CN115773977A - Protein quantification method based on ES-DMA-CPC particle counting - Google Patents
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Abstract
The invention discloses a protein quantification method based on ES-DMA-CPC particle counting, which comprises the following steps: (1) determination of calibration factors; (2) quantification of protein samples of known calibration factors: obtaining the calibration factor of the protein from the protein standard substance according to the step (1), and analyzing the protein sample with the known calibration factor by ES-DMA-CPC when quantifying the protein sample with the known calibration factor to obtain the total number C of the detected particles CPC Calculating the concentration of the protein sample to be detected according to the calibration factor; (3) A method for quantifying a protein sample for which a calibration factor is unknown. The method of the invention is not influenced by factors such as protein activity, marking efficiency and the like in immune reaction; the method can directly measure the protein content in the solution without carrying out pretreatment such as enzyme digestion and the like.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a protein quantification method based on ES-DMA-CPC particle counting.
Background
Protein is a very important biological macromolecule, the existence and expression of which is closely related to the physiological or pathological changes of organisms. In the past decades, with the important role of protein content determination in clinical fields, especially in disease diagnosis, monitoring and drug development, there has been an increasing demand for accurate quantification of proteins. Unlike the structure of DNA, proteins in different biological environments have different structures and functions, and biological samples have great complexity, so it is necessary to develop a protein quantification method based on different principles.
Particle counting is an important technique in environmental monitoring. In recent years, with the continuous development of the chemical and biological analysis fields, the technology is gradually applied to other industries, and currently, single-particle inductively coupled plasma mass spectrometry and digital enzyme-linked immunosorbent assay are commonly used in the aspect of protein quantification. However, at present, the two methods cannot count the protein directly, the concentration of the target protein is obtained indirectly by quantifying the concentration of the marker after the protein is labeled, and factors such as the labeling efficiency and the protein activity in the process influence the determination of the final result.
The applicant discloses in CN110873683B a method for valuing a high-accuracy protein standard substance based on ES-DMA-CPC, comprising the following steps: 1) Preparing a target protein to be detected; 2) Diluting a protein sample to be detected by using an ammonium acetate buffer solution; 3) Preparing a sucrose solution, injecting a sample into an electric spray aerosol generator through a sample injection pump, generating atomized high-charge liquid drops through a capillary with the inner diameter of 25 mu m, evaporating and drying the liquid drops, and allowing the charged particles to pass through a differential electric mobility nano-column; performing droplet size calculation using a sucrose solution; 4) Protein samples are respectively injected into an electric spray aerosol particle generator through a sample injection pump, atomized high-charge liquid drops are generated through a capillary with the inner diameter of 25 mu m, after the liquid drops are evaporated and dried, the charged particles are analyzed through differential electric mobility, and the particle size distribution of the protein samples is obtained through a particle counter; 5) Obtaining absolute concentrations of the monomer Cp1 and the dimer Cp2 by linear regression fitting, and then determining the trimer concentration Cp3; and summing to determine the original concentration of the target protein sample to be tested.
Although the original concentration of the target protein sample can be calculated by the concentrations of the monomer Cp1, the dimer Cp2 and the trimer Cp3, the method needs the protein particles to form the dimer by inducing aggregation, the protein with large particle size distribution is more difficult to form the dimer along with the increase of the molecular weight of the protein, and the application of the method is limited for the protein which can not form the dimer.
In view of the above, in the field of protein quantitative detection technology, there is a need to develop a protein quantitative method which is not affected by factors such as protein activity and labeling efficiency in immune reaction, is applicable to large molecular weight, and is based on single particle counting.
Disclosure of Invention
In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for protein quantification based on ES-DMA-CPC particle counting. The method adopts the electrospray-differential electromigration-particle counting technology to measure the protein content in the solution, and obtains the corresponding calibration factor through the measurement of the standard substance, thereby realizing the measurement of the content of the same unknown protein sample. In addition, for protein samples without standard substances, the particle size distribution of the protein standard substances detected by an instrument and corresponding calibration factors are subjected to linear regression fitting by measuring the calibration factors of three or more protein standard substances with different particle size distributions, and the proteins with different particle size distributions and the corresponding calibration factors thereof are obtained according to a regression equation, so that the content of different protein samples is measured.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
electrospray-differential electromigration-particle counting (ES-DMA-CPC) is mainly performed by a three-part instrument, including an electrospray aerosol generator (ES), a differential electromigration analyzer (DMA) and a condensed nucleation particle counter (CPC), and a charge neutralizer is usually added between the electrospray aerosol generator and the differential electromigration analyzer to balance the charge distribution of the particles, so that more than 90% of the droplets are only charged with a single charge, and the migration of the particles in the DMA is only related to the size of the particles.
The ES-DMA-CPC particle counting working principle of the invention is as follows: injecting the prepared protein sample into an electric spray aerosol particle generator through a sample injection pump, generating atomized high-charge liquid drops through a capillary with the inner diameter of 25 mu m, after the liquid drops are evaporated and dried, analyzing the particle size distribution and the detected particle number of the protein sample through differential electric mobility by the charged particles, and obtaining the protein according to the particle size distribution and the particle number of the protein sample and the obtained calibration factor, thereby realizing the quantitative analysis of the protein.
The invention provides a protein quantification method based on ES-DMA-CPC particle counting, which comprises the following steps:
(1) Determination of calibration factor
Diluting the protein standard substance by adopting an ammonium acetate buffer solution, injecting a prepared protein sample into an electric spray aerosol particle generator through a sample injection pump, generating atomized high-charge liquid drops through a capillary with the inner diameter of 25 mu m, evaporating and drying the liquid drops, analyzing the charged particles through differential electric mobility, and obtaining the particle size distribution of the protein sample by a particle counter; obtaining the total number of detected particles through integration, and obtaining a calibration factor of the protein standard substance according to the ratio of the total number of particles to the number of particles corresponding to the standard value according to the concentration standard value of the protein standard substance, wherein the formula (1) can be seen specifically:
wherein K is a calibration factor, C CPC The total number of particles obtained by the particle counter through integration,
the number of particles corresponding to the concentration standard value of the protein standard substance;
specific formula (2) for calculating particle number concentration corresponding to concentration standard value of protein standard substance
Wherein the content of the first and second substances,
is the concentration standard value of the protein standard substance, MW is molecular weight, N A Is an Avogastron constant;
(2) Quantification of protein samples for known calibration factors
Obtaining the calibration factor of the protein from the protein standard substance according to the step (1), and analyzing the protein sample with the known calibration factor by ES-DMA-CPC when quantifying the protein sample with the known calibration factor to obtain the total number C of the detected particles CPC And calculating the concentration of the protein sample to be detected according to the calibration factor, wherein the formula (3) can be seen specifically:
wherein, C m Is the mass concentration of the protein sample to be tested, C CPC The total number of particles obtained by integrating the particle counter, K is a calibration factor, m is a dilution multiple, MW is the molecular weight of a sample to be detected, and N A Is the Avogastron constant.
(3) When quantifying the protein sample with unknown calibration factors, selecting three or more protein standard substances with different particle size distributions, determining the calibration factors and the particle size distribution of the protein standard substances according to the step (1), and performing linear regression fitting on the particle size distribution of the protein standard substances detected by an instrument and the corresponding calibration factors to obtain the proteins with different particle size distributions and regression equations of the calibration factors; analyzing a protein sample to be detected with unknown calibration factors through ES-DMA-CPC to obtain the particle size distribution of the protein sample and the total number of detected particles; and (4) fitting according to the particle size and a regression equation to obtain a calibration factor, and calculating by using a formula (3) to obtain the concentration of the protein sample to be detected.
In the step (1), the preparation method of the ammonium acetate buffer solution comprises the steps of preparing a buffer solution with a concentration of 20mmol/L by taking a proper amount of ammonium acetate, filtering, and adjusting the pH value of the solution to 8 by using ammonia water. The protein sample was diluted with the configured ammonium acetate buffer solution to ensure that the diluted sample was predominantly in monomeric form in the ES-DMA-CPC assay.
Wherein, in the step (1), the sample injection pump is set to be in a constant flow mode, and the flow rate is set to be 0.20 mu L/min; the flow rates of dry air and carbon dioxide were 1.2slpm and 0.1slpm respectively; the flow rate of the DMA sheath gas is set to be 6L/min, and the particle size scanning range is set to be a corresponding scanning range; CPC sampling flow rate was set to 0.6L/min.
Compared with the prior art, the invention has the outstanding effects that:
(1) The invention provides a brand-new protein quantification method based on ES-DMA-CPC particle counting, which is not influenced by factors such as protein activity, labeling efficiency and the like in immune reaction.
(2) The method can directly measure the protein content in the solution without performing pretreatment such as enzyme digestion and the like.
(3) The method can directly carry out quantification according to the number of detected particles without inducing to generate dimers, and can be suitable for protein samples with monomer particle sizes in a wider range of 4-30nm and quantitative analysis of proteins which can not form dimers due to larger particle sizes.
The method for protein quantification based on ES-DMA-CPC particle count according to the present invention is further described below with reference to the accompanying drawings and the specific examples.
Drawings
FIG. 1 is a linear regression fit of calibration factors to the particle size of protein monomers.
Detailed Description
Example 1
The protein quantification method based on the ES-DMA-CPC particle counting is used for quantitatively determining the bovine serum albumin, and the method is proved to be capable of realizing accurate quantification of the protein.
1. Laboratory instruments and reagents:
FLOW-EZ microfluidic constant FLOW pump (fluent, france); electrospray aerosol particle generator (TSI corporation, usa, model 3482); electrostatic classifier (TSI corporation, usa, model 3082); a nano water-based condensed nucleus particle counter (TSI corporation, usa, model 3788); silica capillary (inner diameter 25 μm, length 4.1cm, TSI Corp., USA).
Ammonium acetate (chromatographic grade, purity greater than or equal to 99.0%, honeywell, germany); bovine serum albumin standard (national institute of metrology science, NIM-RM 3627-11); the experimental water was ultrapure water.
2. Sample preparation and standard solution preparation:
preparing a buffer solution of 20mmol/L with a proper amount of ammonium acetate, filtering, and adjusting the pH of the solution to 8 with ammonia water. Bovine serum albumin solution was diluted to 0.10mg/mL with buffer. The protein sample was diluted with the configured ammonium acetate buffer solution to ensure that the diluted sample was predominantly in monomeric form in the ES-DMA-CPC assay.
3. Experimental conditions and parameters:
the experimental process is as follows: firstly, injecting a sample into an electrospray aerosol particle generator through a FLOW-EZ microfluid constant FLOW pump, generating atomized high-charge liquid drops through a capillary with the inner diameter of 25 mu m, evaporating and drying the liquid drops, enabling charged particles to pass through a DMA nano-column, obtaining the particle size distribution of a protein sample through a particle counter, and obtaining the total number of particles in the solution through integration.
The sample injection speed of the microfluid constant flow pump is set to be in a constant flow mode, and the flow speed is set to be 0.20 mu L/min. Dry air and carbon dioxide gas were used as carrier gases at 1.2slpm and 0.1slpm respectively. The flow rate of the DMA sheath gas is set to be 6L/min, and the particle size scanning range is set to be a corresponding scanning range (3.16-107.5 nm). The CPC sampling flow rate was set to 0.6L/min.
4. Determination of protein content of the sample:
(1) Determination of calibration factor:
the calibration factor for this sample was calculated by using bovine serum albumin at a mass concentration of 0.10 mg/mL:
wherein C CPC The total number of particles obtained by the particle counter through integration,
and the number of particles is corresponding to the concentration standard value of the protein standard substance. The calibration factor K was calculated to be 4.19E-08.
(2) And (3) determination of sample content:
C m is the mass concentration of the protein in the sample solution to be tested, C CPC The total number of particles obtained by integrating the particle counter, K is a calibration factor, m is a dilution multiple, MW is the molecular weight of a sample to be detected, and N A Is the Avogastron constant. Where m =81, mw =66kda, specific measurements are shown in table 1.
TABLE 1 bovine serum albumin quantification results
The standard value of the used bovine serum albumin standard substance is 8.6mg/mL, and the uncertainty is 0.6mg/mL, while the measurement result of protein quantification based on ES-DMA-CPC particle counting by adopting the method is in the uncertainty range of the standard value of the bovine serum albumin standard substance, which proves that the method can accurately quantify the protein.
Example 2
The quantitative determination method of the protein based on the ES-DMA-CPC particle counting is used for quantitatively determining the humanized CD20IgG1 kappa monoclonal antibody, and the method is proved to be capable of accurately quantifying the protein.
The bovine serum albumin standard substance in example 1 was replaced with a humanized CD20IgG1 κ type monoclonal antibody standard substance (chinese institute of metrology science, GBW (E) 091164), which was diluted to 0.10mg/mL with a buffer solution under the same experimental conditions as in example 1.
The calibration factor K of the humanized CD20IgG1 κ monoclonal antibody was found to be 7.44E-08 by formula (1), the dilution factor m =217 of the sample, the molecular weight MW =150kDa, the mass concentration of the sample was found by formula (2), and the specific measurement results are shown in table 2.
TABLE 2IgG 1. Kappa. Monoclonal antibody quantitation results
The standard value of the humanized CD20IgG1 kappa monoclonal antibody standard substance is 21.7mg/mL, and the uncertainty is 1.7mg/mL. The measurement result of the method is in the uncertainty range of the standard value of the humanized CD20IgG1 kappa monoclonal antibody standard substance, and the method is proved to be capable of accurately quantifying the protein.
Example 3
The protein quantification method based on the ES-DMA-CPC particle counting is used for quantitatively determining the bovine serum albumin with different dilution times, and an unknown sample is simulated by the bovine serum albumin with different dilution times, so that the method can be used for quantifying the protein sample with known calibration factors.
The amounts of bovine serum albumin at different concentrations were determined using the calibration factors determined in example 1, keeping all experimental conditions and parameters in agreement with example 1. Bovine serum albumin was diluted 57-fold and 35-fold with buffer to final concentrations of about 0.15 and 0.25mg/mL, and the results of the measurement and the quantification of bovine serum albumin at different dilutions are shown in Table 3.
TABLE 3 results of quantification of bovine serum albumin at different dilution ratios
The standard value of the used bovine serum albumin standard substance is 8.3mg/mL, and the uncertainty is 0.6mg/mL, and the measurement result obtained by adopting the method is in the uncertainty range of the standard value of the bovine serum albumin standard substance, so that the method can realize the content determination of the similar sample through the calibration factor obtained by the standard substance.
Example 4
The protein quantification method based on the ES-DMA-CPC particle counting is used for quantitatively measuring the humanized CD20IgG1 kappa type monoclonal antibody with different dilution times, and simulating an unknown sample by the humanized CD20IgG1 kappa type monoclonal antibody with different dilution times, thereby proving that the method can be used for quantifying the protein sample with known calibration factors.
The concentrations of the IgG1 kappa monoclonal antibodies at different concentrations were measured using the calibration factors determined in example 2, keeping all experimental conditions and parameters in agreement with example 2. The IgG 1. Kappa. Monoclonal antibodies were diluted 630-fold and 524-fold with buffer to final concentrations of about 0.035 and 0.040mg/mL, and the results of the measurement and the quantification of the IgG 1. Kappa. Monoclonal antibodies at different dilutions are shown in Table 4.
TABLE 4 quantification of IgG 1. Kappa. Monoclonal antibodies at different dilution ratios
The standard value of the humanized CD20IgG1 kappa type monoclonal antibody standard substance is 21.7mg/mL, the uncertainty is 1.7mg/mL, and the measurement result obtained by the method is in the uncertainty range of the humanized CD20IgG1 kappa type monoclonal antibody standard value, so that the method can realize the content determination of the similar samples through the calibration factor obtained by the standard substance.
Example 5
Protein quantification method based on ES-DMA-CPC particle counting the NIST monoclonal antibody (NIST 8671) was quantitated, demonstrating that this method can quantify protein samples for which calibration factors are unknown.
1) Four protein standard substances with different particle size distributions or protein samples with known accurate concentrations, including horse heart myoglobin standard substance (Chinese institute of metrology science, NIM-RM 3624), bovine serum albumin (NIM-RM 3627-11), humanized CD20IgG1 kappa monoclonal antibody (GBW (E) 091164), and human fibrinogen (16088, cayman), were selected, and the calibration factors for the four proteins were obtained according to the calibration factor determination method shown in example 1, thereby obtaining linear regression curves between the calibration factors and the measured particle sizes d of the corresponding protein monomers. The specific results are shown in FIG. 1.
2) Diluting NIST 8671 with buffer solution to 0.10mg/mL, performing ES-DMA-CPC analysis to obtain a monomer particle size of the protein of 9.82nm, and calculating to obtain a calibration factor corresponding to the NIST 8671 protein of 7.29E-08 according to an obtained linear regression equation.
3) And calculating the calibration factor of the NIST 8671d protein according to the particle number obtained by ES-DMA-CPC analysis and a linear regression equation, and obtaining the mass concentration of the NIST 8671 protein sample according to the following formula, wherein the specific measurement results are shown in Table 5.
Quantitative results of Table 5NIST 8671
The result of the isotope dilution mass spectrometry on the NIST 8671 is 8.71mg/mL, and the relative error of the result and the average value obtained by the method is about 1 percent, which proves that the method can accurately quantify the protein sample of unknown calibration factors according to the known calibration factors and the protein with the particle size distribution.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (5)
1. A method for protein quantification based on ES-DMA-CPC particle count, comprising the steps of:
(1) Determination of calibration factor
Diluting a protein standard substance by adopting an ammonium acetate buffer solution, injecting a prepared protein sample into an electric spray aerosol particle generator through a sample injection pump, generating atomized high-charge liquid drops through a capillary with the inner diameter of 25 mu m, evaporating and drying the liquid drops, analyzing the charged particles through differential electric mobility, and obtaining the particle size distribution of the protein sample by a particle counter; obtaining the total number of detected particles through integration, and obtaining a calibration factor of the protein standard substance according to the concentration standard value of the protein standard substance and the ratio of the total number of the particles to the number of the particles corresponding to the standard value, which can be seen in a formula (1):
wherein K is a calibration factor, C CPC The total number of particles obtained by the particle counter through integration,
the number of particles corresponding to the concentration standard value of the protein standard substance;
the specific formula (2) for calculating the particle number concentration corresponding to the concentration standard value of the protein standard substance is as follows:
wherein the content of the first and second substances,
is the concentration standard value of the protein standard substance, MW is molecular weight, N A Is an Avogastron constant;
(2) Quantification of protein samples for known calibration factors
Obtaining the calibration factor of the protein from the protein standard substance according to the step (1), and analyzing the protein sample with the known calibration factor by ES-DMA-CPC when quantifying the protein sample to obtain the total number C of the detected particles CPC According to the calibration factorAnd (3) calculating to obtain the concentration of the protein sample to be detected, wherein the formula can be seen specifically as follows:
wherein, C m Is the mass concentration of the protein sample to be tested, C CPC The total number of particles obtained by integrating the particle counter, K is a calibration factor, m is a dilution multiple, MW is the molecular weight of a sample to be detected, N A Is an avogalois constant.
2. The method of claim 1 for protein quantification based on the counting of ES-DMA-CPC particles, characterized in that: also included is the quantification of protein samples of unknown calibration factors, specifically: when quantifying a protein sample with unknown calibration factors, selecting three or more protein standard substances with different particle size distributions, determining the calibration factors and the particle size distribution of the protein standard substances according to the step (1), and performing linear regression fitting on the particle size distribution of the protein standard substances detected by an instrument and the corresponding calibration factors to obtain the proteins with different particle size distributions and regression equations of the calibration factors thereof; analyzing a protein sample to be detected with unknown calibration factors through ES-DMA-CPC to obtain the particle size distribution of the protein sample and the total number of detected particles; and (4) fitting according to the particle size and a regression equation to obtain a calibration factor, and calculating by using a formula (3) to obtain the concentration of the protein sample to be detected.
3. The method of claim 1 for protein quantification based on the counting of ES-DMA-CPC particles, characterized in that: in the step (1), the preparation method of the ammonium acetate buffer solution comprises the steps of preparing a buffer solution with 20mmol/L of ammonium acetate by taking a proper amount of ammonium acetate, filtering, and adjusting the pH value of the solution to 8 by using ammonia water.
4. The method of claim 3 for protein quantification based on the counting of ES-DMA-CPC particles, characterized in that: in the step (1), the protein sample is diluted by using the prepared ammonium acetate buffer solution, and the diluted sample is ensured to exist in a monomer form in the ES-DMA-CPC analysis.
5. Method for protein quantification based on the counting of ES-DMA-CPC particles according to claim 4, characterized in that: in the step (1), the sample injection pump is set to be in a constant flow mode, and the flow rate is set to be 0.20 mu L/min; the flow rates of the dry air and carbon dioxide were 1.2slpm and 0.1slpm, respectively; setting the flow rate of the DMA sheath gas to be 6L/min, and setting the particle size scanning range to be a corresponding scanning range; CPC sampling flow rate was set to 0.6L/min.
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