A liquid chromatography method for simultaneous detection of multiple proteins

Abstract

The application provides a liquid chromatography analysis method for simultaneously detecting multiple proteins, which adopts anion liquid chromatography with non-porous resin as a stationary phase, a Tris gradient eluent phase containing Cl ‑ , the non-porous resin is bonded with a hydrophilic polymer nanolayer on the surface, the nanolayer is modified with a quaternary ammonium group, the proteins have different isoelectric points, and the pH of the eluent phase is greater than the isoelectric points of the proteins. The application can simultaneously separate and purify multiple proteins through one-time anion liquid chromatography, and can approximately achieve baseline separation. The overall method is simple, economical, efficient, and environmentally friendly for the mobile phase, and is suitable for the separation of proteins with different isoelectric points, and is particularly suitable for the detection and separation of proteins such as albumin, transferrin, retinol binding protein, beta 2 microglobulin and alpha 1 microglobulin in urine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein purification, and in particular to a liquid chromatography analysis method for simultaneously detecting multiple proteins. BACKGROUND

[0002] High Performance Liquid Chromatography (HPLC) is also called high pressure, high speed, and modern liquid chromatography, commonly known as high performance liquid chromatography. It is a high-efficiency and rapid separation method of compounds established in the mid-1960s, and has been widely used in protein separation and purification since the late 1970s. It has become one of the most effective methods for separating and purifying proteins. Almost all proteins can be separated and purified by different HPLC methods according to their property differences (isoelectric point, hydrophobicity, molecular weight, charge distribution, etc.). Generally, according to the mechanism of the separation process, liquid chromatography can be divided into adsorption chromatography, partition chromatography, ion exchange chromatography, and volume exclusion chromatography.

[0003] HPLC is mainly used for the separation and purification of single proteins, and is less used for the simultaneous separation and purification of multiple proteins, especially for the separation and purification of albumin, transferrin, retinol binding protein, beta 2 microglobulin, alpha 1 microglobulin, and other proteins in urine. At present, the technologies that can analyze the above-mentioned multiple proteins mainly include electrophoresis technology, highly integrated protein biochemical analysis technology, and chemiluminescence technology. Among them, the electrophoresis technology is complicated to operate, has many interference factors, the results are unstable, and cannot be quantitatively analyzed. The pseudo-turbidity of the biochemical analysis technology can cause inaccurate quantitative results, and different types of proteins usually need to be detected separately. The chemiluminescence technology is complex and has high cost. Therefore, it is particularly important to explore a method for efficiently and economically separating and purifying multiple proteins at the same time, especially for the separation and purification of albumin, transferrin, retinol binding protein, beta 2 microglobulin, and alpha 1 microglobulin. HPLC has the advantages of being able to sample multiple proteins at one time, having low instrument cost, and being able to directly detect immunologically active and non-immunologically active proteins, and thus is considered as a potential effective method for simultaneously separating and purifying multiple proteins. SUMMARY

[0004] The present application provides a liquid chromatography analysis method for simultaneously detecting multiple proteins, which can simply and efficiently separate proteins with different isoelectric points at the same time, especially for the separation and purification of albumin, transferrin, retinol binding protein, beta 2 microglobulin, and alpha 1 microglobulin.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: a non-porous resin is used as the stationary phase, and a Cl -The method simultaneously detects a plurality of proteins with different isoelectric points by anion liquid chromatography with a Tris gradient elution phase, wherein the non-porous resin is surface-bonded with a hydrophilic polymer nanolayer, and the nanolayer is modified with a quaternary ammonium group, and the pH of the elution phase is greater than the isoelectric points of the proteins.

[0006] Further, the isoelectric points of the proteins are all less than 8, and the proteins are one or more of albumin, transferrin, retinol binding protein, beta 2 microglobulin, and alpha 1 microglobulin.

[0007] Further, the non-porous resin is polystyrene / divinylbenzene (PS / DVB) resin particles with a particle size of 1.7-10 μm.

[0008] Further, the non-porous polystyrene / divinylbenzene (PS / DVB) resin particles have a particle size of 5 μm.

[0009] Further, the elution phase is a mixed phase composed of phase A and phase B, the phase A is 20 mM Tris-HCl, pH=8.0, and the phase B is 20 mM Tris-HCl+500 mM NaCl, pH=8.0.

[0010] Further, the elution mode of the elution phase is gradient elution, and the flow rate is 0.5 mL / min.

[0011] Further, the gradient elution conditions are as follows:

[0012] 0-0.5 min 0%B

[0013] 0.5-7.0 min 0-100%B

[0014] 7.0-7.1 min 100-0%B

[0015] 7.1-15 min 0%B.

[0016] Further, the anion liquid chromatography uses a chromatographic column of Proteomix SAX NP5, 4.6×50 mm, Sepax.

[0017] Further, the ultraviolet detection wavelength is 210 nm and 280 nm.

[0018] Further, the analysis method specifically includes the following steps:

[0019] Protein sample configuration: configure a mixed sample of albumin, transferrin, retinol binding protein, beta 2 microglobulin, and alpha 1 microglobulin;

[0020] Chromatographic column: Proteomix SAX NP5, 4.6 x 50 mm, Sepax, anion exchange analytical column;

[0021] Mobile phase A: 20 mM Tris-HCl, pH = 8.0;

[0022] Mobile phase B: 20 mM Tris-HCl, 500 mM NaCl, pH = 8.0;

[0023] Elution gradient:

[0024] 0-0.5 min 0%B

[0025] 0.5-7.0 min 0-100%B

[0026] 7.0-7.1 min 100-0%B

[0027] 7.1-15 min 0%B

[0028] Detector: UV detection wavelength 210, 280 nm;

[0029] Column temperature: room temperature;

[0030] Flow rate: 0.5 mL / min;

[0031] Injection volume: 5 μL.

[0032] Mechanism of the present application: The present application adopts an anion high performance liquid chromatography analysis method, and separates a plurality of proteins with different isoelectric points (pI) by regulating the strength of competitive binding among the stationary phase filler, the proteins, and the eluent. In the present application, the stationary phase filler is a non-porous polystyrene / divinylbenzene (PS / DVB) resin particle, which contains a nanohydrophilic layer on its surface, and a three-dimensional strong anion exchange group, quaternary amine group, is bonded on the hydrophilic layer. The isoelectric points of the proteins involved in the separation are all different and less than 8, and the pH of the eluent is adjusted to be greater than the isoelectric point of the proteins (such as pH = 8.0), so that these proteins are negatively charged and are adsorbed by the positively charged stationary phase. Then in the gradient elution process, the proportion of mobile phase B is increased, and the Cl - ions in the mobile phase B compete with the five proteins to bind to the stationary phase, so that the five proteins will flow out in order from weak to strong binding force, completing the separation.

[0033] Beneficial effects: the present application can simultaneously separate and purify multiple proteins by one anion liquid chromatography, and approximate baseline separation is achieved, the overall method is simple, economical and efficient, the mobile phase is environmentally friendly, and the method is suitable for the separation of proteins with different isoelectric points, and is particularly suitable for the detection and separation of proteins such as albumin, transferrin, retinol binding protein, beta 2 microglobulin and alpha 1 microglobulin in urine. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a spectrum of a detection wavelength of 210 nm;

[0035] Figure 2 is an amplified spectrum of a detection wavelength of 210 nm;

[0036] Figure 3 is a spectrum of a detection wavelength of 280 nm;

[0037] Figure 4 is an amplified spectrum of a detection wavelength of 280 nm. EMBODIMENT

[0038] The present application provides a liquid chromatography analysis method for simultaneously detecting multiple proteins, and the detection method is anion high performance liquid chromatography: Proteomix SAX NP5, 4.6*50 mm, Sepax, anion exchange analysis column (Suzhou Saifen Science and Technology Co., Ltd., model 403NP5-4605) is used, the mobile phase is a mixed phase of phase A and phase B (phase A: 20 mM Tris-HCl, pH=8.0; phase B: 20 mM Tris-HCl+500 mM NaCl, pH=8.0), the elution mode is gradient elution, the flow rate is 0.5 mL / min, the column temperature is room temperature, the ultraviolet detection wavelength is 210 nm and 280 nm, and the five proteins of albumin, transferrin, retinol binding protein, beta 2 microglobulin and alpha 1 microglobulin can be simultaneously separated and purified by anion high performance liquid chromatography.

[0039] The anion exchange analysis column Proteomix SAX NP5 adopts a rigid, spherical, high cross-linking degree, non-porous polystyrene / divinylbenzene (PS / DVB) resin particle as the filler, the particle size can be 1.7 μm, 3 μm, 5 μm, 10 μm, preferably 5 μm, and the surface of the resin is bonded with a highly hydrophilic nanoscale thickness neutral polymer thin layer, the hydrophobic PS / DVB resin surface is completely covered with this hydrophilic material, thereby eliminating the irreversible adsorption of PS / DVB to biological molecules, ensuring that it has very high separation efficiency and biological sample recovery rate; the surface of the polymer thin layer is chemically bonded with strong anion exchange functional groups (quaternary amine groups) in a dense and uniform manner. This stationary phase filler has three characteristics: first, the nanoscale thickness of the hydrophilic layer completely eliminates non-specific interactions between the carrier and the biological sample; second, the non-porous particle structure minimizes the lateral diffusion of the sample while inhibiting its diffusion into the interior of the filler particles; third, using the unique chemical bonding technology of Saifeng, three-dimensional strong anion exchange groups are bonded on the hydrophilic layer, which can provide the best resolution and separation efficiency for proteins, oligonucleotides, carbohydrates and polypeptides, etc.

[0040] Since the stationary phase filler is positively charged, and the isoelectric points (pI) of albumin, transferrin, retinol binding protein, beta 2 microglobulin, alpha 1 microglobulin are all less than 8, under the environment of pH=8.0, these proteins release protons and become negatively charged, so there is an electrostatic interaction between the stationary phase and the proteins. Because the isoelectric points of the five proteins are different, the strength of the electrostatic interaction between the stationary phase and the five proteins is also different. During gradient elution, the proportion of mobile phase B is constantly increasing, and the Cl - ions in the mobile phase B compete with the five proteins to bind to the stationary phase, so that the five proteins will flow out in order from weak to strong according to the binding force, completing the separation. Under normal circumstances, the order of protein flow-out is from high to low according to the isoelectric point.

[0041] Table 1

[0042]

[0043] The technical solutions of the embodiments of the present application will be described below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0044] Embodiment 1

[0045] 1), configure protein samples

[0046] Take 20 μL of 1.0 mg / mL of β2 microglobulin (B2M) and retinol binding protein (RBP4), 10 μL of 2.0 mg / mL of transferrin (TRF), 20 μL of 2.0 mg / mL of albumin (HSA), 40 μL of 2.0 mg / mL of α1 microglobulin (A1M) and 110 μL of dilution solvent ultrapure water respectively using a pipette to obtain mixed sample 1, and the concentrations of the diluted proteins are: the concentrations of β2 microglobulin (B2M), transferrin (TRF) and retinol binding protein (RBP4) are 0.1 mg / mL, the concentration of albumin (HSA) is 0.2 mg / mL, and the concentration of α1 microglobulin (A1M) is 0.4 mg / mL.

[0047] 2) Configure mobile phase A

[0048] Weigh 2.4228 g of Tris and dissolve it in 1 L of ultrapure water, adjust the pH to 8.0 using hydrochloric acid (HCl), and then filter it through a 0.45 μm hydrophilic filter membrane to obtain mobile phase A.

[0049] 3) Configure mobile phase B

[0050] Weigh 2.4228 g of Tris and 29.2214 g of sodium chloride (NaCl) and dissolve them in 1 L of ultrapure water, adjust the pH to 8.0 using hydrochloric acid (HCl), and then filter it through a 0.45 μm hydrophilic filter membrane to obtain mobile phase B.

[0051] 4) High performance liquid chromatography conditions:

[0052] Chromatographic column: Proteomix SAX NP5, 4.6x50 mm, Sepax, anion exchange analysis column

[0053] Mobile phase A: 20 mM Tris-HCl, pH=8.0

[0054] Mobile phase B: 20 mM Tris-HCl, 500 mM NaCl, pH=8.0

[0055] Elution gradient:

[0056] 0-0.5min 0%B

[0057] 0.5-7.0min 0-100%B

[0058] 7.0-7.1min 100-0%B

[0059] 7.1-15min 0%B

[0060] Detector: UV detection wavelength 210, 280 nm

[0061] Column temperature: room temperature

[0062] Injection volume: 5 μL.

[0063] 5 μL of the above mixed sample 1 solution was taken and separated and purified by Proteomix SAX NP5, 4.6x50 mm, Sepax, anion exchange analysis column (Suzhou Saifen Science and Technology Co., Ltd., model 403NP5-4605), the mobile phase was a mixture of phase A and phase B (phase A: 20 mM Tris-HCl, pH=8.0; phase B: 20 mM Tris-HCl+500 mM NaCl, pH=8.0), the elution mode was gradient elution, the flow rate was 0.5 mL / min, the column temperature was room temperature, and the UV detection wavelength was 210 and 280 nm, and the specific steps were as described above for the high performance liquid chromatography conditions.

[0064] Figure 1 and Figure 3 respectively, the detection wavelength spectrum at 210 nm and 280 nm is shown, Figure 2 and Figure 4 respectively, are their enlarged graphs. It can be seen that, except for slight differences in response degree, all five proteins can be well detected, and the outflow order of the five proteins is: beta 2 microglobulin (B2M), transferrin (TRF), retinol binding protein (RBP4), alpha 1 microglobulin (A1M), and albumin (HSA). Among them, beta 2 microglobulin (B2M), transferrin (TRF), retinol binding protein (RBP4), and albumin (HSA) can achieve baseline separation; retinol binding protein (RBP4) and alpha 1 microglobulin (A1M) will have partial overlap, but can be processed by peak cutting using high performance liquid chromatograph analysis software.

[0065] The above examples are only used to help understand the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A liquid chromatography method for simultaneous detection of multiple proteins, characterized in that, The analytical method employs anionic liquid chromatography with a non-porous resin as the stationary phase and a Cl--containing Tris gradient elution phase. The surface of the non-porous resin is bonded with a hydrophilic polymer nanolayer, and the nanolayer is modified with quaternary ammonium groups. The proteins have different isoelectric points, and the pH of the elution phase is greater than the isoelectric point of the protein. The proteins are albumin, transferrin, retinol-binding protein, β2-microglobulin, and α1-microglobulin; Chromatographic column: Proteomix SAX NP5, 4.6×50 mm, Sepax, anion exchange analytical column, particle size 5 μm; Mobile phase A: 20 mM Tris-HCl, pH=8.0; Mobile phase B: 20 ​​mM Tris-HCl, 500 mM NaCl, pH=8.0; Elution gradient: 0-0.5 min 0%B 0.5-7.0 min 0-100%B 7.0-7.1 min 100-0%B 7.1-15 min 0%B Detector: Ultraviolet detection wavelengths 210 and 280 nm.

2. The liquid chromatography method for simultaneous detection of multiple proteins according to claim 1, characterized in that, The isoelectric points of all the proteins are less than 8.

3. The liquid chromatography method for simultaneous detection of multiple proteins according to claim 1, characterized in that, The flow rate of the mobile phase was 0.5 mL / min.

4. A liquid chromatography method for simultaneous detection of multiple proteins according to any one of claims 1-3, characterized in that, The analytical method specifically includes the following steps: Protein sample preparation: Prepare a mixed standard sample of albumin, transferrin, retinol-binding protein, β2-microglobulin and α1-microglobulin; Column temperature: room temperature; Flow rate: 0.5 mL / min; Injection volume: 5 μL.

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