase bonded solid phase extraction method, adsorbing proteins in the sample solution by utilizing ZipTip C4, and carrying out elution by utilizing acetonitrile to obtain a bombyx batryticatus test solution; detecting proteins in the bombyx batryticatus by adopting an MALDI-TOF mass spectrometer, wherein the detecting results show that the molecular mass of the proteins in the bombyx batryticatus mainly ranges between 10-40 kDa, and the protein of which the mass-to-charge ratio (m / z) is 14803 is easy to ionize and higher in content than other proteins. The method has the advantages that the operation is simple, convenient and quick; a plurality of samples can be analyzed at a time; the molecular mass detecting range is wide; the detecting results are accurate; the repeatability is high; the samples are simple and convenient to treat; the time and reagent consumption are low; proteins in the animal drug bombyx batryticatus from different sources and different batches can be analyzed; a specific way for accurately identifying the animal drug bombyx batryticatus is provided.

Description

technical field [0001] The invention relates to a method for rapidly detecting protein of silkworm silkworm by using biological mass spectrometry technology, which belongs to the technical field of traditional Chinese medicine. Background technique [0002] Bombyx mori ( Bombyx Batryticatus ), the Bombyx mori (Bombyx mori) Bombyxmori Linnaeus) 4-5 instar larvae infected (or artificially inoculated) Beauveria bassiana ( Beauveria bassiana Bals. Vuillant), also known as white silkworm, zombie worm, ginger worm, celestial worm, etc. Bombyx mori is mild in nature, salty and pungent in taste; it enters the four meridians of liver, heart, spleen and lung. Silkworm can dispel wind-heat, relieve wind and spasm, dispel wind and relieve pain, activate collaterals and stimulate the meridians, reduce phlegm and relieve cough, detoxify and dissipate stagnation, etc. Indications for infantile convulsions, epilepsy, sore throat, headache, stroke convulsions, hemiplegia, erysipelas, m...

Claims

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Application Information

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IPC IPC(8): G01N30/72G01N30/06
Inventor 沈玉萍杨欢徐莹余江南陈慧卿曹进夏国华
Owner JIANGSU UNIV
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