Method for analyzing molecular weight of ovalbumin and application thereof

Abstract

The invention relates to the field of analysis, in particular to an ovalbumin molecular weight analysis method and application thereof. The method for analyzing the molecular weight of the ovalbumin comprises the following steps: (a) performing denaturation treatment and cooling on an ovalbumin solution, and adding glycosidase for enzyme digestion to obtain an ovalbumin desugaring sample; (b) detecting and analyzing the ovalbumin desugaring sample by using liquid chromatography and mass spectrometry; and (c) carrying out molecular weight analysis according to detection data obtained by the mass spectrum. According to the method for analyzing the molecular weight of the ovalbumin, the desugaring step is simplified, and the desugaring effect is improved; the analysis time is short, the operation is simple, and only several hours are needed from sample pretreatment to result analysis completion.

Description

Technical Field

[0001] The present invention relates to the field of analysis, and in particular to an analysis method for the molecular weight of ovalbumin and an application thereof. Background Art

[0002] Ovalbumins (OVA) is a high-quality protein, accounting for 54%-69% of the total amount of egg white protein. Its structure and properties will significantly affect the functional properties of egg white protein during food processing. Therefore, studying the changes of ovalbumin after heat treatment or Maillard reaction is of great significance to understanding the changes in the structure and properties of egg white protein during food processing. OVA protein is a typical globulin with a molecular weight of about 44.5kDa. It is a phosphorus-containing glycoprotein with four free sulfhydryl groups and 385 amino acid residues. These amino acid residues are entangled and folded to form a spherical structure with a high degree of secondary structure, most of which are α-helices and β-folds. There is a disulfide bond and 4 free sulfhydryl groups in the center of the OVA protein, which are exposed when heated, and the colloidal structure of the OVA protein becomes more stable through intermolecular interactions.

[0003] Due to the very complex post-translational modifications of OVA protein, including N-terminal acetylation, phosphorylation and N-sugar modification, the heterogeneity of OVA protein is very serious, which makes the molecular weight analysis very complicated. Although there is an existing technology for molecular weight analysis of OVA protein after deglycosylation under non-denaturing conditions, the deglycosylation takes a long time and is not thorough, which makes the molecular weight analysis of OVA protein very complicated.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In view of the defects of the existing molecular weight analysis methods of egg white protein after desugaring, the present invention aims to provide an analysis method of the molecular weight of egg white protein and its application, which has the advantages of short analysis time, high analysis accuracy, simple operation, etc., and greatly simplifies the molecular weight analysis of egg white protein after desugaring.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] One aspect of the present invention relates to a method for analyzing the molecular weight of ovalbumin, comprising the following steps:

[0008] (a) denaturing and cooling the ovalbumin solution, adding glycosidase to perform enzymatic digestion, and obtaining an ovalbumin desugared sample;

[0009] (b) using liquid chromatography and mass spectrometry to detect and analyze the ovalbumin desugaring sample;

[0010] (c) performing molecular weight analysis based on the detection data obtained from the mass spectrometry.

[0011] The ovalbumin molecular weight analysis method simplifies the desugaring steps and improves the desugaring effect; the analysis time is short and the operation is simple, and only a few hours are needed from sample pretreatment to completion of result analysis.

[0012] Preferably, the temperature of the denaturation treatment is 90-100°C.

[0013] Preferably, the denaturation treatment time is 10 to 30 minutes.

[0014] Preferably, the addition ratio of the egg white protein solution and the glycosidase is 5-15 μg:1 μL.

[0015] Preferably, the concentration of the ovalbumin solution is 1-10 mg / mL.

[0016] Preferably, the temperature of the enzyme cleavage is 35-39°C.

[0017] Preferably, the enzyme cleavage time is 2 to 4 hours.

[0018] Preferably, the glycosidase comprises: N-glycosidase (PNGase F enzyme).

[0019] Preferably, the elution mode of the liquid chromatography includes: isocratic elution.

[0020] Preferably, the elution time of the liquid chromatography is 5 to 10 minutes.

[0021] Preferably, the mobile phase of the liquid chromatography comprises: ammonium acetate and / or ammonium formate.

[0022] Preferably, the concentration of the mobile phase of the liquid chromatography is 8-12 mM.

[0023] Preferably, the liquid chromatography column comprises: an ACQUITY UPLC Protein BEH SEC chromatography column.

[0024] Preferably, the detection conditions of the liquid chromatography are: detection wavelength of 270-290 nm; flow rate of 0.1-0.2 mL / min; injection volume of 5-20 μL; column temperature of 30-40° C.; injection plate temperature of 5-10° C.

[0025] Preferably, the detection conditions of the mass spectrometer are: the detection mode is the positive mode; the scanning range is 2000 to 6000 m / z; and the collision energy in the source is 10.0 to 20.0 eV.

[0026] Another aspect of the present invention also relates to a method for analyzing the structure of ovalbumin, including the method for analyzing the molecular weight of ovalbumin.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The method for analyzing the molecular weight of ovalbumin provided by the present invention is combined with the special spatial structure of ovalbumin to design a desugaring method with a high degree of adaptability, thereby simplifying the desugaring steps and improving the desugaring effect; combined with the liquid chromatography-mass spectrometry method, the molecular weight analysis of the desugared sample is performed, the analysis time is short, the operation is simple, and it only takes a few hours from sample pretreatment to completion of result analysis.

[0029] (2) The method for analyzing the molecular weight of ovalbumin provided by the present invention denatures the ovalbumin before enzyme cleavage to expose more glycosylation sites and improve the deglycosylation effect.

[0030] (3) The method for analyzing the molecular weight of ovalbumin provided by the present invention is easy to operate, the analysis is fully automated, and the detection is completed by instruments. Regardless of whether one has experimental foundation or not, the operation can be performed after training. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is the molecular weight analysis result of the ovalbumin after desugaring provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0034] One aspect of the present invention relates to a method for analyzing the molecular weight of ovalbumin, comprising the following steps:

[0035] (a) denaturing and cooling the ovalbumin solution, adding glycosidase to perform enzymatic digestion, and obtaining an ovalbumin desugared sample;

[0036] (b) using liquid chromatography and mass spectrometry to detect and analyze the ovalbumin desugaring sample;

[0037] (c) performing molecular weight analysis based on the detection data obtained from the mass spectrometry.

[0038] The ovalbumin molecular weight analysis method is comprehensively improved and perfected from multiple perspectives such as instrument selection, detection condition setting, sample processing and detection, thereby improving analysis accuracy, reducing operational difficulty and simplifying operational steps.

[0039] The ovalbumin molecular weight analysis method is combined with the special spatial structure of ovalbumin to design a desugaring method with a higher degree of adaptability, thereby simplifying the desugaring steps and improving the desugaring effect; combined with the liquid chromatography-mass spectrometry method, the molecular weight analysis of the desugared sample is performed, the analysis time is short, the operation is simple, and it only takes a few hours from sample pretreatment to completion of result analysis.

[0040] Glycosidase cannot completely remove the N-sugar of OVA protein under non-denaturing conditions. It is speculated that the glycosylation sites are not exposed under non-denaturing conditions. However, under denaturing conditions, glycosidase shows a good deglycosylation effect, and the N-sugar of OVA protein is basically removed. Therefore, the present invention denatures the ovalbumin before enzyme cleavage to expose more glycosylation sites and improve the deglycosylation effect.

[0041] Furthermore, the temperature of the denaturation treatment is 90-100°C, including but not limited to any point value of 90°C, 92°C, 94°C, 96°C, 98°C or 100°C or a range value between any two of them.

[0042] Furthermore, the denaturation treatment time is 10 to 30 minutes, including but not limited to any point value of 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes, or a range value between any two of them.

[0043] The present invention designs the temperature and time of the denaturation treatment in combination with the special spatial structure of ovalbumin. Under such conditions, the denaturation treatment can expose most of the glycosylation sites of the ovalbumin, which is beneficial to the deglycosidation of glycosidase.

[0044] Furthermore, the addition ratio of the egg white protein solution and the glycosidase is 5 to 15 μg:1 μL, including but not limited to 5 μg:1 μL, 7 μg:1 μL, 9 μg:1 μL, 11 μg:1 μL, 13 μg:1 μL or 15 μg:1 μL.

[0045] Furthermore, the concentration of the egg white protein solution is 1-10 mg / mL, including but not limited to any point value of 1 mg / mL, 3 mg / mL, 5 mg / mL, 7 mg / mL or 10 mg / mL or a range value between any two of them.

[0046] The concentration and added mass of the egg white protein solution can be used to determine the addition ratio of egg white protein and glycosidase. Limiting the addition ratio of egg white protein and glycosidase within a certain range can ensure that there is enough glycosidase to act on the glycosylation sites of egg white protein to successfully complete desugaring, and also avoid waste of reagents caused by excessive addition of glycosidase.

[0047] Furthermore, the solvent of the egg white protein solution includes ultrapure water.

[0048] Furthermore, the glycosidase includes but is not limited to: N-glycosidase (PNGase F enzyme).

[0049] PNGase F is also known as peptide N-glycosidase F. PNGase F can catalyze the amide bond between N-acetylglucosamine (GlcNAc) and asparagine residues on the inner side of glycoproteins, converting aspartyl on the enzymatically hydrolyzed protein into aspartic acid. PNGase F can be used to remove N-linked sugar chains from glycoproteins, which is essential for studying the underlying protein structure and function of glycoproteins.

[0050] Furthermore, the enzyme cleavage temperature is 35-39° C., including but not limited to any point value of 35° C., 36° C., 37° C., 38° C. or 39° C. or a range value between any two of them. Under the current temperature conditions, the glycosidase has a higher activity and a good enzyme cleavage effect.

[0051] Furthermore, the enzyme cleavage time is 2 to 4 hours, including but not limited to any point value of 2 hours, 2.5 hours, 3 hours, 3.5 hours or 4 hours or any range value between two of them. The enzyme cleavage time is determined according to the characteristics of ovalbumin, and the glycosidase can complete the desugaring within this time.

[0052] Furthermore, the mobile phase of the liquid chromatography includes but is not limited to: ammonium acetate and / or ammonium formate.

[0053] Further, the concentration of the mobile phase of the liquid chromatography is 8-12 mM, including but not limited to any point value of 8 mM, 9 mM, 10 mM, 11 mM or 12 mM or a range value between any two of them. The concentration of the mobile phase will affect the peak time and the mass spectrometry response intensity.

[0054] Furthermore, the mobile phase of the liquid chromatography is filtered and ultrasonically degassed.

[0055] Furthermore, the pore size of the filtering microporous membrane is 0.22-0.45 μm.

[0056] Furthermore, the ultrasonic degassing time is 13 to 17 minutes, including but not limited to any one of 13 minutes, 14 minutes, 15 minutes, 16 minutes or 17 minutes, or a range between any two of them.

[0057] Furthermore, the chromatographic column of the liquid chromatography includes but is not limited to: UPLC BEH SEC chromatographic column.

[0058] Furthermore, the specifications of the chromatographic column are as follows: length 150 mm, inner diameter 2.1 mm, filler particle size 1.7 μm, pore size

[0059] Furthermore, the elution mode of the liquid chromatography includes: isocratic elution.

[0060] The isocratic elution mentioned in the present invention refers to elution with a constant composition ratio and flow rate of the mobile phase.

[0061] Furthermore, the elution time of the liquid chromatography is 5 to 10 minutes, including but not limited to any point value of 5 minutes, 6 minutes, 7 minutes, 8 minutes or 10 minutes, or a range value between any two of them.

[0062] Further, the detection conditions of the liquid chromatography are: the detection wavelength is 270-290 nm (for example, it can be but not limited to any one of 270 nm, 275 nm, 280 nm, 285 nm or 290 nm, or a range between any two of them); the flow rate is 0.1-0.2 mL / min (for example, it can be but not limited to any one of 0.1 mL / min, 0.12 mL / min, 0.14 mL / min, 0.16 mL / min, 0.18 mL / min or 0.2 mL / min, or a range between any two of them); The injection volume is 5-20 μL (for example, it can be but not limited to any point value of 5 μL, 7 μL, 9 μL, 11 μL, 13 μL, 15 μL, 17 μL or 20 μL, or a range value between any two); the column temperature is 30-40°C (for example, it can be but not limited to any point value of 30°C, 32°C, 34°C, 36°C, 38°C or 40°C, or a range value between any two); the injection plate temperature is 5-10°C (for example, it can be but not limited to any point value of 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, or a range value between any two). The above liquid chromatography detection conditions are defined to separate the ovalbumin in the sample well.

[0063] Furthermore, the detection conditions of the mass spectrometer are: the detection mode is the positive mode; the scanning range is 2000-6000 m / z; the collision energy in the source is 10.0-20.0 eV (for example, it can be but not limited to any point value of 10.0 eV, 12.0 eV, 14.0 eV, 16.0 eV, 18.0 eV or 20.0 eV or a range value between any two of them).

[0064] Furthermore, the ovalbumin desugaring sample is detected and analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0065] Liquid chromatography-mass spectrometry (HPLC-MS), or liquid chromatography-mass spectrometry, is an analytical technique that combines the high separation capability of liquid chromatography with the high selectivity, high sensitivity, and structural information-providing capability of mass spectrometry. Liquid chromatography-mass spectrometry uses liquid chromatography as the separation system and mass spectrometry as the detection system. After the sample is separated in the liquid chromatography part, the mobile phase carries the sample into the mass spectrometer. In the mass spectrometer, the sample is ionized to produce ions with a certain charge and different mass numbers. These ions are detected due to their different motion behaviors in the electromagnetic field, and ultimately a mass spectrum is formed based on the mass-to-charge ratio of the ions. By analyzing the mass spectrum, the qualitative and quantitative results of the sample can be obtained.

[0066] The present invention does not specifically limit the instrument used for LC-MS, and any instrument that can realize LC-MS in the art can be used to implement the technical solution of the present invention. In some specific embodiments, the instrument used for LC-MS includes but is not limited to: Thermo Fisher Vanquish-QE Plus.

[0067] The present invention does not specifically limit the software for molecular weight analysis, and any software in the art that can perform molecular weight analysis based on mass spectrometry data can be used to implement the technical solution of the present invention. In some specific embodiments, the molecular weight analysis is performed using Thermo Fisher Biopharma Finder software, and the analysis time is 4 to 6 minutes, including but not limited to any point value of 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes or 6 minutes, or a range value between any two of them.

[0068] Another aspect of the present invention also relates to a method for analyzing the structure of ovalbumin, including the method for analyzing the molecular weight of ovalbumin.

[0069] In the present invention, the instruments used include:

[0070] (1) Liquid chromatography-mass spectrometry (LC-MS) was performed using the Thermo Fisher Scientific Vanquish-QE Plus;

[0071] (2) The chromatographic column was ACQUITY UPLC Protein BEH SEC (150×2.1 mm, 1.7 μm, );

[0072] (3) Ultrasonic degassing device.

[0073] The diluent or blank involved in the present invention is ultrapure water.

[0074] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0075] Example 1

[0076] The method for analyzing the molecular weight of ovalbumin provided in this embodiment comprises the following steps:

[0077] 1) Take 10 μg of 10 mg / mL ovalbumin solution, then add ultrapure water to make the final volume 50 μL, then denature at 95°C for 30 min, cool to room temperature, add 1 μL PNGase F enzyme, mix well, and digest at 37°C for 4 h to obtain an ovalbumin desugaring sample;

[0078] 2) The prepared 10 mM ammonium acetate solution was first filtered through a 0.22 μm membrane, and then ultrasonically degassed for 15 minutes for use; the ovalbumin desugaring sample was detected and analyzed by liquid chromatography and mass spectrometry, wherein the elution mode of the liquid chromatography was isocratic elution, the elution time was 5 min, the chromatographic column was an ACQUITY UPLC Protein BEH SEC chromatographic column, and the detection conditions of the liquid chromatography were: the detection wavelength was 280 nm, the flow rate was 0.2 mL / min, the injection volume was 10 μL, the column temperature was 35° C., and the injection plate temperature was 10° C.; the detection conditions of the mass spectrometry were: the detection mode was positive mode; the scanning range was 2000-6000 m / z; the Insource CID was 10.0 eV;

[0079] 3) After the sample test is completed, the molecular weight analysis is performed using the Thermo Fisher Biopharma Finder software. The desugaring ratio is calculated (desugaring ratio % = molecular weight mass spectrometry response intensity after desugaring / molecular weight mass spectrometry response intensity of the highest undesugared glycoform ratio * 100). The desugaring effect is judged by the size of the desugaring ratio value. The larger the desugaring ratio value, the better the desugaring effect.

[0080] Figure 1This is the molecular weight analysis result of the ovalbumin after desugaring in this example. It can be seen that the desugaring effect of this example is good, which greatly simplifies the molecular weight analysis of the OVA protein. From the molecular weight results of the OVA protein after desugaring, it can be seen that the OVA protein has different degrees of phosphorylation modification, as well as some unknown modifications.

[0081] Example 2

[0082] The method for analyzing the molecular weight of ovalbumin provided in this embodiment comprises the following steps:

[0083] 1) Take 15 μg of 1 mg / mL ovalbumin solution, then add ultrapure water to make the final volume 50 μL, then denature at 90°C for 20 min, cool to room temperature, add 1 μL PNGase F enzyme, mix well, and digest at 35°C for 2.5 h to obtain an ovalbumin desugaring sample;

[0084] 2) The prepared 8 mM ammonium acetate solution was first filtered through a 0.22 μm membrane, and then ultrasonically degassed for 15 minutes for use; the ovalbumin desugaring sample was detected and analyzed by liquid chromatography and mass spectrometry, wherein the elution mode of the liquid chromatography was isocratic elution, the elution time was 10 min, the chromatographic column was an ACQUITY UPLC Protein BEH SEC chromatographic column, and the detection conditions of the liquid chromatography were: the detection wavelength was 280 nm, the flow rate was 0.2 mL / min, the injection volume was 10 μL, the column temperature was 35° C., and the injection plate temperature was 10° C.; the detection conditions of the mass spectrometry were: the detection mode was positive mode; the scanning range was 2000-6000 m / z; the Insource CID was 10.0 eV;

[0085] 3) Same as Example 1.

[0086] Example 3

[0087] The method for analyzing the molecular weight of ovalbumin provided in this embodiment comprises the following steps:

[0088] 1) Take 13 μg of 3 mg / mL ovalbumin solution, then add ultrapure water to make the final volume 50 μL, then denature at 100°C for 1 min, cool to room temperature, add 1 μL PNGase F enzyme, mix well, and digest at 36°C for 2 h to obtain an ovalbumin desugaring sample;

[0089] 2) The prepared 12 mM ammonium acetate solution was first filtered through a 0.22 μm membrane, and then ultrasonically degassed for 15 minutes for use; the ovalbumin desugaring sample was detected and analyzed by liquid chromatography and mass spectrometry, wherein the elution mode of the liquid chromatography was isocratic elution, the elution time was 8 min, the chromatographic column was an ACQUITY UPLC Protein BEH SEC chromatographic column, and the detection conditions of the liquid chromatography were: the detection wavelength was 280 nm, the flow rate was 0.2 mL / min, the injection volume was 10 μL, the column temperature was 35° C., and the injection plate temperature was 10° C.; the detection conditions of the mass spectrometry were: the detection mode was positive mode; the scanning range was 2000-6000 m / z; the Insource CID was 10.0 eV;

[0090] 3) Same as Example 1.

[0091] Example 4

[0092] The method for analyzing the molecular weight of ovalbumin provided in this embodiment comprises the following steps:

[0093] 1) Take 5 μg of 5 mg / mL ovalbumin solution, then add ultrapure water to make the final volume 40 μL, then denature at 97°C for 5 min, cool to room temperature, add 1 μL PNGase F enzyme, mix well, and digest at 39°C for 3 h to obtain an ovalbumin desugaring sample;

[0094] 2) The prepared 11 mM ammonium acetate solution was first filtered through a 0.22 μm membrane, and then ultrasonically degassed for 15 minutes for use; the ovalbumin desugaring sample was detected and analyzed by liquid chromatography and mass spectrometry, wherein the elution mode of the liquid chromatography was isocratic elution, the elution time was 6 min, the chromatographic column was an ACQUITY UPLC Protein BEH SEC chromatographic column, and the detection conditions of the liquid chromatography were: the detection wavelength was 280 nm, the flow rate was 0.2 mL / min, the injection volume was 10 μL, the column temperature was 35° C., and the injection plate temperature was 10° C.; the detection conditions of the mass spectrometry were: the detection mode was positive mode; the scanning range was 2000-6000 m / z; the Insource CID was 10.0 eV;

[0095] 3) Same as Example 1.

[0096] Comparative Example 1

[0097] The difference between this comparative example and Example 1 is that no denaturation treatment is performed.

[0098] Comparative Example 2

[0099] The difference between this comparative example and Example 1 is that the temperature of the denaturation treatment is 80°C.

[0100] Comparative Example 3

[0101] The difference between this comparative example and Example 1 is that the enzyme digestion time is 6 h.

[0102] Comparative Example 4

[0103] The difference between this comparative example and Example 1 is that the addition amount of the egg white protein solution is 30 μg.

[0104] Comparative Example 5

[0105] The difference between this comparative example and Example 1 is only that the concentration of the mobile phase is 6 mM.

[0106] Experimental example

[0107] The desugaring ratio values ​​of each embodiment and the comparative example were compared to determine the desugaring effect. The specific results are shown in Table 1.

[0108] Table 1

[0109]

[0110]

[0111] It can be seen from Table 1 that (1) the desugaring effect of comparative example 1, i.e., under the condition of no denaturation, is the worst, indicating that denaturation is required to improve the desugaring effect; (2) it can be seen from Examples 3 and 4 that the denaturation time also has a relatively large impact on the desugaring effect. Combined with comparative example 2, a lower denaturation temperature will also lead to a worse desugaring effect. Overall, the denaturation conditions of a denaturation temperature of 90-100°C and a denaturation time of 10-30 min are most suitable and have a better desugaring effect; (3) Combined with comparative example 2, when the denaturation temperature and time are both reached, the desugaring ratio can reach 95% after enzyme digestion for about 2 hours. Although comparative example 3, i.e., extending the desugaring time, can make the desugaring ratio higher, the longer the enzyme digestion time, the more likely it is to cause side reactions and it is more time-consuming. Therefore, on the whole, the enzyme digestion time of 2 to 4 is the best.

[0112] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A method for analyzing the molecular weight of ovalbumin, characterized in that: The following steps are involved: (a) denaturing and cooling the ovalbumin solution, adding glycosidase to perform enzymatic digestion, and obtaining an ovalbumin desugared sample; (b) using liquid chromatography and mass spectrometry to detect and analyze the ovalbumin desugaring sample; (c) performing molecular weight analysis based on the detection data obtained from the mass spectrometry.

2. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: The temperature of the denaturation treatment is 90-100°C; And / or, the denaturation treatment time is 10 to 30 minutes.

3. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: The addition ratio of the ovalbumin solution and the glycosidase is 5-15 μg:1 μL; And / or, the concentration of the ovalbumin solution is 1-10 mg / mL.

4. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: The temperature of the enzyme digestion is 35-39°C; And / or, the enzyme cleavage time is 2 to 4 hours.

5. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: The glycosidase includes: N-glycosidase F.

6. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: The elution mode of the liquid chromatography includes: isocratic elution; Preferably, the elution time of the liquid chromatography is 5 to 10 minutes.

7. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The mobile phase of the liquid chromatography comprises: ammonium acetate and / or ammonium formate; (2) The concentration of the mobile phase of the liquid chromatography is 8 to 12 mM; (3) The chromatographic column of the liquid chromatography comprises: ACQUITY UPLC Protein BEH SEC chromatographic column.

8. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: The detection conditions of the liquid chromatography are: detection wavelength of 270-290 nm; flow rate of 0.1-0.2 mL / min; injection volume of 5-20 μL; column temperature of 30-40° C.; injection plate temperature of 5-10° C.

9. The method for analyzing the molecular weight of ovalbumin according to claim 1, characterized in that: The detection conditions of the mass spectrometer are: the detection mode is the positive mode; the scanning range is 2000-6000 m / z; and the collision energy in the source is 10.0-20.0 eV.

10. A method for analyzing the structure of ovalbumin, characterized in that: A method for analyzing the molecular weight of ovalbumin comprising the method according to any one of claims 1 to 9.

Images