Biomarker for freshness of royal jelly and screening method of biomarker
A technology of biomarkers and screening methods, which is applied in the field of biomarkers and screening methods for the freshness of royal jelly, can solve the problems of single, lack of comprehensive evaluation methods and detection methods for multiple protein combinations, and achieve specificity and accurate quantitative capabilities Effect
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Embodiment 1
[0035] Example 1: Screening of specific marker peptides of MRJP1-3 in royal jelly
[0036] Pretreatment: 0.1 g royal jelly sample was weighed and fully dissolved in 8 mL ammonium bicarbonate solution (0.1 M), then transferred to a 100 mL volumetric flask to constant volume. Take 930 μL of the above lysate and 10 μL DTT (100 mM) in a 2 mL centrifuge tube, shake and mix well, and then incubate in a constant temperature water bath at 50 °C for 30 min. Subsequently, 30 μL of IAA (100 mM) was added to the above mixture, shaken and mixed, and placed in a dark environment for 30 min at room temperature. Subsequently, 10 μL of recombinant bovine trypsin solution (500 μg / mL) was added to the above mixed solution for shaking and mixing, and then placed in a constant temperature water bath at 37 °C for 2 h for enzymatic hydrolysis. After 2 h, 10 μL of 10% formic acid was added to terminate the enzymatic hydrolysis reaction. After filtering with a 0.22 μm membrane, it can be analyzed on...
Embodiment 2
[0049] Embodiment 2: the detection method of MRJP1-3 content in royal jelly
[0050] Pretreatment: with the pretreatment method in example 1.
[0051] Mass spectrometry detection: the reference conditions are as follows: a Xevo-TQ-MS system equipped with an electrospray ion source is used for data collection. The specific detection conditions of Xevo-TQ-MS are as follows: Mode ESI + , capillary voltage 3.5 kV, ion source temperature 150°C, desolvation temperature 350°C, cone backflush flow 50 L / h, desolvation flow 650 L / h, argon collision pressure 0.35pa. The parameter reference conditions of the mass spectrometry multiple reaction monitoring method are shown in Table 3.
[0052] Table 3 MRM method for marker peptides and their internal standard peptides
[0053]
[0054] Note: * Quantifier ion, L * for isotope labeling [ 13 C 6 , 15 N]-leucine.
[0055] Liquid chromatography separation: the reference conditions are as follows: use a C18 protein analysis column (1....
Embodiment 3
[0060] Example 3: A method for detecting the freshness of royal jelly with MRJP1 as the detection target
[0061] Experimental sample: In order to simulate the degradation process of royal jelly affected by temperature in the general storage process, the royal jelly sample is now subjected to aging treatment according to the following method: the royal jelly sample is stored at a constant temperature of 40°C, and a part is taken out every 7 days. Stored in -80°C refrigerator, numbered A-F respectively, that is, samples numbered A-F are royal jelly stored at 40°C for 0, 7, 14, 21, 28, and 35 days at a constant temperature. All samples were stored at -80°C after extraction until further identification.
[0062] Detection method of MRJP1 in royal jelly: as described in Example 2.
[0063] Experimental results: The experimental results (Table 4) show that compared with frozen fresh royal jelly, the MRJP1 content in fresh royal jelly kept for 7d, 14d, 21d, 28d, and 35d decreased b...
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