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Method of establishing liquid chromatography-mass spectrometry (LC-MS) analysis of royal jelly sensitized protein

A royal jelly protein, liquid chromatography-mass spectrometry technology, applied in the field of liquid mass spectrometry analysis of royal jelly allergenic proteins, can solve the problems of poor linear range, difficulty in large-scale promotion, high detection limit, etc., and achieve high accuracy and repeatability Good, highly specific effect

Active Publication Date: 2017-10-24
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the quantitative analysis of MRJP1 in royal jelly at home and abroad mostly uses ELISA technology, which has a high detection limit and poor linear range, making it difficult to promote on a large scale

Method used

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  • Method of establishing liquid chromatography-mass spectrometry (LC-MS) analysis of royal jelly sensitized protein
  • Method of establishing liquid chromatography-mass spectrometry (LC-MS) analysis of royal jelly sensitized protein
  • Method of establishing liquid chromatography-mass spectrometry (LC-MS) analysis of royal jelly sensitized protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Extraction of protein in RJ

[0054] Take an appropriate amount of pure RJ sample and dilute it with ultrapure water at a mass ratio of 4:5 (RJ / Water), extract overnight at 4°C, then centrifuge at 12000g for 30min; after centrifugation, take the supernatant and continue centrifuging for 20min, then take the upper The supernatant was frozen at -18°C for later use. The protein content in the supernatant was determined by the Coomassie brilliant blue G-250 method.

[0055] Pretreatment of MRJP1 quantitative samples

[0056] Take 50 μL of the above supernatant and wash with 40 mM NH 4 HCO 3 Dilute the solution to 1 mL, take 900 μL of the diluted solution (at this time, the protein content is about 2.5 mg), first add a certain amount of internal standard peptide standard solution, then add 10 μL of 500 mM dithiothreitol (DTT), and heat in a water bath at 56 °C 30min; after cooling, add 30μL of 500mM iodoacetamide (IAA), and react at room temperature in the dark for 30min...

Embodiment 2

[0108] The LC-MS / MS method is as follows:

[0109] Chromatographic conditions:

[0110] Chromatographic column: Acquity BEH C18 column (2.1mm x 100mm, 1.7μm); column temperature: 40°C; injection volume: 10μL; mobile phase A: 0.1% formic acid / water solution; mobile phase B: 0.1% formic acid / acetonitrile solution; flow rate: 0.3mL / min; gradient elution, elution program: within 10min, 97%A to 60%A; within 0.1min, 60%A to 100%A, and maintain 100%A for 1min, then within 1min To 97% A. Finally, the column was equilibrated under the condition of 97% A for 3 minutes.

[0111] Mass Spectrometry Conditions:

[0112] Electrospray ion source ionization mode: ESI+; capillary voltage: 4.0kV; cone voltage: 25V; extractor voltage: 2.5V; entrance lens voltage: 0.2V; ion source temperature: 110°C; desolvation temperature: 360°C; desolvation Air flow: 550L / h; Cone blowback airflow: 45L / h; Scanning mode: Multi-ion reaction monitoring mode (MRM).

[0113] According to the experimental results,...

Embodiment 3

[0115] 1 mg peptide standard, first dissolved in 125 μL of acetonitrile into a suspension, and then added 500 μL of water.

[0116] At this time, the dissolution of the standard is not complete, and the quantitative result is inaccurate, the detection limit rises, and the sensitivity drops.

[0117] It can be seen that the quantitative method for allergenic proteins invented by us has strong specificity, high sensitivity (the detection limit LOD of MRJP1 in the method is 7.04 μg / g, the limit of quantification LOQ is 23.07 μg / g), good repeatability (relative standard deviation RSD range 4.6~6.3%), high accuracy (recoveries of three spiked concentrations are 88.65%, 90.11% and 87.26%), etc., can effectively carry out accurate quantitative analysis of certain allergenic proteins in actual samples .

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Abstract

The invention discloses a method of establishing liquid chromatography-mass spectrometry (LC-MS) analysis of a royal jelly sensitized protein. The method comprises the following steps: carrying out electrophoresis analysis on royal jelly proteins, preprocessing the sensitized protein bands, analyzing sensitized protein bands by LC-MS, screening the specific peptide fragments of sensitized proteins; extracting proteins from a royal jelly sample, digesting the proteins by enzymes, and quantitatively measuring the sensitized proteins by LC-MS / MS. The provided sensitized protein quantitative method has the advantages of high specificity and sensitivity, good repeatability, high accuracy, and wide linear range, and is capable of precisely and quantitatively analyzing a sensitized protein in a sample.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a method for establishing liquid chromatography-mass spectrometry analysis of royal jelly allergenic proteins. Background technique [0002] Royal jelly is referred to as RJ, also known as royal jelly, royal jelly, royal jelly, and bee milk. It is the secretion of the pharyngeal glands of young workers who cultivate larvae in the honeycomb. food. [0003] The so-called royal jelly is a milky substance secreted by young and middle-aged bees after eating pollen. This milky substance is like mammalian milk and has great nutritional value and immune function and contains extremely high longevity factors. Honey bees (worker bees or drones) and queen bees It is the same at the egg stage. After hatching, eat royal jelly for three days and then change to honey and pollen to grow into bees. After hatching, eat royal jelly and grow into queen bees. The life span...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/88
CPCG01N30/06G01N30/88
Inventor 张虹林娜陈思
Owner ZHEJIANG GONGSHANG UNIVERSITY
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