CN109342627B - Method for detecting amino acid in cell culture - Google Patents
Method for detecting amino acid in cell culture Download PDFInfo
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- CN109342627B CN109342627B CN201811506295.4A CN201811506295A CN109342627B CN 109342627 B CN109342627 B CN 109342627B CN 201811506295 A CN201811506295 A CN 201811506295A CN 109342627 B CN109342627 B CN 109342627B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
Abstract
The invention relates to the technical field of chemical analysis and detection, in particular to a method for detecting amino acids in cell culture. The method for detecting amino acids in the cell culture comprises the following steps: centrifuging the cell culture to be detected, taking supernatant, and diluting by 100-fold to 500-fold to obtain a sample to be detected; detecting a sample to be tested by a capillary electrophoresis-mass spectrometry method to obtain an ion current chromatogram; and acquiring a peak area corresponding to the extracted ion current chromatogram of the amino acid to be detected in the sample to be detected, and substituting the peak area into a standard curve established by a corresponding amino acid standard substance to obtain the content of the amino acid to be detected in the cell culture. The method can carry out certain pretreatment on the cell culture, combines the capillary electrophoresis-mass spectrometry combined technology, utilizes an external standard method for quantification, can carry out rapid detection on the content of the amino acid in the cell culture, can test the content of the amino acid in the cell culture aiming at different stages of the cell culture, and knows the consumption of the cell on the amino acid.
Description
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a method for detecting amino acids in cell culture.
Background
In the pharmaceutical industry, biomedical technology is vigorously developed. Drug proteins are expressed by cells or microorganisms. For example, in continuous culture and perfusion culture processes, it is important to know the amino acid consumption of cells to further optimize the process, so that the amino acid content of the culture can be determined. In addition, the culture medium development process is an important component of an upstream cell culture process, and the detection of the amino acid content can also assist the optimization of the culture medium process and promote the improvement of the target protein expression of cells. Since cell culture media, particularly commercial culture media, are expensive, have unknown components, and the influence on the quality of pharmaceutical proteins cannot be evaluated, biopharmaceutical enterprises need to develop culture media by themselves. The content of amino acid in the culture can be rapidly and timely obtained, and the development process of the culture medium can be greatly accelerated.
The existing method for determining the content of amino acid in the culture is mainly high performance liquid chromatography. The traditional high performance liquid chromatography has complex pretreatment process, needs complicated derivative labeling, wastes time and labor, has high toxicity of derivative reagents, has serious interference of culture matrixes, and has the disadvantages of small amount of detection and analysis samples in unit time, low efficiency and low detection flux and automation degree due to long analysis and test time of a single sample. In addition, asparagine, glutamine and tryptophan cannot be measured by the traditional high performance liquid chromatography, glycine cannot be separated from arginine, cysteine mostly exists in the form of cystine in a culture, and cystine cannot be measured by the traditional high performance liquid chromatography.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting amino acids in cell cultures, which aims to solve the technical problems that the detection method is complex and various components cannot be accurately detected in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for detecting amino acids in a cell culture comprising the steps of:
centrifuging the cell culture to be detected, taking supernatant, and diluting by 100-fold to 500-fold to obtain a sample to be detected; detecting a sample to be tested by a capillary electrophoresis-mass spectrometry method to obtain an ion current chromatogram; and acquiring a peak area corresponding to the extracted ion current chromatogram of the amino acid to be detected in the sample to be detected, and substituting the peak area into a standard curve established by a corresponding amino acid standard substance to obtain the content of the amino acid to be detected in the cell culture.
In the capillary electrophoresis-mass spectrometry combined technology, a capillary electrophoresis apparatus separates and ionizes an analyte according to the charge condition and the molecular size of the analyte, the sample loading amount reaches nanoliter level, the sensitivity and the detection limit of mass spectrometry can be greatly improved, the interference of a matrix in a complex cell culture is further reduced, the requirement of sample pretreatment is simplified, and complicated derivatization operation is avoided. And the analysis speed is extremely high, the sample detection can be completed within a few minutes, so that high-throughput detection is realized, and compared with the traditional high performance liquid chromatography, the mass spectrum utilization rate is greatly improved within at least 2 days. The method applies the capillary electrophoresis-mass spectrometry technology to the amino acid detection of cell cultures, and has the advantages of strong specificity, high accuracy, high sensitivity and high analysis flux.
The method provided by the invention has the advantages that the cell culture is subjected to certain pretreatment, a capillary electrophoresis-mass spectrometry combined technology is combined, and the quantification is realized by using an external standard method, so that the content of amino acid in a cell process culture sample is rapidly detected, the content of amino acid in the cell culture can be tested according to different stages of cell culture, and the consumption of the cell on the amino acid is known.
Moreover, glutamine, tryptophan, cystine, glycine and arginine cannot be detected by conventional high performance liquid chromatography, and cysteine will be mostly present in the form of cystine in the culture. The detection method of the present invention can detect various amino acids.
The method carries out quantification by an external standard method, does not need to additionally use an internal standard substance, and avoids the influence on the test result caused by poor mixing, reaction or variable purity of the internal standard substance and the sample.
Through carrying out centrifugal treatment on the cell culture, discarding the precipitate and collecting the supernatant, the interference of other components in the cell culture is reduced, the ion flow chromatogram resolution is improved, and the detection accuracy is improved.
The standard curve of the peak area and the concentration of the ion current chromatogram of the amino acid has a better linear relation in a certain concentration range, and the invention can ensure that the content of each amino acid in the obtained sample to be tested is in the linear range and the correlation coefficient is more than 0.99 by diluting the supernatant of the culture by 500 times, thereby ensuring the detection precision.
Preferably, the method for establishing the standard curve comprises the following steps: the method comprises the steps of carrying out gradient dilution on an amino acid standard substance to obtain an amino acid standard substance solution with gradient concentration, detecting the amino acid standard substance solution with gradient concentration by a capillary electrophoresis-mass spectrometry combination method to obtain the ion flow chromatogram peak area of the corresponding amino acid under each concentration, and establishing a standard curve of the corresponding amino acid according to the corresponding relation between the extraction ion flow chromatogram peak area and the concentration of the corresponding amino acid.
The gradient concentration includes at least five concentrations arranged in steps, and the number of the gradient concentrations can be increased for further improving the accuracy, for example, gradient concentration settings of 62.5. mu.M, 31.25. mu.M, 15.625. mu.M, 7.813. mu.M, 5.208. mu.M, 3.906. mu.M, 3.125. mu.M, 2.604. mu.M, 2.232. mu.M, 0.833. mu.M, 0.250. mu.M and the like can be adopted.
The peak area can be obtained by integration, and software with an integration function is adopted. The data are preferably processed by adopting a quantitative module of the Xcalibur software, the ion flow diagram of the amino acid to be detected can be automatically extracted and integrated, the data are rapidly processed in batches, the working efficiency is greatly improved, and meanwhile, the human errors are reduced.
Preferably, the amino acid includes any one or more of lysine, arginine, histidine, glycine, alanine, valine, isoleucine, leucine, serine, threonine, methionine, asparagine, proline, phenylalanine, tryptophan, glutamine, glutamic acid, cysteine, tyrosine, aspartic acid, cystine. The amino acid of the invention can be selected according to actual needs, for example, only the content of a certain amino acid component in a cell culture is concerned, only the amino acid standard substance can be selected to construct a standard curve, for example, all kinds of amino acid standard substances can be selected to construct a standard curve, for example, the content of all amino acid components in a cell culture is concerned.
In the actual test process, amino acid can be mixed in advance to obtain an amino acid mixed standard substance, and standard curves of various amino acids can be obtained only by detecting the amino acid mixed standard substance with gradient concentration. Preferably, the standard solution is prepared by mixing 21 amino acids of lysine, arginine, histidine, glycine, alanine, valine, isoleucine, leucine, serine, threonine, methionine, asparagine, proline, phenylalanine, tryptophan, glutamine, glutamic acid, cysteine, tyrosine, aspartic acid and cystine. Wherein the amount of each amino acid in the standard solution is the same.
The cystine can be mixed with the rest 20 kinds of amino acids, or can be independently prepared into standard cystine solution, which can avoid the influence of cystine on the detection of partial basic amino acids due to the acidic property of cystine solution.
The preparation method of the cystine standard solution preferably comprises the following steps: dissolving the cystine standard substance in water, adding one thousandth volume of 4 +/-1M sodium hydroxide solution, adjusting the pH value to 5.0 by using formic acid, and adding water to dilute to obtain the cystine standard substance solution with target concentration. The cystine standard solution with the target concentration of 1mM can be prepared firstly, and then diluted to obtain the cystine standard solution with the gradient concentration.
Preferably, the amino acid standard substance is prepared into a mother solution with the concentration of 0.1-10mM, and the amino acid standard substance solution with the gradient concentration is obtained by gradient dilution.
Preferably, the gradient concentration of the amino acid standard solution is in the range of 0.1 to 100. mu.M, preferably 0.25 to 62.5. mu.M. So as to ensure good linear relation and ensure that the correlation coefficient of each amino acid can reach more than 0.99.
Preferably, the supernatant and the mother liquor are diluted with a diluent comprising methanol, water and ammonium acetate at a volume ratio of (45-50): 45-50 (0.5-5). The concentration of the ammonium acetate solution is preferably 0.5 to 1.5mol/L, preferably 1 mol/L. All components in the diluent are mass spectrum levels so as to meet the requirement of mass spectrum detection.
Preferably, in the capillary electrophoresis-mass spectrometry method, the buffer solution comprises methanol, water and formic acid at a volume ratio of (45-50): 0.5-5.
Preferably, the conditions of the centrifugation treatment include: the centrifugal speed is 10000-. More preferably, the centrifugation speed is 120000-14000rpm, and the centrifugation time is 12-18 min. Further preferably, the centrifugation speed is 13000rpm, and the centrifugation time is 15 min.
Under the above centrifugation conditions, interfering substances in the cell culture can be sufficiently reduced and influence on the actual content of amino acids in the cell culture can be avoided.
The supernatant may be diluted 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, etc., as in various embodiments, to ensure that the concentration of amino acids in the diluted supernatant is within a good linear relationship.
Preferably, the supernatant is diluted by 400 times, preferably by 200 times. By diluting the above range, the concentration of amino acids in the obtained test sample can be further ensured to be in a good linear relation range.
In order to further improve the accuracy of the detection result and avoid errors of manual operation and instrument operation, the supernatant is taken out, two or more dilution times are selected to obtain two or more test samples, the data of the plurality of test samples are tested, the average value is taken, and the accuracy is improved.
Preferably, the test conditions of the capillary electrophoresis-mass spectrometry combined method comprise:
capillary electrophoresis parameters: the field strength is 500-1000V/cm, the sample injection amount is 1.0-7.0nL, the chip type is HS or HSX, the auxiliary pressurization starting time is 0.5-1.0min, the multi-hole delay time is 30s, and the detection time is 2.5-3.0 min;
mass spectrum parameters: the scanning range m/z is 70-500, the flow rate of the sheath gas is 2L/min, the temperature of the ion transmission tube is 180-.
The test conditions of the present invention are capable of detecting various types of amino acids in cell culture within the above-mentioned ranges, e.g., in various embodiments, the field strength of capillary electrophoresis may be 500V/cm, 600V/cm, 700V/cm, 800V/cm, 900V/cm, 1000V/cm, etc., preferably 1000V/cm; the temperature of the ion transfer tube can be 180 deg.C, 190 deg.C, 200 deg.C, 210 deg.C, 220 deg.C, 230 deg.C, 240 deg.C, 250 deg.C, etc., preferably 200 deg.C.
The mass-to-charge ratio m/z of each amino acid to be detected in the invention is respectively as follows: lysine 147.1128, arginine 175.1190, histidine 156.0768, glycine 76.0393, alanine 90.0550, valine 118.0863, isoleucine 132.1019, leucine 132.1019, serine 106.0499, threonine 120.0655, methionine 150.0583, asparagine 133.0608, proline 116.0706, phenylalanine 166.0863, tryptophan 205.0972, glutamine 147.0764, glutamic acid 148.0604, cysteine 122.0270, tyrosine 182.0812, aspartic acid 134.0448, and cystine 241.0311.
Preferably, the detection sequence is: firstly, detecting an amino acid standard substance solution from low concentration to high concentration; then, the test sample is detected, and if a plurality of test samples exist, the detection is performed from a low concentration to a high concentration. Preferably, the mixed standard solution of 20 amino acids is detected from low concentration to high concentration, and then the standard solution of cystine is detected from low concentration to high concentration.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method is characterized in that the cell culture is subjected to certain pretreatment, a capillary electrophoresis-mass spectrometry combined technology is combined, and an external standard method is used for quantification, so that the content of amino acid in a culture sample with the cell process is rapidly detected, the content of amino acid in the cell culture can be tested according to different stages of cell culture, and the consumption of the cell on the amino acid is known;
(2) the invention adopts the capillary electrophoresis-mass spectrometry combined technology, the sample loading amount is only nano liter level, the interference of the complex culture substrate can be reduced to a lower level, and the sensitivity of the method is greatly improved; meanwhile, the requirements of sample pretreatment are simplified, the analysis flux is improved, and complicated derivatization operation is avoided;
(3) the detection method has extremely high analysis speed, can finish sample detection within 2-3min, thereby realizing high-throughput detection, greatly improving the efficiency compared with the traditional high performance liquid chromatography for at least 2 days, and providing a research basis for the development of upstream cell processes;
(4) glutamine, tryptophan and cystine cannot be detected by the traditional high performance liquid chromatography, glycine and arginine cannot be separated, and cysteine mostly exists in the form of cystine in a culture; the detection method of the invention can realize the detection of various amino acids; and the correlation coefficient values of the standard curves of the 21 amino acids are all larger than 0.99, the linear range can reach 1000 times to the maximum, and the repeatability and the sensitivity of the method are greatly improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a total ion chromatogram of a test sample obtained from a cell culture according to an embodiment of the present invention;
FIG. 2 is an extracted ion flux chromatogram of lysine, arginine, histidine, glycine, alanine, valine, isoleucine, leucine obtained from the total base peak ion chromatogram of FIG. 1;
FIG. 3 is an extracted ion flux chromatogram of serine, threonine, methionine, proline, asparagine, phenylalanine obtained from the total base peak ion chromatogram of FIG. 1;
FIG. 4 is an extracted ion flux chromatogram of glutamine, tryptophan, cysteine, glutamic acid, tyrosine, aspartic acid, cystine obtained from the total base peak ion chromatogram of FIG. 1.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
The method for detecting amino acids in a cell culture of this embodiment comprises the steps of:
(1) preparing 1mol/L ammonium acetate solution, sample diluent and electrophoresis buffer solution in advance, wherein the concrete steps are as follows:
preparing 1mol/L ammonium acetate solution: taking a 15mL centrifuge tube, accurately weighing 0.77g of ammonium acetate, adding 10mL of water, performing vortex for 30s, and fully and uniformly mixing to obtain a 1mol/L ammonium acetate solution for later use;
preparation of sample diluent: taking a 15mL centrifuge tube, respectively adding 5mL methanol, 5mL water and 0.1mL ammonium acetate solution of 1mol/L, swirling for 30s, and fully and uniformly mixing to obtain a sample diluent for later use;
preparing an electrophoresis buffer solution: respectively adding 50mL of mass spectrum methanol, 50mL of mass spectrum water and 1mL of mass spectrum formic acid into a 100mL clean reagent bottle, and fully shaking up;
(2) preparing an amino acid standard solution with gradient concentration:
taking a mixed standard solution (manufacturer: Promega, model: L4461) of 20 amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), wherein the concentration of each amino acid is 1mM, and diluting the mixed standard solution of 1mM of 20 amino acids into a gradient series of standard working solutions SD1, SD2, SD3, SD4, SD5, SD6, SD7, SD8, SD9, SD10 and SD11 by using the sample diluent in the step (1), wherein the concentration of each amino acid is respectively 62.5. mu.M, 31.25. mu.M, 15.625. mu.M, 7.813. mu.M, 5.208. mu.M, 3.906. mu.M, 3.125. mu.M, 2.604. mu.232. mu.M, 2.232. mu.M, 0.250. mu.M 833 and 0.250. mu.M;
preparing a cystine standard solution: accurately weighing a cystine standard substance, dissolving the standard substance by using water, adding one thousandth volume of 4mol/L sodium hydroxide solution to completely dissolve the standard substance, adjusting the pH value to 5.0 by using formic acid, and adding water to dilute the standard substance to obtain a cystine standard substance solution with the concentration of 1 mmol/L; diluting 1mmol/L cystine standard solution into a gradient series of standard working solutions by using the sample diluent in the step (1), wherein the concentrations of cystine are respectively 62.5. mu.M, 31.25. mu.M, 15.625. mu.M, 7.813. mu.M, 5.208. mu.M, 3.906. mu.M, 3.125. mu.M, 2.604. mu.M, 2.232. mu.M, 0.833. mu.M and 0.250. mu.M;
(3) centrifuging the cell culture to be detected for 15min at the rotation speed of 13000rpm, discarding the precipitate, taking 20 mu L of the centrifuged cell culture supernatant, adding 180 mu L of sample diluent, and fully and uniformly mixing by swirling for 30s to obtain a cell culture sample to be detected which is diluted by 10 times; taking 20 mu L of the centrifuged cell culture sample to be tested which is diluted by 10 times, adding 180 mu L of sample diluent, and fully and uniformly mixing by swirling for 30s to obtain the cell culture sample to be tested which is diluted by 100 times and is used as a test sample A; then taking 50 mu L of the test sample A, adding 50 mu L of the sample diluent, and fully and uniformly mixing by swirling for 30s to obtain a cell culture sample to be tested which is diluted by 200 times and is used as a test sample B;
(4) detecting the amino acid standard solution with gradient concentration by adopting a capillary electrophoresis-mass spectrometer method and testing parameters shown in tables 1 and 2, wherein the mass spectrum acquires data by adopting a primary scanning mode, the capillary electrophoresis is ZipChip of 908devices in America, a ZipChip HS chip is matched for separating and ionizing samples, and the mass spectrometer is a Thermoscientific QE mass spectrometer; firstly, detecting a mixed standard solution of 20 amino acids with gradient concentration from low concentration to high concentration; detecting the cystine standard substance solution with gradient concentration from low concentration to high concentration; then detecting a test sample B and then detecting a test sample A; the total radical peak ion chromatogram map of the test sample B is shown in FIG. 1, wherein the extracted ion current chromatograms of the amino acids are shown in FIGS. 2-4;
(5) processing data by adopting a quantitative module of Xcalibur software, and integrating according to an extracted ion current chromatogram of each amino acid parent ion; the [ M + H ] monoisotopic mass-to-charge ratio M/z of each amino acid parent ion is as follows: lysine 147.1128, arginine 175.1190, histidine 156.0768, glycine 76.0393, alanine 90.0550, valine 118.0863, isoleucine 132.1019, leucine 132.1019, serine 106.0499, threonine 120.0655, methionine 150.0583, asparagine 133.0608, proline 116.0706, phenylalanine 166.0863, tryptophan 205.0972, glutamine 147.0764, glutamic acid 148.0604, cysteine 122.0270, tyrosine 182.0812, aspartic acid 134.0448, cystine 241.0311; establishing a standard curve by using an external standard method, wherein as shown in Table 3, the linear relation of 21 amino acids is good in a concentration range of 0.250-62.5 mu M, and the linear correlation coefficients are all above 0.996, wherein Y is a peak area, and X is concentration (unit mu M);
and acquiring peak areas corresponding to the extracted ion current chromatograms of the amino acids to be detected in the samples A and B, substituting the peak areas into corresponding standard curves to obtain the content of the amino acids to be detected in the samples A and B, multiplying the content by the corresponding dilution times, and taking the average value of the two to obtain the content of each amino acid in the cell culture to be detected.
Through a plurality of tests of the test method of the embodiment, the precision of the method can reach RSD less than 15%.
TABLE 1 capillary electrophoresis apparatus parameter settings
| ZipChip parameter | Parameter value |
| Field strength | 1000V/cm |
| Sample volume | 4.0nL |
| Chip type | HS/HSX |
| Electrophoresis buffer type | Metabolites |
| Auxiliary pressurization start time | 1.0min |
| Time delay of hole drilling | 30sec |
| Time of detection | 2.5min |
TABLE 2 Mass spectrometer Primary Scan related parameter settings
TABLE 3 results of standard curves for each amino acid
Example 2
This example refers to the detection method of example 1, except that only one test sample was prepared, with a dilution factor of 150.
Experimental example 1
To illustrate the detection method of the present invention, the content of amino acids in the same cell culture was measured by the detection method of example 1 and conventional high performance liquid chromatography, and the results are shown in Table 4. Wherein the high performance liquid chromatography technology is an amino acid detection kit of Waters company.
TABLE 4 comparison of amino acid detection results in cell cultures
As can be seen from the above table, the detection method of the present invention can effectively detect glutamine, tryptophan and cystine, distinguish glycine from arginine, and truly reflect the content of cystine in the culture. The technical problems that glutamine, tryptophan and cystine cannot be detected by high performance liquid chromatography, and partial amino acids cannot be effectively separated are solved.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (11)
1. A method for detecting an amino acid in a cell culture, comprising the steps of:
centrifuging the cell culture to be detected, taking supernatant, and diluting by 100-fold to 500-fold to obtain a sample to be detected; detecting the sample to be tested by a capillary electrophoresis-mass spectrometry combined method to obtain an ion current chromatogram; acquiring a peak area corresponding to an extracted ion current chromatogram of the amino acid to be detected in the test sample, and substituting the peak area into a standard curve established by a corresponding amino acid standard substance to obtain the content of the amino acid to be detected in the cell culture;
diluting the supernatant and the mother liquor by using a diluent, wherein the diluent is a solution of methanol, water and ammonium acetate at a volume ratio of 50: 1; the concentration of the ammonium acetate solution is 1 mol/L;
in the capillary electrophoresis-mass spectrometry combined method, the mass spectrometry adopts a positive ion mode;
in the capillary electrophoresis-mass spectrometry combined method, an electrophoresis buffer solution comprises methanol, water and formic acid in a volume ratio of 50: 1;
the test conditions of the capillary electrophoresis-mass spectrometry combined method comprise:
capillary electrophoresis parameters: the field strength is 500-1000V/cm, the sample injection amount is 1.0-7.0nL, the chip type is HS or HSX, the auxiliary pressurization starting time is 0.5-1.0min, the multi-hole delay time is 30s, and the detection time is 2.5-3.0 min;
mass spectrum parameters: the scanning range m/z is 70-500, the flow rate of sheath gas is 2L/min, and the temperature of an ion transmission tube is 180-;
the gradient concentration range of the amino acid standard substance solution is 0.1-100 mu M;
the amino acids include lysine, arginine, histidine, glycine, alanine, valine, isoleucine, leucine, serine, threonine, methionine, asparagine, proline, phenylalanine, tryptophan, glutamine, glutamic acid, cysteine, tyrosine, aspartic acid and cystine.
2. The method according to claim 1, wherein the standard curve is established by a method comprising: and carrying out gradient dilution on the amino acid standard substance to obtain an amino acid standard substance solution with gradient concentration, detecting the amino acid standard substance solution with gradient concentration by a capillary electrophoresis-mass spectrometry method to obtain the ion flow chromatogram peak area of the corresponding amino acid under each concentration, and establishing a standard curve of the corresponding amino acid according to the corresponding relation between the extracted ion flow chromatogram peak area and the concentration of the corresponding amino acid.
3. The method of claim 2, wherein the gradient concentration comprises at least five steps of concentration.
4. The method according to claim 3, wherein the amino acid standard is prepared as a stock solution having a concentration of 0.1 to 10mM, and the amino acid standard solution is subjected to gradient dilution to obtain a gradient concentration of the amino acid standard solution.
5. The method according to claim 1, wherein the gradient concentration of the amino acid standard solution is in the range of 0.25 to 62.5. mu.M.
6. The method according to claim 1, wherein the amino acid mixed standard is obtained by mixing an amino acid standard to be tested in advance.
7. The method according to claim 1, wherein the conditions for the centrifugation treatment comprise: the centrifugal speed is 10000-.
8. The method according to claim 7, wherein the centrifugation speed is 120000-14000rpm and the centrifugation time is 12-18 min.
9. The method according to claim 8, wherein the centrifugation speed is 13000rpm and the centrifugation time is 15 min.
10. The method of claim 1, wherein the supernatant is diluted 100-fold to 400-fold.
11. The method according to claim 10, wherein the supernatant is diluted 100-fold to 200-fold.
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