CN107091885B - Method for determining sialic acid content of protein - Google Patents
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- CN107091885B CN107091885B CN201610091196.9A CN201610091196A CN107091885B CN 107091885 B CN107091885 B CN 107091885B CN 201610091196 A CN201610091196 A CN 201610091196A CN 107091885 B CN107091885 B CN 107091885B
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Abstract
The invention provides a method for determining sialic acid content of protein, which comprises the following steps: (1) digesting the protein sample with a sialidase to obtain a digest containing sialic acid; (2) subjecting the digest to a precipitation treatment and separating a supernatant containing the sialic acid; (3) analyzing the supernatant by ultra performance liquid chromatography-mass spectrometry so as to obtain a mass spectrum; and (4) determining the sialic acid content of the protein based on the mass spectrum. The method provided by the embodiment of the invention can be used for quickly and quantitatively detecting the total sialic acid content in the protein sample without derivatization of sialic acid, is simple to operate, high in sensitivity, high in accuracy and short in time consumption, and can be used for intermediate quality control in biomacromolecule production or quality inspection of final products.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for determining sialic acid content of protein.
Background
Sialic acid is a class of monosaccharide derivatives of neuraminic acid with a 9 carbon backbone. The most common sialic acid is N-acetylneuraminic acid (Neu5Ac), widely distributed in animal tissues, mainly in the form of glycoproteins and gangliosides. Sialic acid exerts a variety of functions in the body, including promoting brain development, enhancing memory, promoting the absorption of vitamins and minerals by the intestinal tract, and the like. Meanwhile, sialic acid is negatively charged, so that a relatively concentrated negative charge region is formed on cells, and the sialic acid is also a binding receptor of viruses such as influenza.
In recent years, biomacromolecules represented by monoclonal antibodies and recombinant proteins have been rapidly developed in the medical field, and many macromolecular drugs have become heavy-pound bombs and are sold in excess of billions of dollars in size in the world every year. The biomacromolecule medicine is mostly expressed by a mammalian cell line and has abundant glycosylation modifications, including sialic acid modification. Sialic acid with strong negative electricity can affect the half-life period of protein macromolecules in vivo and affect the pharmacokinetics of macromolecular drugs in vivo. Therefore, sialic acid degree is often one of the key quality attributes of biomacromolecule drugs, and occupies a very important position in the development and production processes of biomacromolecule drugs.
However, methods for determining sialic acid content in monoclonal antibodies and recombinant protein biomacromolecule drugs are still under development.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention provides a method for determining the sialic acid content, which has simple operation, high sensitivity and short time consumption and can be used for intermediate quality control of biomacromolecule production or quality inspection of final products.
In a first aspect of the invention, a method is provided for determining the sialic acid content of a protein. According to an embodiment of the invention, the method comprises: (1) digesting the protein sample with a sialidase to obtain a digest containing sialic acid; (2) subjecting the digest to a precipitation treatment and separating a supernatant containing the sialic acid; (3) analyzing the supernatant by ultra performance liquid chromatography-mass spectrometry so as to obtain a mass spectrum; and (4) determining the sialic acid content of the protein based on the mass spectrum. In the existing method for determining the sialic acid content in a protein sample by using a high performance liquid chromatography or a gas chromatography, as sialic acid does not have ultraviolet absorption, the high performance liquid chromatography or the gas chromatography needs to carry out pretreatment on the protein sample, and only derivatized sialic acid can be detected, so that the whole process is complex in operation and consumes a long time; the existing method for measuring the sialic acid content in the protein sample by using a spectrophotometry method has higher detection limit and quantification limit. Compared with the prior art, the method provided by the embodiment of the invention has the advantages that the total sialic acid content in the protein sample is rapidly and quantitatively detected under the condition of not deriving sialic acid, the method is simple to operate, high in sensitivity, high in accuracy and short in time consumption, and the method can be used for intermediate quality control of biomacromolecule production or quality inspection of final products.
According to an embodiment of the present invention, the method for determining sialic acid content of a protein may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the mass spectrum uses the following conditions: in the negative ion scanning mode, the ion source temperature is 150 ℃, the capillary voltage is 2.5kV, the taper hole voltage is 12V, the desolvation temperature is 450 ℃, the collision energy is 18eV, the ion collection mode is a multi-reaction detection mode, and the collected ions are 308.7 parent ions and 87.02 child ions. According to the embodiment of the invention, under the mass spectrum condition, the obtained sialic acid has good mass spectrum peak shape, good peak separation degree, high detection sensitivity and high accuracy.
According to an embodiment of the present invention, the ultra high performance liquid chromatography employs the following conditions: the chromatographic column is ACQUITYUPLC BEH C18, the particle size of the chromatographic column is 1.7 microns, the size is 2.1 × 50mm, the column temperature is 30 ℃, the sample chamber temperature is 10 ℃, the sample injection amount is 1 microliter, the phase A of the mobile phase is 0.1% ammonia water/water solution, the phase B of the mobile phase is acetonitrile, the elution procedure is 0-2 min, and the elution procedure is 90% A; 2-4 min, 20% A; 4-6 min, 90% A. According to the embodiment of the invention, under the condition of the liquid chromatography, sialic acid and other impurities in the supernatant are separated efficiently, the ionization degree is good, and the accuracy and the sensitivity of the next step of mass spectrometry on sialic acid are further improved.
According to an embodiment of the present invention, the amount of sialidase is 50U based on 4 microgram of the protein sample. The inventors have found that the amount of sialidase is excessive, so that adequate digestion of the protein sample can be ensured in terms of the amount of enzyme.
According to an embodiment of the invention, the digestion treatment is carried out at 37 ℃ for 1-12 h, preferably 4 h. The inventor experimentally found that when the amount of sialidase is excessive, the digestion time is longer than 12h, which results in partial sialic acid degradation, the digestion time is shorter than 1h, which results in insufficient hydrolytic digestion, preferably the digestion time is 4h, and the release of the sialic acid bound to the protein sample is the most complete.
According to an embodiment of the present invention, the step (2) includes: the digest is contacted with acetonitrile and the digest is subjected to a precipitation treatment and the supernatant containing the sialic acid is separated by centrifugation. Acetonitrile is used to change the hydrogen bonds within and between molecules of the protein to aggregate and separate out the protein (including protein components and sialidase in the protein sample), and the supernatant containing sialic acid is collected by removing the protein precipitate by centrifugation. Protein impurities in the obtained supernatant are removed completely, interference on mass spectrum detection is eliminated, and the accuracy of mass spectrum detection results and the sensitivity of mass spectrum detection are further improved.
According to the embodiment of the invention, the acetonitrile is pre-cooled for 1h at-20 ℃. Precooled acetonitrile can obviously improve the denaturation and precipitation efficiency of acetonitrile on protein, so that protein impurities in the obtained supernatant can be removed more thoroughly, and the accuracy of a mass spectrum detection result and the sensitivity of mass spectrum detection are further effectively improved.
According to an embodiment of the invention, said contacting is carried out at-20 ℃. The denaturation and precipitation efficiency of acetonitrile on protein can be obviously improved by contacting at the temperature of-20 ℃, so that protein impurities in the obtained supernatant can be removed more thoroughly, and the accuracy of a mass spectrum detection result and the sensitivity of mass spectrum detection are further effectively improved.
According to an embodiment of the invention, the precipitation treatment is carried out at-20 ℃ for 1 h. In the embodiment of the invention, after the digestive juice is contacted with acetonitrile, the mixture is kept stand and precipitated for 1h at the temperature of-20 ℃, and protein impurities in the supernatant are completely precipitated after detection, so that the interference of the protein impurities on the subsequent mass spectrometry detection is effectively eliminated, and the accuracy of the mass spectrometry detection result and the sensitivity of the mass spectrometry detection can be further effectively improved.
According to an embodiment of the invention, the centrifugation is performed at 10000 Xg, 4 ℃ for 10 min. Under the centrifugal condition, the precipitated protein impurities are effectively removed, so that the interference of the protein impurities on the subsequent mass spectrum detection is effectively eliminated, and the accuracy of the mass spectrum detection result and the sensitivity of the mass spectrum detection can be further effectively improved.
According to an embodiment of the invention, the volume ratio of the digest to the acetonitrile is 1: 3. The inventor finds that when the volume ratio of the digestive juice to the acetonitrile is 1:3, the efficiency of precipitating the protein by the acetonitrile can be further improved, the interference of protein impurities on the subsequent mass spectrum detection can be further effectively eliminated, and the accuracy of the mass spectrum detection result and the sensitivity of the mass spectrum detection can be further effectively improved.
According to an embodiment of the present invention, before the step (3), the supernatant is diluted with ultrapure water in advance. According to the embodiment of the invention, at the stage of digesting the protein sample by sialic acid, the used enzymolysis buffer solution contains nonvolatile salt (including calcium chloride and sodium acetate), and before the step (3), ultrapure water is used for diluting the supernatant in advance, so that the nonvolatile salt entering the mass spectrometer can be effectively reduced, the damage to the mass spectrometer caused by direct sample injection is avoided, and the sensitivity of the mass spectrometer and the accuracy of the mass spectrometer detection result are further effectively improved.
According to an embodiment of the invention, the diluting treatment is to dilute the supernatant until the content of sialic acid is in the range of 0.004935-0.0987 micrograms/ml. Experiments show that the content of sialic acid in diluted supernatant is in the range of 0.004935-0.0987 micrograms/ml, the peak area of the obtained sialic acid has a linear dependence on the concentration of the sialic acid, the linear relation between the peak area y and the concentration x of the sialic acid is 40002.4613x-24.5366, R is 40002.4613x-24.536620.9991, namely the content of sialic acid in the diluted supernatant is 0.004935-0.0987 micrograms/ml, the content of sialic acid in the diluted supernatant can be calculated through the dependence of the peak area and the concentration, and the obtained content value of sialic acid is high in reliability and accuracy.
According to an embodiment of the invention, in step (4), the sialic acid content of the protein is determined based on the following formula:
wherein Y represents the sialic acid content of the protein, X represents the peak area corresponding to sialic acid, N represents the dilution factor of the supernatant, M represents the volume of the supernatant, and A represents the total weight of the protein sample. According to the embodiment of the invention, when the concentration of sialic acid in the diluted supernatant is in the range of 0.004935-0.0987 micrograms/ml, the percentage of sialic acid in the protein sample to be detected can be calculated through the formula, and the obtained percentage of sialic acid in the protein sample to be detected is high in reliability and accuracy.
In a second aspect of the invention, a method of determining the sialic acid content of a protein is provided. According to an embodiment of the invention, the method comprises:
1) preparing the sample
a) Taking 4 micrograms of protein samples to be detected, adding 50U of sialidase, enzyme digestion buffer solution and ultrapure water to prepare a 25 microliter reaction system, incubating for 4 hours at 37 ℃, hydrolyzing sialic acid combined on the protein samples to be detected, and forming free total sialic acid in the solution;
b) adding 75 microliters of precooled acetonitrile into a reaction system after sialidase hydrolysis, precooling the acetonitrile at-20 ℃ for 1h, carrying out vortex oscillation for 1min, standing at-20 ℃ for 1h, then centrifuging at 10000 Xg and 4 ℃ for 10min, discarding the precipitate, and placing the precipitate at-20 ℃ until free total sialic acid exists in supernatant for subsequent detection.
2) Analyzing the supernatant by ultra performance liquid chromatography-mass spectrometry to determine the content of sialic acid in the supernatant
a) Diluting the supernatant containing free total sialic acid with ultrapure water by multiple times, placing in a 1.5ml loading bottle of ultra performance liquid chromatography, separating by 2.1 × 50mm, controlling the column temperature at 30 ℃, the sample chamber temperature at 10 ℃, the sample inlet amount at 1 μ l, controlling the phase A of the mobile phase to be 0.1% ammonia water/water solution, controlling the phase B of the mobile phase to be acetonitrile, and eluting: 0-2 min, 90% A; 2-4 min, 20% A; and 4-6 min, 90% A, and 1min later, switching the flow path to waste liquid.
b) The mass spectrum is set to be in a negative ion scanning mode, the ion source temperature is 150 ℃, the capillary voltage is 2.5kV, the taper hole voltage is 12V, the desolvation temperature is 450 ℃, the collision energy is 18eV, the ion collection mode adopts a multi-reaction detection mode, the collected ions are parent ions 308.7 and daughter ions 87.02, and the collected mass spectrum data is analyzed by MassLynx V4.1 software.
c) The concentration of sialic acid in the diluted supernatant is in the range of 0.004935-0.0987 micrograms/ml, and the sialic acid content of the protein is determined based on the following equation:
wherein Y represents the sialic acid content of the protein, X represents the peak area corresponding to sialic acid, N represents the dilution factor of the supernatant, M represents the volume of the supernatant, and A represents the total weight of the protein sample. The method provided by the embodiment of the invention can be used for quickly and quantitatively detecting the total sialic acid content in the protein sample under the condition of not derivatizing sialic acid, is simple to operate, high in sensitivity, high in accuracy and short in time consumption, and can be used for intermediate quality control of biomacromolecule production or quality inspection of final products.
Drawings
FIG. 1 is an SDS-PAGE detection of protein precipitates after sialidase hydrolysis of protein samples according to embodiments of the invention;
FIG. 2 is a typical sialic acid mass spectrum obtained by ultra performance liquid chromatography-mass spectrometry (UPLC-MS) quantitative determination according to an embodiment of the present invention;
FIG. 3 is a quantitative graph of UPLC-MS quantitative determination of sialic acid content according to an embodiment of the present invention;
FIG. 4 is a diagram of detection limit and quantitative limit review for a UPLC-MS method for quantitatively determining sialic acid content in accordance with an embodiment of the present invention;
FIG. 5 is a mass spectrum and quantitative results obtained after a measurement sample is diluted 200 times according to an embodiment of the present invention; and
FIG. 6 is a mass spectrum and quantitative results obtained after a measurement sample is diluted 1000 times according to an embodiment of the present invention.
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, but not limiting, of the invention. It is noted that materials and methods not described in detail in the following examples are all conventionally used in the art unless explicitly stated otherwise.
The sialidase used in the following examples was α 2-3,6,8 sialidase from New England Biolab inc (cat # P0720S), the sialic acid standard (A187000, lot # 4-SCC-101-1) was purchased from TRC (Canada), the protein to be tested was a recombinant fusion protein made by the applicant, the ultra high performance liquid chromatography-mass spectrometry (UPLC-MS) system used was the UPLC-MS Xevo TQ-S system from Waters, the analytical column used for separation was ACQUITY UPLCBEH C181.7 μm, 2.1 × 50mm separation column from Waters, and the data processing system was MassLynx V4.1.
Example 1 method for determining sialic acid content of protein and corresponding Condition screening
1) Preparing the sample
During the course of sialidase digestion hydrolysis, the hydrolysis time is the decisive factor for the efficiency of hydrolysis (when the sialidase is in excess). The inventors compared 3 different hydrolysis times in total, namely 1h at 37 ℃ (sample No. 01), 4h at 37 ℃ (sample No. 02) and overnight hydrolysis at 37 ℃ (sample No. 03). To facilitate the subsequent precipitation of the protein, the hydrolysis volume was adjusted to 25. mu.l. Meanwhile, the inventors carried out a protein-only sample, a sialidase-free control (sample No. 04), and a sialidase-free control (sample No. 05) in parallel. Specific sample processing protocols are shown in table 1.
TABLE 1
| Sample (I) | 01 | 02 | 03 | 04 | 05 |
| 0.25 mg/ml VEGF-Trap (microliter) | 16 | 16 | 16 | 16 | 0 |
| Sialidase (microliter) | 1(50U) | 1(50U) | 1(50U) | 0 | 1(50U) |
| 10X reaction buffer (microliter) | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| Deionized water (microliter) | 5.5 | 5.5 | 5.5 | 6.5 | 21.5 |
| Incubation time (hours) | 1 | 4 | Overnight | Overnight | Overnight |
After sialidase hydrolysis, the sialic acid bound to the protein to be tested is present in free form in solution. To avoid interference with subsequent mass spectrometry analysis, the inventors removed proteins and non-volatile salts by precipitation with an organic reagent. The specific operation is as follows: adding ice precooling acetonitrile with the volume of 3 times into the hydrolysis system, carrying out vortex oscillation for 1min, uniformly mixing, and placing at the temperature of minus 20 ℃ for 1h, thereby improving the protein precipitation efficiency and the desalting efficiency. After 1h of precipitation, the sample was placed in a refrigerated centrifuge and centrifuged at 10000 Xg for 10min at 4 ℃. After centrifugation, the supernatant was carefully aspirated and placed at 4 ℃ as a sample for subsequent quantitative detection by UPLC-MS. After the protein precipitate is collected, the protein precipitate is re-dissolved in SDS-PAGE sample buffer for SDS-PAGE detection, and then whether the sialidase hydrolysis is complete or not is preliminarily determined, the specific SDS-PAGE detection result is shown in figure 1, and the result in figure 1 shows that the molecular weight of all protein samples added with sialidase hydrolysis is obviously reduced compared with the molecular weight of protein samples not added with sialidase, which indicates that the protein samples to be detected can be efficiently hydrolyzed by sialidase under the hydrolysis condition, and then the supernatant is preliminarily determined to be used for next accurate UPLC-MS quantitative analysis of sialic acid.
Furthermore, in the step of hydrolysis of sialic acid, as the enzymolysis buffer solution contains nonvolatile salts (calcium chloride and sodium acetate), in order to avoid the harm to the instrument caused by directly feeding the supernatant, the inventor uses more protein samples in the hydrolysis stage, thereby providing enough free total sialic acid; after protein precipitation, further fold-by-fold dilution was performed, thereby reducing the amount of non-volatile salts entering the mass spectrometer. In this example, the supernatant samples after protein precipitation (samples No. 01, 02, 03, 04, and 05) were diluted 10-fold, 100-fold, 200-fold, and 1000-fold with MilliQ water (ultrapure water), respectively. The sample salt concentration after dilution by multiple ratios is already tolerated by the mass spectrometer, since most of the non-volatile salts have been removed during protein precipitation.
2) Analyzing the supernatant by ultra performance liquid chromatography-mass spectrometry to determine the content of sialic acid in the supernatant
Free total sialic acid samples were diluted in MilliQ water at appropriate fold ratios and placed in 1.5ml UPLC loading vials for 2.1 x 50mm separation by ACQUITY UPLC BEH C181.7 microns in a Waters UPLC-MS Xevo TQ-S system, column temperature controlled at 30 ℃, sample chamber temperature controlled at 10 ℃, sample loading 1 microliter. Since sialic acid is not bound to the analytical column of C18, sialic acid is retained on the column for a very short time (<1min), based on which the inventors adjusted the chromatographic conditions as follows: mobile phase: phase A: 0.1% ammonia/water solution, phase B: acetonitrile, elution procedure was: 0-2 min, 90% A; 2-4 min, 20% A; 4-6 min, 90% A. After 1min, the flow path was switched to waste, reducing as much as possible the impurities and non-volatile salt components that were brought into the mass spectrum.
Furthermore, the inventors dissolved sialic acid standard (A187000, lot 4-SCC-101-1) in MilliQ ultrapure water to prepare a stock solution with a concentration of 1mg/mL, and diluted it to obtain a control solution with a concentration of 0.1. mu.g/mL for exploring mass spectrometry conditions.
Under the MassLynx V4.1 operation interface, a Mass tune submenu is selected, and the ionization mode is set to ESI (-). Firstly, carrying out parent ion scanning in an MS mode, and obtaining an intensity value of 10 by adjusting and scanning parameters such as capillary voltage, taper hole voltage and the like6-7308.07. Then, the product ions are scanned in an MS mode, and the scanning strength value is 10 by setting collision energy6-7The daughter ion m/z of (1) is 87.02. Further, the parameter file is saved, the automatic tuning is performed in a submenu MassConsole, and the optimization of the mass spectrum is further confirmed by the automatic tuningAnd (4) conditions. The resulting mass spectral conditions are as follows: the typical sialic acid mass spectrum obtained by using the precursor ion 308.07, the daughter ion 87.02, the taper hole voltage of 12V, the collision energy of 18eV, the capillary voltage of 2.5kV, the desolvation temperature of 450 ℃ and the ion source temperature of 150 ℃ is shown in FIG. 2.
Example 2 establishment of a quantitative Curve
Before carrying out mass spectrum quantitative analysis on a protein sample to be detected, establishing a quantitative curve by using an international sialic acid standard substance. Sialic acid standard: shipment number a187000, lot number: 4-SCC-101-1, molecular weight 309.27, available from TRC (Canada). Sialic acid standards were dissolved in MilliQ ultrapure water to prepare 1mg/ml stock solutions, which were then frozen at-80 ℃. The 1mg/ml sialic acid standard solution was serially diluted with ultrapure water to 0.1 mg/ml, 0.01 mg/ml, 1 μ g/ml, 0.1 μ g/ml, 0.08 μ g/ml, 0.04 μ g/ml, 0.02 μ g/ml, 0.01 μ g/ml, 0.005 μ g/ml, and blank zero point was ultrapure water.
The quantitative curve established is shown in FIG. 3, and the results in FIG. 3 show that: the method has good specificity and no interference in blank (such as detecting No. 04, 05 of a sample); sialic acid has a good linear relationship in the concentration range of 0.004935-0.0987 micrograms/ml, and the linear equation is that y is 40002.4613x-24.5366, R20.9991 (wherein x represents the concentration of sialic acid in the sample, y represents the area of the peak corresponding to sialic acid, and R2Representing the degree of fitting), meets the requirement of accurate quantitative analysis of sialic acid; when sialic acid is measured by the method, no sialic acid is detected in a blank sample after the control solution with the concentration of 0.0987 micrograms/milliliter is measured, which shows that no residue exists in the sample when the method is used for detection.
Example 3 determination of detection and quantitation limits
As can be seen from the results in fig. 4, the signal-to-noise ratio (S/N) of the standard solution at a concentration of 0.04 μ g/ml was 104.6991 (1 μ l was injected), which resulted in a detection limit (LOD — S/N — 3) of about 1.0pg for this method; the limit of quantitation (LQD ═ S/N ═ 10) is about 3.5 pg. Therefore, the method meets the quantitative detection requirement of the total sialic acid of the protein biomacromolecule medicine.
EXAMPLE 4 sample testing
Samples (No. 01, 02, 03, 04, 05) were diluted 10-fold, 100-fold, 200-fold and 1000-fold with MilliQ water, respectively, with the 200-fold and 1000-fold dilutions being quantitatively determined as described in example 1. The quantitative detection spectrum and the result of the sample diluted by 200 times are shown in FIG. 5, and the quantitative detection spectrum and the result of the sample diluted by 1000 times are shown in FIG. 6. The results in FIG. 5 and FIG. 6 show that the samples 04 and 05 have no signal, the specificity of the method is good, the peak areas of the samples No. 01, 02 and 03 diluted by 200 times are all in the linear range obtained in example 2, and the method can be used for measuring the sialic acid content of the samples, and further can be used according to the formula
(wherein Y represents the sialic acid content of the protein to be tested, X represents the peak area corresponding to sialic acid, N represents the dilution factor of the supernatant, M represents the volume of the supernatant, and A represents the total weight of the protein sample to be tested) to determine the sialic acid content of the protein to be tested. The peak area of sialic acid in a sample diluted 1000 times is small, and the concentration of the sialic acid is 0.0022-0.0087 microgram/ml. Thus the final dilution factor for the sample of the example of the invention was determined to be 200 fold.
In addition, as a result of quantitative detection of samples obtained under different sialidase hydrolysis conditions, it is found that, in a sample hydrolyzed for 4 hours (a 02 sample), the release of sialic acid bound to a protein sample is the most complete, and the quantitative determination of sialic acid in the supernatant reaches 7.2 micrograms/ml, and the quantitative determination of sialic acid content in the corresponding supernatant of a sample hydrolyzed for 1 hour (a 01 sample) and a sample hydrolyzed overnight (a 03 sample) is respectively 1.8 micrograms/ml and 3.0 micrograms/ml, so that 4 hours of hydrolysis should be selected as the sialidase hydrolysis time for the protein sample to be detected in the embodiment of the present invention.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A method for determining the sialic acid content of a protein, comprising:
(1) carrying out digestion treatment on a protein sample for 1-12 h at 37 ℃ by using sialidase so as to obtain a digestive juice containing sialic acid;
(2) the digestive juice is contacted with acetonitrile at the temperature of-20 ℃, the digestive juice is subjected to precipitation treatment, and a supernatant containing the sialic acid is separated by centrifugation, wherein the acetonitrile is pre-cooled for 1h at the temperature of-20 ℃, and the volume ratio of the digestive juice to the acetonitrile is 1: 3;
(3) analyzing the supernatant by ultra performance liquid chromatography-mass spectrometry so as to obtain a mass spectrogram; and
(4) determining a sialic acid content of the protein based on the mass spectrum;
wherein the ultra-high performance liquid chromatography adopts the following conditions:
the column was an ACQUITY UPLC BEH C18, the particle size of the column was 1.7 microns, the size was 2.1 x 50mm,
the column temperature was 30 c,
the temperature of the sample chamber was 10 c,
the sample feeding amount is 1 microliter,
the mobile phase A is 0.1% ammonia water/water solution,
the phase B of the mobile phase is acetonitrile,
the elution procedure is 0-2 min, 90% A; 2-4 min, 20% A; 4-6 min, 90% A;
the mass spectrum adopts the following conditions:
in the negative ion scanning mode, the ion scanning mode is adopted,
the ion source temperature was 150 c,
the capillary voltage was 2.5kV,
the voltage of the taper hole is 12V,
the temperature of the desolventizing agent is 450 ℃,
the collision energy is 18eV,
the ion collection mode is a multi-reaction detection mode,
the collected ions are the parent ion 308.7 and the daughter ion 87.02.
2. The method of claim 1, wherein the amount of sialidase is 50U based on 4 micrograms of the protein sample.
3. The method according to claim 1, wherein the digestion treatment is carried out at 37 ℃ for 4 h.
4. The method according to claim 1, wherein the precipitation treatment is carried out at-20 ℃ for 1 h.
5. The method of claim 1, wherein the centrifugation is performed at 10000 Xg, 4 ℃ for 10 min.
6. The method according to claim 1, wherein the supernatant is diluted with ultrapure water in advance before the step (3).
7. The method according to claim 6, wherein the dilution treatment is to dilute the supernatant to a sialic acid content in the range of 0.004935-0.0987 μ g/ml.
8. The method according to claim 7, wherein in step (4), the sialic acid content of the protein is determined based on the following formula:
wherein Y represents the sialic acid content of the protein, X represents the peak area corresponding to sialic acid, N represents the dilution factor of the supernatant, M represents the volume of the supernatant, and A represents the total weight of the protein sample.
9. A method for determining the sialic acid content of a protein, comprising:
1) preparing the sample
Taking 4 micrograms of protein samples to be detected, adding 50U of sialidase, enzyme digestion buffer solution and ultrapure water to prepare a 25 microliter reaction system, incubating for 4 hours at 37 ℃, hydrolyzing sialic acid combined on the protein samples to be detected, and forming free total sialic acid in the solution;
adding 75 microliters of precooled acetonitrile into a reaction system after sialidase hydrolysis, precooling the acetonitrile at-20 ℃ for 1h, carrying out vortex oscillation for 1min, standing at-20 ℃ for 1h, then centrifuging at 10000 Xg and 4 ℃ for 10min, discarding the precipitate, and placing the precipitate at-20 ℃ until free total sialic acid exists in supernatant for subsequent detection;
2) analyzing the supernatant by ultra performance liquid chromatography-mass spectrometry to determine the content of sialic acid in the supernatant
Diluting the supernatant containing free total sialic acid with ultrapure water by multiple times, placing in a 1.5ml loading bottle of ultra performance liquid chromatography, separating by 2.1 × 50mm, controlling the column temperature at 30 ℃, the sample chamber temperature at 10 ℃, the sample inlet amount at 1 μ l, controlling the phase A of the mobile phase to be 0.1% ammonia water/water solution, controlling the phase B of the mobile phase to be acetonitrile, and eluting: 0-2 min, 90% A; 2-4 min, 20% A; 4-6 min, 90% A, 1min later, switching the flow path to waste liquid;
the mass spectrum is set to be in a negative ion scanning mode, the ion source temperature is 150 ℃, the capillary voltage is 2.5kV, the taper hole voltage is 12V, the desolvation temperature is 450 ℃, the collision energy is 18eV, the ion collection mode adopts a multi-reaction detection mode, the collected ions are parent ions 308.7 and daughter ions 87.02, and the collected mass spectrum data is analyzed by MassLynx V4.1 software;
the concentration of sialic acid in the diluted supernatant is in the range of 0.004935-0.0987 micrograms/ml, and the sialic acid content of the protein is determined based on the following equation:
wherein Y represents the sialic acid content of the protein, X represents the peak area corresponding to sialic acid, N represents the dilution factor of the supernatant, M represents the volume of the supernatant, and A represents the total weight of the protein sample.
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