A liquid chromatography tandem mass spectrometry method for the detection of royal jelly major protein 5 in Italian honey and Chinese honey
A royal jelly main protein and tandem mass spectrometry technology, which is applied in the field of food detection, can solve problems such as amino acid sequence differences, and achieve the effects of strong specificity, good accuracy and precision, high specificity and sensitivity
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Embodiment 1
[0052] 1. Sample source
[0053] A total of 20 samples of real honey were purchased from the market or beekeepers.
[0054] 2. Experimental steps
[0055] 2.1 Solution preparation
[0056] 5M Urea solution: Weigh 4.5 g of Urea, and dilute to 15 mL with ultrapure water;
[0057] 100 mM DTT: Weigh 308.5 mg DTT, 40 mM NH 4 HCO 3 Dilute the solution to 20 mL, dispense 1 mL into each tube, and store in a -20°C refrigerator until use.
[0058] 40 mM NH 4 HCO 3 Solution: weigh 0.316 g NH 4 HCO 3 , dilute to 100 mL with ultrapure water, and store in a 4°C refrigerator until use.
[0059] 100 mM IAA solution: Weigh 0.39 g of IAA, add 40 mM NH 4 HCO 3 The solution was fixed to 20 mL, and stored in a -20°C refrigerator until use.
[0060] Activation solution: 500 μL ACN, 3 μL TFA, dilute to 1 mL with ultrapure water, store at 4°C.
[0061] Equilibrium solution: 1 μL TFA, dilute to 1 mL with ultrapure water, store at 4°C.
[0062] Eluent: 800 μL ACN, 1 μL TFA, dilute to 1 mL...
Embodiment 2
[0080] Example 2 Detection of honey adulteration
[0081] 1. Sample source
[0082] Buy real samples of Chinese or Italian honey from the market or beekeepers.
[0083] 2. Experimental steps
[0084] (1) Solution preparation
[0085] With embodiment 1.
[0086] (2) Blending of honey: Italian honey is mixed into medium honey in different proportions.
[0087] Mix Italian honey into the honey according to the ratio of 5%, 10%, 20%, 30%, 40%, 50%.
[0088] (3) Pretreatment of honey samples
[0089] ① Accurately weigh 10 g of honey to be tested evenly into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new 2 mL centrifuge tube.
[0090] ② Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mix. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IAA solut...
Embodiment 3
[0096] Example 3 Detection of honey in commodities
[0097] 1. Sample source
[0098] 30 bottles of medium-sized honey from different manufacturers purchased from the mall.
[0099] 2. Experimental steps
[0100] (1) Solution preparation
[0101] With embodiment 1.
[0102] (2) Pretreatment of samples to be tested
[0103] ① Accurately weigh 10 g of honey to be tested evenly into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new centrifuge tube.
[0104] ② Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mix. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IAA solution and react in dark at room temperature for 60 min.
[0105] ③Add 30 μL of trypsin solution to each sample, and digest at 37°C overnight. When the digestion reaction is complete, ...
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