Non-denaturing mass spectrometry detection method of lactoferrin

Abstract

The invention relates to a non-denaturing mass spectrometry method for characterizing the active structure and stability of lactoferrin, which is characterized by the purity, molecular weight, structural compactness, conformational stability and glycosylation heterogeneity of lactoferrin by combining a static micro-spraying ion source with non-denaturing mass spectrometry. The structural difference of lactoferrin from different sources and the influence of different processing modes on the structure and the stability of the lactoferrin can be evaluated. The method has the advantages of rapidness, no damage, high sensitivity, accuracy and the like, and a new method can be provided for structure and activity monitoring and product quality control of lactoferrin in the production process.

Description

Technical Field

[0001] The present invention relates to a non-denaturing mass spectrometry detection method for lactoferrin structure, which is suitable for evaluating the purity, conformational stability, activity and degradation of lactoferrin during production, storage and processing, can be used for quality control of lactoferrin production and processing, and can provide a new method and new idea for improving the production and application of lactoferrin in my country and the establishment of quality standards, thereby more effectively achieving quality control of lactoferrin. Background Art

[0002] Lactoferrin is a multifunctional glycoprotein present in many biological secretions of mammals. It has multiple biological activities, such as anti-inflammatory, antibacterial, antiviral and anticancer properties. All lactoferrin discovered so far are glycosylated, and the glycosylation sites and occupancy are highly heterogeneous. However, the isolation, extraction, storage and processing of lactoferrin may affect its structural stability and thus lead to changes in biological activity. At present, the quality control standards for lactoferrin production only involve reversed-phase liquid chromatography and particle size exclusion chromatography. However, the quality control strategy based on liquid chromatography cannot detect the conformation and glycosylation heterogeneity of lactoferrin. The traditional liquid chromatography method has limitations for the detection of lactoferrin. Therefore, it is urgent to develop a rapid, non-destructive, sensitive and effective method to detect the purity, conformational stability and glycosylation heterogeneity of lactoferrin.

[0003] Non-denaturing electrospray ionization mass spectrometry is a powerful tool for exploring protein structural changes and protein interactions. By using non-denaturing buffer solutions, intact proteins and protein complexes can be transferred from solution to the gas phase while retaining their compact structure and protein-protein interactions. Non-denaturing mass spectrometry has been widely used to evaluate protein conformational stability, protein assembly, and protein-ligand interactions. In addition, non-denaturing mass spectrometry has also been successfully applied in the biopharmaceutical field for quantification and characterization of the structural and glycosylation heterogeneity of monoclonal antibodies. Secondly, non-denaturing mass spectrometry has the advantages of simple sample preparation, small sample volume, no need for complex pretreatment, simple operation, high reproducibility, and high accuracy. Moreover, non-denaturing mass spectrometry allows high-throughput analysis, which is very beneficial for rapid detection in large-scale production processes. However, traditional liquid chromatography has certain limitations in analytical efficiency. Therefore, in view of the problem that the current quality control standards for lactoferrin are difficult to effectively evaluate its activity and conformational stability, this patent adopts a non-denaturing mass spectrometry strategy to establish a method for detecting the purity, molecular weight, structural stability, glycosylation heterogeneity, activity and degradation of lactoferrin, which can achieve rapid and effective detection of the source of lactoferrin and changes in the production process, establish a real-time non-destructive quality control method, and thus provide a new method for the production application of lactoferrin and the establishment of quality standards. Summary of the invention

[0004] The present invention provides a non-denaturing mass spectrometry detection method for lactoferrin structure, which evaluates the structural stability of lactoferrin by combining a static micro-spray ion source with a non-denaturing mass spectrometry, including the differences in product purity, conformation, activity, glycosylation heterogeneity, etc. during its production, storage, and processing. The single-time single-sample analysis mode of the static micro-spray ion source equipment realizes real-time monitoring of lactoferrin structural changes, effectively avoids sample contamination, and has the advantages of high sensitivity, high accuracy, small sample loading, and rapid detection. It can be promoted and applied in industrial production in the future to realize rapid monitoring of lactoferrin quality.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] The method for detecting the conformation and activity of lactoferrin by non-denaturing mass spectrometry comprises the following steps:

[0007] (1) Preparation of lactoferrin sample solution: Accurately weigh a certain amount of lactoferrin and dissolve it in an aqueous solution of ammonium acetate, stirring until completely dissolved.

[0008] (2) Ultrafiltration of lactoferrin was performed using an ultrafiltration tube (manufacturer: Merck Millipore, volume: 500 μl) at 12,000 × g for 10 min. This was repeated three times and the flow-through was collected.

[0009] (3) The experiment was conducted using a static micro-spray ion source. First, the lactoferrin solution was injected into the glass capillary spray needle and the wire was connected to the platinum wire. The platinum wire was inserted into the glass capillary spray needle to ensure that the platinum wire was immersed in the protein solution and did not touch the tip of the glass capillary. The three-axis translation stage was adjusted to align the spray needle tip with the injection cone coaxially and keep a distance from the injection cone. Finally, a high voltage was applied through a high-voltage power supply to ionize the protein solution and enter the mass spectrometer. Figure 1 .

[0010] (4) Adjust the voltage of the high-voltage power supply to stabilize the spray state and produce continuous peaks in the mass spectrum.

[0011] (5) Adjust the mass spectrometry parameters to obtain a non-denaturing mass spectrum.

[0012] In step (1), ammonium acetate solution is used as a buffer to dissolve lactoferrin, because under non-denaturing conditions, the buffer must not only be volatile, but also have sufficient ionic strength to maintain the folding of biomolecules in the solution to maintain the native conformation of the protein, and ammonium acetate solution has all these characteristics, so it is a commonly used buffer.

[0013] In step (1), the concentration range of the ammonium acetate solution is 5-300 mmol.

[0014] In step (1), the concentration of lactoferrin is in the range of 1-100 mM.

[0015] In step (1), the molecular weight of lactoferrin is 70,000-90,000 Daltons.

[0016] In step (2), the molecular weight cut-off of the ultrafiltration tube can be selected from 3000 Daltons to 30000 Daltons.

[0017] In step (2), the protein amount required by the ultrafiltration tube is 1-200 μg.

[0018] In step (3), the voltage of the static micro-spray ion source is 0.1-2.0 kV.

[0019] In step (3), the static micro-spray ion source current is 0.1-0.5 microamperes.

[0020] In step (3), the static micro-spray ion source flow rate is 10-100 nanoliters / minute.

[0021] In step (3), the signal-to-noise ratio of the static micro-spray ion source is improved by 1-40 times

[0022] In step (3), the inner diameter of the glass capillary spray needle is 0.2-3 mm.

[0023] In step (3), the outlet diameter of the needle tip is 50-2000 nanometers.

[0024] In step (3), the tip (a truncated cone, the top angle of the cone) is 1-30°

[0025] In step (3), the volume of the protein sample injected into the glass capillary needle is 1-100 μl.

[0026] In step (3), ensure that the platinum wire is immersed in the protein solution and does not touch the inner wall of the glass capillary.

[0027] In step (3), the distance between the glass capillary spray needle and the injection cone is 5-20 mm, and the tip of the spray needle and the injection cone are at a coaxial center position.

[0028] In step (3), the voltage applied for protein ionization is in the range of 0.1-2.0 kV.

[0029] In step (4), the spray state stable voltage range is 0.1-2.0 kV.

[0030] In step (5), scanning range: 200-10000 mass-to-charge ratio; resolution: 17500-140000 (e.g., 17500, 35000, 70000 or 140000); induced collision dissociation: 0-30 electron volts; collision energy 0-30; polarity: positive ion mode

[0031] In step (5), mass spectrometry is applicable to proteins with a molecular weight of 200 Daltons to 200,000 Daltons.

[0032] The present invention uses a static micro-spray ion source combined with a non-denaturing mass spectrometer to characterize the purity, molecular weight, structural compactness, conformational stability, and glycosylation heterogeneity of lactoferrin, and can evaluate the structural differences of lactoferrin from different sources and the effects of different processing methods on the structure and stability of lactoferrin. This method has the advantages of being rapid, non-destructive, highly sensitive, and accurate, and can provide a new method for monitoring the structure and activity of lactoferrin in the production process and controlling product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the static micro-spray ion source method and its advantages. Among them: 1 is a high-voltage power supply, 2 is a wire, 3 is a three-axis translation stage, 4 is a platinum wire, 5 is a glass capillary spray needle, 6 is a protein solution, and 7 is an injection cone;

[0034] Figure 2 The mass spectra of the static micro-spray ion source at different voltages;

[0035] Figure 3 This is the non-denaturing mass spectrum of recombinant human lactoferrin using a static microspray ion source;

[0036] Figure 4 This is the non-denaturing mass spectrum of bovine lactoferrin using a static microspray ion source;

[0037] Figure 5 This is the non-denaturing mass spectrum of bovine lactoferrin after heat treatment using a static microspray ion source. DETAILED DESCRIPTION

[0038] The present invention is further illustrated below with reference to specific embodiments

[0039] The invention discloses a new static micro-spray ion source device ( Figure 1 A): including glass capillary needle,

[0040] The inner diameter of the glass capillary needle is 0.58 mm, the outlet diameter of the needle tip is 600 nm, and the tip (a truncated cone with a cone angle of 10°) is connected to one end of a platinum wire electrode through a wire. The other end of the platinum wire is inserted into the glass capillary needle to charge the lactoferrin loaded therein, ensuring that the platinum wire is immersed in the protein solution and does not touch the inner wall of the glass capillary.

[0041] The glass capillary spray needle is fixed on an XYZ three-axis translation stage (model: Exactive Plus EMR, manufacturer: Thermo Fisher). The three-axis translation stage is adjusted to make the spray needle tip outlet coaxially aligned with the middle injection hole of the injection cone (the injection cone set at the mass spectrometer injection port), and the spray needle tip outlet faces the middle injection hole of the injection cone and is kept at a distance of 10 mm from the opening end of the middle injection hole of the injection cone. The injection cone is at 0 potential (grounded).

[0042] The preparation process of the enriched material click maltose: a silane coupling agent (structure: X3Si-(CH2)3-A, wherein X is -OCH2CH3, A is Br-), sodium azide and a catalyst (potassium iodide) are mixed in an acetonitrile solvent in a molar ratio of 1:2:0.08, with each gram of silane coupling agent corresponding to 20 ml of acetonitrile solvent volume, and reacted at 110°C for 16 hours; then microspherical silica gel (particle size 5 microns) is added, with each gram of silica gel corresponding to 8 mmol / L of silane coupling agent, heated at 160°C for 16 hours, and then heated at 120°C for 16 hours. After filtering the product, it is washed with dichloromethane, ethanol, water, and acetone in turn to obtain azidized silica; maltose and propargylamine are mixed, with each gram of maltose corresponding to 80 ml of propargylamine, and heated at 80°C The reaction was carried out for 24 hours, and then the reaction system was poured into an acetonitrile solvent for precipitation, with each gram of maltose corresponding to 80 ml of acetonitrile solvent volume to obtain alkynyl-derivative maltose; then the azidation-derivative silica prepared above was added to a water / ethanol mixed solvent (volume ratio of 1:1), with each gram of silica gel corresponding to 30 ml of the mixed solvent, and then alkynyl-derivative maltose (the mass ratio of alkynyl-derivative maltose and azidation-derivative silica was 2:1) and a catalyst (the catalyst was copper sulfate and sodium ascorbate, with a molar ratio of 1:1; the molar amount of the catalyst was 30% of the molar amount of the alkynyl-derivative maltose), and the reaction was carried out at 60°C for 60 hours, and the modified alkynyl-derivative maltose was bonded to the silica gel surface through a click chemistry reaction to obtain click maltose microspheres with a particle size of 5 microns.

[0043] Example 1

[0044] 1). Weigh 1 mg of commercial recombinant human lactoferrin (LF, manufacturer: Beijing Jinming Biotechnology Co., Ltd.) and dissolve it in 50 mmol / L ammonium acetate aqueous solution, stir and dissolve thoroughly to prepare a protein sample solution with a concentration of 1 mg / mL. The prepared sample solution is ultrafiltered using a 3000 Dalton molecular weight cutoff ultrafiltration tube at 12000×g for 10 minutes, repeated three times, and the intercepted liquid in the tube is collected to obtain the lactoferrin solution, waiting for injection.

[0045] 2). Using the new static micro-spray ion source device of the present invention ( Figure 1A) Recombinant human lactoferrin is analyzed, and the sample solution is ionized and enters the mass spectrometer. 10 microliters of recombinant human lactoferrin solution are injected into a glass capillary spray needle, the glass capillary inner diameter is 0.58 mm, the spray needle tip outlet diameter is 600 nanometers, and the tip angle is 10 °. A high-voltage DC power supply is connected to one end of a platinum wire electrode through a wire, and the other end of the platinum wire is inserted into the glass capillary spray needle and contacts the lactoferrin solution therein, so that the protein solution is charged. The glass capillary spray needle is fixed on an XYZ three-axis displacement stage, and the three-axis displacement stage is adjusted to align the spray needle tip with the middle injection hole of the injection cone (the injection cone provided at the mass spectrometer injection port) coaxially, and the spray needle tip and the middle injection hole opening end of the injection cone are maintained at a distance of 10 mm, and the injection cone is 0 potential (grounding). A high voltage DC power supply was used to apply a voltage of 1000 volts through the platinum wire electrode, and the current was 0.12 microamperes, so that the protein solution was ionized and entered the mass spectrometer. It was observed that the protein peak state was discontinuous, the signal intensity was low (1.34E4), and no stable mass spectrum was formed ( Figure 2 A). At this time, the voltage was adjusted back to 800 V, the current was 0.1 μA, and the flow rate was 30 nL / min. It was observed that the protein peak was continuous and the mass spectrum was clear ( Figure 2 B), the signal intensity was significantly increased (1.56E6).

[0046] 3). Mass spectrometry data acquisition (model: Exactive Plus EMR, manufacturer: Thermo Fisher), scanning range: 1000-7000 mass-to-charge ratio, resolution: 17500, induced collision dissociation: 30 electron volts, collision energy 10, polarity: positive ion mode, obtain a non-denaturing mass spectrum with continuous peaks and a signal intensity of 1.5E6.

[0047] 4). Analysis results: Figure 3 It can be seen that the lactoferrin peak was detected by non-denaturing mass spectrometry, and no other substances were detected ( Figure 3 A), indicating that the commercial recombinant human lactoferrin has a high purity. Lactoferrin is mainly concentrated between the mass-to-charge ratio of 3600-4600, and the charge valence state is mainly concentrated between 18+ and 21+. The narrower charge state indicates that lactoferrin has a compact structure, a dense structure is retained, and has a high conformational stability ( Figure 3 B) After deconvolution of lactoferrin using UniDec deconvolution software, its molecular weight is 78620 Daltons ( Figure 3 C).

[0048] 5). Lactoferrin was hydrolyzed into peptides, the process was as follows: 1 mg of recombinant human lactoferrin was weighed, dissolved in 20 mmol / L ammonium bicarbonate solution, and a protein sample solution with a concentration of 1 mg / mL was prepared; 10 μL of 5 mmol / L tris(2-carboxyethyl)phosphine was added, and the solution was shaken at 25°C for 30 minutes; 1.85 mg of iodoacetic acid was added, and the solution was shaken at 25°C in the dark for 30 minutes; 10 μL of 1 mg / mL chymosin was added, and the solution was shaken at 30°C for 16 hours; 10% formic acid was added to adjust the pH to 3.0, and the solution was divided into 50 μg / tube and freeze-dried. The process of enriching glycopeptides is as follows: weigh 2 mg of maltose in the enrichment material; add 200 μl of 30% acetonitrile / 0.1% trifluoroacetic acid aqueous solution by volume, and wash the material twice; add 200 μl of 80% acetonitrile / 1% trifluoroacetic acid aqueous solution by volume, and wash the material twice; add 50 μl of 80% acetonitrile / 0.1% trifluoroacetic acid aqueous solution by volume to the freeze-dried lactoferrin to dissolve it, take 40 μl of lactoferrin solution and add it to the enrichment material, add 200 μl of 80% acetonitrile / 1% trifluoroacetic acid aqueous solution by volume, shake at 25°C for 10 minutes, centrifuge at 16000×g for 5 minutes, and discard the supernatant solution; add 200 μl of 30% acetonitrile / 0.1% trifluoroacetic acid aqueous solution by volume, centrifuge at 16000×g for 5 minutes, and collect the supernatant solution. LC-MS / MS analysis was performed using pGlyco3.0 software to obtain the glycoforms Hex(3)HexNAc(2) and Hex(3)HexNAc(2)Fuc(1).

[0049] 6) Analysis results: The 19+ charge state is amplified, and two peaks can match the glycoforms obtained by enzymatic hydrolysis and enrichment ( Figure 3 D), and the mass spectrometry peaks with different abundances have different glycoform structures, indicating that this lactoferrin has glycosylation heterogeneity.

[0050] Comparative Example 1

[0051] 1): The process and conditions are the same as those of step 1) in Example 1;

[0052] 2) Analyze recombinant human lactoferrin using a commercial ion source (model: direct injection ion source provided by Exactive Plus EMR, manufacturer: Thermo Fisher Scientific). Take 50 μL of lactoferrin solution, apply a high voltage of 4.5 kV, a current of 0.6 μA, and a flow rate of 2 μL / min ( Figure 1 B).

[0053] 3): Mass spectrometry data acquisition (model: Exactive Plus EMR, manufacturer: Thermo Fisher) to obtain a non-denaturing mass spectrum with a low signal-to-noise ratio and a signal intensity of the fourth power.

[0054] 4) Analysis results: The commercial ion source has high voltage, large current and large sample consumption (2 μl / min) during the experiment. The signal intensity of the non-denaturing mass spectrum is 4 times that of the original mass spectrum. The signal-to-noise ratio of this method is 3137:1 ( Figure 1 B); and the new static micro-spray ion source of the present invention has a voltage of 800 volts, a current of 0.1 microamperes, a flow rate of 30 nanoliters / minute, and a signal-to-noise ratio of 24054:1, ( Figure 1 C); Compared with commercial ion sources, the voltage and current have been greatly reduced, which significantly reduces sample consumption, increases signal strength to the sixth power, and increases the signal-to-noise ratio by 8 times.

[0055] Example 2

[0056] 1). Weigh 1 mg of bovine lactoferrin and dissolve it in 200 mmol / L ammonium acetate aqueous solution. Stir thoroughly to dissolve and prepare a protein sample solution with a concentration of 1 mg / mL. Ultrafilter the prepared sample solution using a 3000 Dalton molecular weight cutoff ultrafiltration tube at 12000×g for 10 minutes. Repeat three times and collect the intercepted liquid in the tube to obtain the lactoferrin solution, waiting for injection.

[0057] 2). Using the new static micro-spray ion source device of the present invention ( Figure 1 A) bovine lactoferrin is analyzed, and the sample solution is ionized and enters the mass spectrometer. 20 microliters of recombinant human lactoferrin solution are injected into a glass capillary spray needle, the inner diameter of the glass capillary is 0.58 mm, the outlet diameter of the spray needle tip is 600 nanometers, and the tip angle is 10 °. A high-voltage DC power supply is connected to one end of a platinum wire electrode through a wire, and the other end of the platinum wire is inserted into the glass capillary spray needle and contacts the lactoferrin solution therein to make the protein solution charged. The glass capillary spray needle is fixed on an XYZ three-axis displacement stage, and the three-axis displacement stage is adjusted to align the spray needle tip with the middle injection hole of the injection cone (the injection cone provided at the mass spectrometer injection port) coaxially, and the spray needle tip is maintained at a distance of 10 mm from the middle injection hole opening end of the injection cone, and the injection cone is 0 potential (grounded). A high-voltage DC power supply applied 800 volts through the platinum wire electrode and a current of 0.1 microamperes to ionize the protein solution and enter the mass spectrometer at a flow rate of 30 nanoliters per minute. It was observed that the protein peak was continuous and the mass spectrum was clear.

[0058] 3). Mass spectrometry data acquisition (model: Exactive Plus EMR, manufacturer: Thermo Fisher), scanning range: 1000-7000 mass-to-charge ratio, resolution: 17500, induced collision dissociation: 30 electron volts, collision energy 10, polarity: positive ion mode, obtain a non-denaturing mass spectrum with continuous peaks and a signal intensity of 1.4E6.

[0059] 4). Analysis results: Figure 4 It can be seen that in addition to lactoferrin, other substances were detected in the mass-to-charge ratio range of 1000-2000 by non-denaturing mass spectrometry ( Figure 4 A), indicating that this sample contains other substances besides bovine lactoferrin, and the purity of this sample is affected. In the mass-to-charge ratio range of 3800-4700, compared with recombinant human lactoferrin ( Figure 3 B), the peak shape of this sample is more complex and the structure is more compact. The charge valence state is mainly concentrated in 18+ to 21+. The conformation is not affected by purity, and no denaturation peak appears, and the high conformational stability is still maintained. The molecular weight of lactoferrin after deconvolution using UniDec deconvolution software is 82916 Daltons ( Figure 4 B).

[0060] 5). Lactoferrin was hydrolyzed into peptides, the process was as follows: 1 mg of bovine lactoferrin was weighed, dissolved in 20 mmol / L ammonium bicarbonate solution, and a protein sample solution with a concentration of 1 mg / mL was prepared; 10 μL of 5 mmol / L tris(2-carboxyethyl)phosphine was added, and the solution was shaken at 25°C for 30 minutes; 1.85 mg of iodoacetic acid was added, and the solution was shaken at 25°C in the dark for 30 minutes; 10 μL of 1 mg / mL chymosin was added, and the solution was shaken at 30°C for 16 hours; 10% formic acid was added to adjust the pH to 3.0, and the solution was divided into 50 μg / tubes and freeze-dried. The process of enriching glycopeptides is as follows: weigh 2 mg of maltose in the enrichment material; add 200 μl of 30% acetonitrile / 0.1% trifluoroacetic acid aqueous solution by volume, and wash the material twice; add 200 μl of 80% acetonitrile / 1% trifluoroacetic acid aqueous solution by volume, and wash the material twice; add 50 μl of 80% acetonitrile / 0.1% trifluoroacetic acid aqueous solution by volume to the freeze-dried lactoferrin to dissolve it, take 40 μl of lactoferrin solution and add it to the enrichment material, add 200 μl of 80% acetonitrile / 1% trifluoroacetic acid aqueous solution by volume, shake at 25°C for 10 minutes, centrifuge at 16000×g for 5 minutes, and discard the supernatant solution; add 200 μl of 30% acetonitrile / 0.1% trifluoroacetic acid aqueous solution by volume, centrifuge at 16000×g for 5 minutes, and collect the supernatant solution. LC-MS / MS analysis was performed using pGlyco3.0 software to obtain the glycoforms Hex(3)HexNAc(3), Hex(3)HexNAc(4)Fuc(3), Hex(3)HexNAc(6)Fuc(1), Hex(4)HexNAc(4), Hex(5)HexNAc(3), Hex(5)HexNAc(3)NeuAc(1), Hex(5)HexNAc(4), Hex(5)HexNAc(5), Hex(6)HexNAc(5), Hex(7)HexNAc(2)Hex(8)HexNAc(2) and Hex(9)HexNAc(2).

[0061] 6) Analysis results: The glycoforms obtained by enzymatic hydrolysis and enrichment can confirm that the substances degraded in the mass-to-charge ratio range of 1000-2000 are sugar chains ( Figure 4 C). And the 20+ charge state is amplified, and the four peaks with higher abundance can match the glycoforms obtained by enzymatic hydrolysis and enrichment ( Figure 4 D), and each high-abundance peak contains three different glycoforms, and the glycoforms contained in the high-abundance peaks are all different, indicating that the protein sample tested is a glycosylated bovine lactoferrin with rich glycoforms, indicating that this protein sample has a high degree of glycosylation heterogeneity.

[0062] Example 3

[0063] 1). Weigh 1 mg of bovine lactoferrin and dissolve it in 200 mmol / L ammonium acetate aqueous solution, stir and dissolve thoroughly to prepare a protein sample solution with a concentration of 1 mg / mL. Ultrafilter the prepared sample solution using a 3000 Dalton molecular weight cutoff ultrafiltration tube at 12000×g for 10 minutes, repeat three times, collect the intercepted liquid in the tube, and obtain the lactoferrin solution, put it into a water bath for heat treatment to simulate the thermal processing process, select a temperature of 70 degrees Celsius, and a heating time of 30 minutes, and wait for injection.

[0064] 2). Using the new static micro-spray ion source device of the present invention ( Figure 1 A) bovine lactoferrin is analyzed, and the sample solution is ionized and enters the mass spectrometer. 20 microliters of recombinant human lactoferrin solution are injected into a glass capillary spray needle, the inner diameter of the glass capillary is 0.58 mm, the outlet diameter of the spray needle tip is 600 nanometers, and the tip angle is 10 °. A high-voltage DC power supply is connected to one end of a platinum wire electrode through a wire, and the other end of the platinum wire is inserted into the glass capillary spray needle and contacts the lactoferrin solution therein to make the protein solution charged. The glass capillary spray needle is fixed on an XYZ three-axis displacement stage, and the three-axis displacement stage is adjusted to align the spray needle tip with the middle injection hole of the injection cone (the injection cone provided at the mass spectrometer injection port) coaxially, and the spray needle tip is maintained at a distance of 10 mm from the middle injection hole opening end of the injection cone, and the injection cone is 0 potential (grounded). A high-voltage DC power supply applied 800 volts through the platinum wire electrode and a current of 0.1 microamperes to ionize the protein solution and enter the mass spectrometer at a flow rate of 30 nanoliters per minute. It was observed that the protein peak was continuous and the mass spectrum was clear.

[0065] 3). Mass spectrometry data acquisition (model: Exactive Plus EMR, manufacturer: Thermo Fisher), scanning range: 1000-7000 mass-to-charge ratio, resolution: 17500, induced collision dissociation: 30 electron volts, collision energy 10, polarity: positive ion mode, obtain a non-denaturing mass spectrum with continuous peaks and a signal intensity of 1.2E6.

[0066] 4). Analysis results: Figure 5 It can be seen that the heat-treated bovine lactoferrin and Figure 4 Similar, except for lactoferrin, there are still sugar chains in the mass-to-charge ratio range of 1000-2000 ( Figure 5A), and most of them exist in the natural state of lactoferrin, indicating that heating does not affect the purity of lactoferrin. The charge state is between 18+ and 21+, and the dense structure of lactoferrin is observed in the mass-to-charge ratio range of 3800-4700. It is not affected by heat treatment, and it still maintains a high degree of conformational stability. After deconvolution using the software UniDec deconvolution, its molecular weight is 82916 Daltons, which is also consistent with that before heating. ( Figure 5 B). However, in the mass-to-charge ratio range of 1000-2000, compared with unheated lactoferrin ( Figure 4 C) has a higher peak intensity, indicating that the heat treatment at 70 degrees Celsius for 30 minutes has an effect on the lactoferrin content. In order to further verify the effect of heating on lactoferrin before and after heating, the absolute peak intensities of lactoferrin and sugar chains were compared ( Figure 5 C), it was found that the peak intensity of lactoferrin decreased after heating, and heating caused lactoferrin to precipitate and reduce its content. The feasibility of using non-denaturing mass spectrometry to detect the difference in lactoferrin was verified by comparing the peak intensities.

[0067] 5). The process, conditions and experimental results are the same as those in step 5 of Example 2;

[0068] 6). Analysis results: After heating, the types of sugar forms obtained by enzymatic hydrolysis and enrichment of lactoferrin remained the same as before heating, and the types of sugar chains dropped in the mass-to-charge ratio range of 1000-2000 did not change ( Figure 5 D). When the 20+ charge state is enlarged, the types of sugar forms on the high-abundance peak are exactly the same as those without heating, indicating that heating will not affect the glycosylation heterogeneity of lactoferrin. The heated lactoferrin still has a high degree of glycosylation heterogeneity ( Figure 5 E).

Claims

1. A non-denaturing mass spectrometry method for detecting lactoferrin, characterized in that : Using static micro-spray electrospray ion source combined with non-denaturing mass spectrometry to detect lactoferrin; The static micro-spray ion source comprises a glass capillary spray needle, a high voltage DC power supply is connected to one end of a platinum wire electrode through a wire, and the other end of the platinum wire is inserted into the glass capillary spray needle to charge the lactoferrin solution filled therein; The tip outlet of the glass capillary spray needle faces the middle injection hole of the injection cone, and the tip outlet of the glass capillary spray needle is coaxial with the middle injection hole of the injection cone arranged at the mass spectrometer injection port.

2. The method for detecting lactoferrin by non-denaturing mass spectrometry according to claim 1, characterized in that The electrospray ion source uses a static micro-spray ion source, which can accurately characterize one or more of the purity, molecular weight, structural compactness, conformational stability, and glycosylation heterogeneity of lactoferrin at the molecular level; the glycosylation heterogeneity is analyzed by the following process: lactoferrin is hydrolyzed with chymosin, clicked on maltose material enrichment, LC-MS / MS analysis, and PGlyco3.0 is used to identify the glycoform.

3. The non-denaturing mass spectrometry detection method of lactoferrin according to claim 1 or 2, characterized in that : The sample is all kinds of lactoferrin, including one or more of bovine lactoferrin, porcine lactoferrin, goat lactoferrin, human lactoferrin, recombinant human lactoferrin, etc.; The molecular weight of lactoferrin is 70,000 Daltons-90,000 Daltons.

4. The method for detecting lactoferrin by non-denaturing mass spectrometry according to claim 1, characterized in that : The inner diameter of the glass capillary needle is 0.2-3 mm (preferably 0.3-0.8 mm), the outlet diameter of the needle tip is 50-2000 nm (preferably 500-700 nm), and the tip (a truncated cone, the top cone angle of the cone) has an angle of 1-30° (preferably 5-20°); The glass capillary spray needle is fixed on an XYZ three-axis translation stage. The three-axis translation stage is adjusted to make the spray needle tip outlet coaxial with the middle injection hole of the injection cone set at the mass spectrometer injection port, and the spray needle tip outlet faces the middle injection hole of the injection cone and is kept at a distance of 1-30 mm (preferably 5-10 mm) from the opening end of the middle injection hole of the injection cone. The injection cone is at 0 potential (grounded).

5. The non-denaturing mass spectrometry detection method of lactoferrin according to any one of claims 1 to 4, characterized in that Specifically, (1) preparing a lactoferrin sample solution: weighing lactoferrin and dissolving it in an aqueous ammonium acetate solution, stirring until completely dissolved; the concentration of the ammonium acetate solution is 5-300 mmol / L (preferably 50-200 mmol / L); the concentration of lactoferrin is in the range of 1-200 mmol / mL (preferably 10-100 mmol / mL); (2) ultrafiltration of lactoferrin at 5000-14000×g for 1-20 minutes using an ultrafiltration tube, repeating 2-5 times, and collecting the intercepted liquid; the molecular weight cut-off of the desalting ultrafiltration tube is 3000 Daltons to 30000 Daltons (preferably 3000-10000 Daltons); (3) Conducting an experiment using a static micro-spray ion source; first, injecting a lactoferrin solution into a glass capillary spray needle, connecting a high-voltage DC power supply to one end of a platinum wire via a wire; inserting the other end of the platinum wire into the glass capillary spray needle to ensure that the platinum wire is immersed in the protein solution and does not touch the inner wall of the glass capillary; adjusting the position of the spray needle so that the outlet of the spray needle tip faces the middle injection hole of the injection cone, the outlet of the spray needle tip is coaxial with the middle injection hole of the injection cone, and is kept at a distance of 5-20 mm (preferably 8-12 mm) from the opening end of the middle injection hole of the injection cone; finally, applying a high voltage to the lactoferrin solution in the glass capillary spray needle through the high-voltage DC power supply to ionize the protein solution and enter the mass spectrometer; (4) Adjust the voltage of the high-voltage power supply to stabilize the spray state and produce continuous peaks in the mass spectrum; (5) Adjust the mass spectrometry parameters: scanning range: 200-10000 mass-to-charge ratio; resolution: 17500-140000 (e.g., 17500, 35000, 70000 or 140000); induced collision dissociation: 0-30 electron volts; collision energy 0-30; polarity: positive ion mode; obtain a non-denaturing mass spectrum.

6. The non-denaturing mass spectrometry detection method for lactoferrin according to claim 5, characterized in that: The desalting ultrafiltration tube requires a protein mass of 1-200 micrograms (preferably 50-200 micrograms).

7. The non-denaturing mass spectrometry detection method for lactoferrin according to claim 5, characterized in that: The static micro-spray ion source voltage is 0.1-2.0 kV (preferably 0.8-1.5 kV); The static micro-spray ion source current is 0.1-0.5 micro-amperes (preferably 0.1-0.2 micro-amperes); The static microspray ion source flow rate is 10-100 nanoliters / minute (preferably 20-50 nanoliters / minute).

8. The non-denaturing mass spectrometry detection method for lactoferrin according to claim 5, characterized in that: The volume of protein sample injected into the glass capillary needle is 1-100 μl (preferably 10-50 μl).

9. The non-denaturing mass spectrometry detection method for lactoferrin according to claim 5, characterized in that: Step (3) protein ionization with a voltage in the range of 0.1-2.0 kV (preferably 0.9-1.5 kV); The spray state stable voltage range of step (4) is 0.1-2.0 kV (preferably 0.8-1.2 kV).

10. The non-denaturing mass spectrometry detection method for lactoferrin according to claim 1 or 5, characterized in that: Mass spectrometry is applicable to proteins with a molecular weight of 200 Daltons to 200,000 Daltons (preferably 10,000 to 100,000 Daltons).

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