CE-MS detection method for host cell protein coverage rate detection

Abstract

The invention discloses a CE-MS detection method for host cell protein coverage rate detection, which comprises the following steps: centrifuging host cells to be detected to obtain supernate, and diluting the supernate to a set concentration to obtain a test sample; detecting the sample to be tested by a capillary electrophoresis-mass spectrometry method to obtain an ion flow chromatogram; acquiring the peak area corresponding to the extracted ion flow chromatogram of the host cell to be tested in the sample to be tested, and comparing with the existing database to obtain the quantity and the type of the protein in the host cell to be tested; dissecting the to-be-detected host cell to obtain the distribution condition of internal proteins of the to-be-detected host cell, and then obtaining the surface protein coverage rate of the to-be-detected host cell according to the distribution condition of the proteins and the quantity and the variety of the proteins in the to-be-detected host cell; the sample pretreatment method adopted by the invention is simple in process, low in cost and low in sample demand.

Description

Technical Field

[0001] The present invention relates to the technical field related to analysis and detection, and specifically to a CE-MS detection method for detecting the coverage of host cell proteins. Background Art

[0002] Capillary electrophoresis-mass spectrometry (CE-MS) technology has the advantages of small sample volume, high separation efficiency, fast analysis speed, and high sensitivity. It has gradually attracted attention in the field of proteomics research, and plays an important role in the separation and identification of peptides, proteins, and protein complexes with its unique advantages. It has been widely used in the field of separation and analysis research. At present, the main method for determining the coverage of host cell proteins is high performance liquid chromatography. The traditional high performance liquid chromatography has a complex pretreatment process, requires cumbersome derivatization labeling, is time-consuming and labor-intensive, and the derivatization reagents are highly toxic. The culture matrix interferes seriously, and the single sample analysis test time is long, resulting in a small amount of sample detection and analysis per unit time, low efficiency, and low detection throughput and automation. Summary of the invention

[0003] In order to solve the existing problems in the prior art, the main method for determining the coverage of host cell proteins is high performance liquid chromatography. The traditional high performance liquid chromatography has a complex pre-treatment process, requires cumbersome derivatization labeling, is time-consuming and labor-intensive, has high toxicity of the derivatization reagent, has serious interference from the culture matrix, and has a long single sample analysis test time, resulting in a small amount of samples to be analyzed per unit time, low efficiency, and low detection throughput and automation. The present invention provides a host cell protein coverage detection CE-MS detection method.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] The present invention discloses a CE-MS detection method for detecting the coverage of host cell proteins. The method comprises the following steps: centrifuging host cells to be tested to obtain a supernatant, and diluting the supernatant to a set concentration to obtain a test sample; detecting the test sample by capillary electrophoresis-mass spectrometry to obtain an ion flow chromatogram; obtaining the peak area corresponding to the extracted ion flow chromatogram of the host cells to be tested in the test sample, and comparing the peak area with an existing database to obtain the number and type of proteins in the host cells to be tested; dissecting the host cells to be tested to obtain the distribution of internal proteins in the host cells to be tested, and then obtaining the surface protein coverage of the host cells to be tested according to the distribution of the proteins combined with the number and type of proteins in the host cells to be tested;

[0006] The supernatant and the mother liquor are diluted with a diluent, and the diluent used for dilution is:

[0007] A mixture of acetonitrile: water: formic acid: 1.1 mol / L ammonium acetate solution, with a volume ratio of 45-50: 45-49.5: 0.5-10;

[0008] In the capillary electrophoresis-mass spectrometry method, the parameters of the capillary electrophoresis instrument are as follows: injection volume is 3.0-4.0 nL, auxiliary pressurization start time is 1-2 min, detection time is 4-6 min, HR or HRX chip;

[0009] The mass spectrometry conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: positive ion scanning mode, drying gas flow rate of 5L / min, drying temperature of 340°C, fragmentation voltage of 360V, splitter voltage of 68V, MS scanning range of m / z750-3300, and scanning rate of 1spectrum / s.

[0010] As a preferred technical solution of the present invention, the gradient concentration of the test sample in step 1 is 3000, 2000, 10 / 0, 500, 250, and 125 μg / mL, and the peak area is integrated and calculated using Chemstation software.

[0011] As a preferred technical solution of the present invention, the centrifugal speed is 120000-14000 rpm, and the centrifugal time is 12-18 min.

[0012] As a preferred technical solution of the present invention, the ion source conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: an electroosmotic flow-driven coaxial sheath liquid electrospray ion source, the sheath liquid is a mixed aqueous solution containing 0.4% by volume formic acid and 8% isopropanol.

[0013] As a preferred technical solution of the present invention, the ion source conditions in the capillary electrophoresis-mass spectrometry online analysis and detection include: the material of the electrospray needle is borosilicate glass; the spray voltage is 2.2 kV, the distance between the electrospray needle and the mass spectrometer is 5.0 mm, and the distance between the capillary and the spray needle is 0.5 mm.

[0014] The beneficial effects of the present invention are:

[0015] This host cell protein coverage detection CE-MS detection method directly performs online analysis of capillary electrophoresis and mass spectrometry after mixing and diluting the host cells with the diluent (the sample is first separated by CE, and the separated protein is ionized and becomes ions, which are directly subjected to mass spectrometry analysis and detection). The effective separation of various proteins in the host cell is achieved by capillary electrophoresis separation. The online analysis technology of capillary electrophoresis and mass spectrometry is used to ionize each protein through a mass spectrometry ion source for mass spectrometry analysis, which can effectively reduce the sample demand, reduce the difficulty of sample pretreatment, increase the online separation degree of protein, accurately identify the number and type of host cells, and thus facilitate the calculation of the host cell protein coverage. The sample pretreatment method adopted by the present invention has a simple process, low cost, and small sample demand. At the same time, the capillary electrophoresis-time of flight mass spectrometry technology of the CE-MS combined ion source can realize efficient separation and high-sensitivity detection of proteins in the host cell, and realize efficient and high-accuracy identification of proteins in the host cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0017] In the attached picture:

[0018] Figure 1 It is a schematic diagram of the process of a CE-MS detection method for detecting host cell protein coverage of the present invention. DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0020] Example: Figure 1 As shown, the present invention provides a CE-MS detection method for detecting the coverage of host cell proteins, comprising the following steps:

[0021] The host cells to be tested are centrifuged to obtain a supernatant, and the supernatant is diluted to a set concentration to obtain a test sample; the test sample is detected by capillary electrophoresis-mass spectrometry to obtain an ion flow chromatogram; the peak area corresponding to the extracted ion flow chromatogram of the host cells to be tested in the test sample is obtained, and the number and type of proteins in the host cells to be tested are obtained by comparing with an existing database; the host cells to be tested are dissected to obtain the distribution of internal proteins in the host cells to be tested, and then the surface protein coverage of the host cells to be tested is obtained according to the distribution of proteins combined with the number and type of proteins in the host cells to be tested;

[0022] The supernatant and the mother liquor are diluted with a diluent, and the diluent used for dilution is:

[0023] A mixture of acetonitrile: water: formic acid: 1.1 mol / L ammonium acetate solution, with a volume ratio of 45-50: 45-49.5: 0.5-10;

[0024] In the capillary electrophoresis-mass spectrometry method, the parameters of the capillary electrophoresis instrument are as follows: the injection volume is 3.0-4.0 nL, the auxiliary pressurization start time is 1-2 min, the detection time is 4-6 min, and the HR or HRX chip is used; the host cell protein coverage detection CE-MS detection method directly performs online analysis of capillary electrophoresis and mass spectrometry after the host cells are mixed and diluted with the diluent (the sample is first separated by CE, and the separated protein is ionized to become ions, which are directly subjected to mass spectrometry analysis and detection). The effective separation of various proteins in the host cells is achieved through capillary electrophoresis separation. The online analysis technology of capillary electrophoresis and mass spectrometry is used to ionize each protein through a mass spectrometry ion source for mass spectrometry analysis, which can effectively reduce the sample demand, reduce the difficulty of sample pre-treatment, increase the online separation degree of proteins, and accurately identify the number and type of host cells, so that the host cell protein coverage can be easily calculated. The sample pretreatment method adopted in the present invention is simple in process, low in cost, and requires little sample. At the same time, the capillary electrophoresis-time-of-flight mass spectrometry technology of the CE-MS combined ion source is adopted to realize the efficient separation and high-sensitivity detection of proteins in the host cells, and realize the efficient and high-accuracy identification of proteins in the host cells.

[0025] The mass spectrometry conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: positive ion scanning mode, drying gas flow rate of 5 L / min, drying temperature of 340°C, fragmentation voltage of 360V, splitter voltage of 68V, MS scanning range of m / z750-3300, and scanning rate of 1 spectrum / s;

[0026] The gradient concentration of the test sample in step 1 is 3000, 2000, 10 / 0, 500, 250, and 125 μg / mL, and the peak area is integrated and calculated using Chemstation software.

[0027] The centrifugal speed is 120000-14000rpm, and the centrifugal time is 12-18min.

[0028] The ion source conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: an electroosmotic flow-driven coaxial sheath liquid electrospray ion source, the sheath liquid is a mixed aqueous solution containing 0.4% by volume formic acid and 8% by volume isopropanol,

[0029] The ion source conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: the material of the electrospray needle is borosilicate glass; the spray voltage is 2.2kV, the distance between the electrospray needle and the mass spectrometer is 5.0mm, and the distance between the capillary and the spray needle is 0.5mm.

[0030] This host cell protein coverage detection CE-MS detection method directly performs online analysis of capillary electrophoresis and mass spectrometry after mixing and diluting the host cells with the diluent (the sample is first separated by CE, and the separated protein is ionized and becomes ions, which are directly subjected to mass spectrometry analysis and detection). The effective separation of various proteins in the host cell is achieved by capillary electrophoresis separation. The online analysis technology of capillary electrophoresis and mass spectrometry is used to ionize each protein through a mass spectrometry ion source for mass spectrometry analysis, which can effectively reduce the sample demand, reduce the difficulty of sample pretreatment, increase the online separation degree of protein, accurately identify the number and type of host cells, and thus facilitate the calculation of the host cell protein coverage. The sample pretreatment method adopted by the present invention has a simple process, low cost, and small sample demand. At the same time, the capillary electrophoresis-time of flight mass spectrometry technology of the CE-MS combined ion source can realize efficient separation and high-sensitivity detection of proteins in the host cell, and realize efficient and high-accuracy identification of proteins in the host cell.

[0031] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A host cell protein coverage detection CE-MS detection method, characterized in that: The following steps are involved: The host cells to be tested are centrifuged to obtain a supernatant, and the supernatant is diluted to a set concentration to obtain a test sample; the test sample is detected by capillary electrophoresis-mass spectrometry to obtain an ion flow chromatogram; the peak area corresponding to the extracted ion flow chromatogram of the host cells to be tested in the test sample is obtained, and the number and type of proteins in the host cells to be tested are obtained by comparing with an existing database; the host cells to be tested are dissected to obtain the distribution of internal proteins in the host cells to be tested, and then the surface protein coverage of the host cells to be tested is obtained according to the distribution of proteins combined with the number and type of proteins in the host cells to be tested; The supernatant and the mother liquor are diluted with a diluent, and the diluent used for dilution is: A mixture of acetonitrile: water: formic acid: 1.1 mol / L ammonium acetate solution, with a volume ratio of 45-50: 45-49.5: 0.5-10; In the capillary electrophoresis-mass spectrometry method, the parameters of the capillary electrophoresis instrument are as follows: injection volume is 3.0-4.0 nL, auxiliary pressurization start time is 1-2 min, detection time is 4-6 min, HR or HRX chip; The mass spectrometry conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: positive ion scanning mode, drying gas flow rate of 5L / min, drying temperature of 340°C, fragmentation voltage of 360V, splitter voltage of 68V, MS scanning range of m / z750-3300, and scanning rate of 1spectrum / s.

2. A host cell protein coverage detection CE-MS detection method according to claim 1, characterized in that: The gradient concentration of the test sample in step 1 is 3000, 2000, 10 / 0, 500, 250, and 125 μg / mL, and the peak area is integrated and calculated using Chemstation software.

3. A host cell protein coverage detection CE-MS detection method according to claim 2, characterized in that: The centrifugal speed is 120000-14000rpm, and the centrifugal time is 12-18min.

4. A host cell protein coverage detection CE-MS detection method according to claim 3, characterized in that: The ion source conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: an electroosmotic flow-driven coaxial sheath liquid electrospray ion source, and the sheath liquid is a mixed aqueous solution containing 0.4% by volume formic acid and 8% isopropanol.

5. A host cell protein coverage detection CE-MS detection method according to claim 1, characterized in that: The ion source conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: the material of the electrospray needle is borosilicate glass; the spray voltage is 2.2kV, the distance between the electrospray needle and the mass spectrometer is 5.0mm, and the distance between the capillary and the spray needle is 0.5mm.

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