An analysis method for simultaneously detecting multiple proteins in urine

Abstract

The application provides an analysis method for simultaneously detecting multiple proteins in urine, which sequentially filters the urine through a hydrophilic MCE filter membrane, a volume exclusion gel column containing dextran, and a high performance anion exchange chromatography column containing a non-porous resin filler with a functional group of + (CH3)3, to remove impurities and concentrate target proteins, and then uses high performance anion exchange chromatography of the non-porous resin filler with a functional group of + (CH3)3 to perform detection analysis. The method is simple in operation and few in steps, and can quickly and accurately perform qualitative and quantitative analysis on proteins such as albumin, transferrin, retinol binding protein, beta 2 microglobulin and alpha 1 microglobulin in urine.

Description

Technical Field

[0001] This invention relates to the field of protein extraction and purification, and more particularly to an analytical method for simultaneously detecting multiple proteins in urine. Background Technology

[0002] Early screening for kidney disease often requires detecting markers in urine such as urinary albumin (HSA), urinary transferrin (TRF), urinary retinol-binding protein (RBP4), urinary alpha-1 microglobulin (A1M), urinary β2-microglobulin (B2M), and NAG. These indicators are sensitive markers of early damage to the glomeruli, renal tubules, and proximal convoluted tubules, as well as early diabetic nephropathy. In clinical diagnosis, various urinary proteins often need to be tested for screening and diagnosis. However, due to limitations in testing methods, each marker needs to be tested individually, which is not only time-consuming, labor-intensive, and expensive, but also more prone to sample contamination and operational errors due to the large number of procedures, leading to inaccurate results and inaccurate diagnosis.

[0003] Based on literature review, analyzing and determining one to three proteins with significantly different physicochemical properties in urine is relatively easy. However, separating multiple proteins in urine simultaneously requires more complex separation techniques. Currently, commonly used techniques include electrophoresis, highly integrated protein biochemical analysis techniques, chemiluminescence, and mass spectrometry. Among these, electrophoresis is cumbersome to operate, has many interfering factors, produces unstable results, and cannot provide quantitative analysis; the false turbidity of biochemical analysis techniques can lead to inaccurate quantitative results, and different types of proteins usually need to be detected separately; chemiluminescence is complex and costly; and mass spectrometry involves expensive equipment that relies on imports and is difficult to operate.

[0004] Therefore, there is an urgent need for an analytical method that can be simple, rapid, economical, and accurate to simultaneously detect and separate multiple proteins in urine. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an analytical method that can simultaneously detect multiple proteins in urine. This method is simple to process urine samples and can quickly and accurately detect proteins such as albumin, transferrin, retinol-binding protein, β2-microglobulin, and α1-microglobulin in urine.

[0006] The present invention adopts the following technical solution:

[0007] S1, Filtration: Filter urine using a hydrophilic MCE filter membrane to obtain filtrate 1;

[0008] S2, First solid-phase extraction: The filtrate 1 is processed through a dextran-containing volumetric gel column to obtain filtrate 2;

[0009] S3, Second solid-phase extraction: The filtrate 2 is subjected to extraction with -N+ The solution was treated with an anion exchange column based on a porous hydrophilic polymethacrylate matrix containing (CH3)3 functional groups to obtain filtrate 3.

[0010] S4, High Performance Anion Liquid Chromatography: Using anion exchange chromatography containing -N... + An anion exchange column containing a (CH3)3 functional group was used to detect filtrate 3. The elution method employed was a Cl-containing... - Tris gradient mobile phase.

[0011] Furthermore, the hydrophilic MCE filter membrane has a pore size of 0.22 micrometers.

[0012] Furthermore, the volumetric columnar gel column is a Hitrap. TM Desalting, 5 mL, cytiva.

[0013] Furthermore, the specific method for the first solid-phase extraction is as follows:

[0014] Replacement: Completely replace the ethanol solution in the volumetric gel column with 25 mL of Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0).

[0015] Equilibration: 5.0 mL Tris-HCl buffer C (20 mM Tris-HCl, 25 mM NaCl, pH=8.0)

[0016] Sample loading: 1.5 mL of the filtrate 1

[0017] Elution: 2.0 mL of the Tris-HCl buffer C was used to collect the eluent, which is the filtrate 2.

[0018] Flow rate: 1.0-10.0 mL / min

[0019] Column temperature: room temperature.

[0020] Furthermore, the anion exchange column for the second solid-phase extraction is one of Monomix Mab60-Q, Monomix HC60-Q-II, and Polar MC60-Q9.

[0021] Furthermore, the specific method for the second solid-phase extraction is as follows:

[0022] Activation: 3.0 mL ultrapure water

[0023] Equilibration: 1.5 mL of the Tris-HCl buffer A

[0024] Sample loading: The filtrate 2

[0025] Wash: 1.5 mL Tris-HCl buffer D (20 mM Tris-HCl, 50 mM NaCl, pH=8.0)

[0026] Elution: 0.15 mL Tris-HCl buffer B (20 mM Tris-HCl, 500 mM NaCl, pH=8.0), collect the eluent as filtrate 3.

[0027] Flow rate: not exceeding 1 d / s

[0028] Column temperature: room temperature.

[0029] Furthermore, the non-porous resin is polystyrene / divinylbenzene (PS / DVB) resin particles with a particle size of 1.7-10 μm, and a hydrophilic polymer nanolayer bonded to the surface, wherein the nanolayer is modified with the -N + (CH3)3 functional group.

[0030] Furthermore, the high-performance anion exchange liquid chromatography column is a Proteomix SAX NP5, 4.6 × 50 mm, Sepax.

[0031] Furthermore, the specific method for the high-performance anion exchange liquid chromatography analysis is as follows:

[0032] Mobile phase: buffer A and buffer B

[0033] Sample loading: The filtrate 3

[0034] Elution gradient:

[0035] 0-0.5min 0%B

[0036] 0.5-7.0 min 0-100% B

[0037] 7.0-7.1 min 100-0% B

[0038] 7.1-15min 0%B

[0039] Detector: Ultraviolet detection wavelengths 210 and 280 nm

[0040] Column temperature: room temperature

[0041] Flow rate: 0.5 mL / min.

[0042] Furthermore, the analytical method mainly detects one or more of albumin, transferrin, retinol-binding protein, β2-microglobulin, and α1-microglobulin in urine.

[0043] Urine samples require pretreatment through filtration and solid-phase extraction. This is because urine contains many impurities and the concentrations of the five target proteins are low, necessitating pretreatment to remove most impurities (mainly small molecules) and enrich the target proteins. First, a syringe filter (containing a hydrophilic membrane with a 0.22-micron pore size) is used to filter the urine, removing sediment and bacteria. Then, utilizing the volume difference between the target proteins and small molecule impurities, a dextran gel column based on the size exclusion principle is selected to remove small molecule impurities while simultaneously displacing the urine to a low-salt concentration and weakly alkaline environment, preparing for subsequent concentration. Next, utilizing the isoelectric point difference between the target proteins and other proteins, an anion exchange column is used to selectively adsorb the target proteins, which are then eluted by a small volume of liquid, completing concentration, enrichment, and further impurity removal.

[0044] Urine samples, after filtration and two solid-phase extractions, were analyzed by high-performance anion exchange chromatography (HPLC). At pH 8.0, the stationary phase was positively charged, while albumin, transferrin, retinol-binding protein, β2-microglobulin, and α1-microglobulin were negatively charged. Electrostatic interactions existed between the stationary phase and the proteins. Due to the different isoelectric points of the five proteins, the electrostatic forces between the stationary phase and the proteins also varied. During gradient elution, the proportion of mobile phase B was continuously increased, and the Cl- in mobile phase B... - The five proteins compete with the stationary phase for binding, causing them to flow out sequentially in order of increasing binding strength, thus enabling qualitative and quantitative detection.

[0045] Compared with existing technologies, the present invention provides an analytical method for simultaneously detecting multiple proteins in urine. It is simple to operate and has fewer steps, and can quickly and accurately perform qualitative and quantitative analysis of proteins such as albumin, transferrin, retinol-binding protein, β2-microglobulin, and α1-microglobulin in urine. Attached Figure Description

[0046] Figure 1 The spectrum of the original urine sample 1 after filtration through a 0.22-micron membrane is detected at 210 nm.

[0047] Figure 2 The spectrum of the original urine sample 1 after filtration through a 0.22-micron membrane is detected at 280 nm.

[0048] Figure 3 The spectrum of the original urine sample 1 after filtration through a 0.22-micron membrane and the first solid-phase extraction is detected at 210 nm.

[0049] Figure 4 The spectrum of the original urine sample 1 after filtration through a 0.22-micron membrane and the first solid-phase extraction is detected at 280 nm.

[0050] Figure 5The spectrum of the original urine sample 1 after filtration, two solid-phase extractions, and high-performance anion chromatography is the detection wavelength at 210 and 280 nm.

[0051] Figure 6 The magnified spectra of the original urine sample 1 after filtration, two solid-phase extractions, and high-performance anion chromatography at detection wavelengths of 210 and 280 nm are shown.

[0052] Figure 7 The spectrum of the original urine sample 2 after filtration, two solid-phase extractions, and high-performance anion chromatography is the detection wavelength at 210 and 280 nm.

[0053] Figure 8 The magnified spectrum of the original urine sample 2 after filtration, two solid-phase extractions, and high-performance anion chromatography is obtained at a detection wavelength of 210 nm.

[0054] Figure 9 The magnified spectrum of the original urine sample 2 after filtration, two solid-phase extractions, and high-performance anion chromatography is obtained at the detection wavelength of 280 nm.

[0055] Figure 10 The detection wavelength spectra of β2 microglobulin (B2M) single-labeled samples at 210 and 280 nm are shown.

[0056] Figure 11 The spectra of the transferrin (TRF) single-labeled sample at detection wavelengths of 210 and 280 nm are shown.

[0057] Figure 12 The spectra of a single-labeled retinol-binding protein (RBP4) sample at 210 and 280 nm are shown.

[0058] Figure 13 The detection wavelength spectra of α1 microglobulin (A1M) single-labeled samples at 210 and 280 nm are shown.

[0059] Figure 14 The spectrum of albumin (HSA) single-labeled samples at detection wavelengths of 210 and 280 nm is shown. Detailed Implementation

[0060] The technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0061] Preparation of elution solution

[0062] Prepare Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0): Weigh 2.4228 g of tris(hydroxymethyl)aminomethane (Tris), dissolve it in 1 L of ultrapure water, adjust the pH to 8.0 with hydrochloric acid (HCl), and then filter it through a 0.45 μm hydrophilic membrane.

[0063] Prepare Tris-HCl buffer B (20 mM Tris-HCl, 500 mM NaCl, pH=8.0): Weigh 2.4228 g of tris(hydroxymethyl)aminomethane (Tris) and 29.2214 g of sodium chloride (NaCl), dissolve them in 1 L of ultrapure water, adjust the pH to 8.0 with hydrochloric acid (HCl), and then filter through a 0.45 μm hydrophilic membrane.

[0064] Prepare Tris-HCl buffer C (20 mM Tris-HCl, 25 mM NaCl, pH=8.0): Mix Tris-HCl buffer A and Tris-HCl buffer B in a 95:5 ratio.

[0065] Prepare Tris-HCl buffer D (20 mM Tris-HCl, 50 mM NaCl, pH=8.0): Mix Tris-HCl buffer A and Tris-HCl buffer B in a 90:10 ratio.

[0066] Example 1

[0067] 1) Filtering

[0068] The original urine sample 1 was filtered using a needle filter (containing a hydrophilic filter membrane with a pore size of 0.22 micrometers), and the filtrate 2 of the original urine was collected.

[0069] Figure 1 and Figure 2 The spectra of filtrate 2 after being filtered by a needle filter at 210 nm and 280 nm are shown. It can be seen that the composition of urine after filtration through a 0.22-micron membrane is still very complex and impurities need to be further removed.

[0070] 2) First solid-phase extraction

[0071] Column selection: Hitrap TM Desalting, 5 mL, cytiva (a dextran gel column packed with Sephadex™ G-25 Superfine filler).

[0072] Replacement: For first-time use, the ethanol solution in the dextran gel column needs to be completely replaced with 25 mL of Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0).

[0073] Equilibration: 5.0 mL Tris-HCl buffer C (20 mM Tris-HCl, 25 mM NaCl, pH=8.0)

[0074] Sample loading: 1.5 mL urine filtrate 2

[0075] Collection: After sample loading, add 2.0 mL of Tris-HCl buffer C (20 mM Tris-HCl, 25 mM NaCl, pH=8.0) and collect the eluent.

[0076] Washing: 10.0 mL Tris-HCl buffer C (20 mM Tris-HCl, 25 mM NaCl, pH=8.0)

[0077] Flow rate: 1.0-10.0 mL / min

[0078] Column temperature: room temperature.

[0079] Figure 3 and Figure 4 The spectra of urine 1 (i.e. effluent 3) after the first solid-phase extraction are shown at 210 nm and 280 nm. It can be seen that after the first solid-phase extraction, most small molecules are removed and the interfering components are significantly reduced.

[0080] 3) Second solid-phase extraction

[0081] Column selection: Anion exchange columns packed with Monomix Mab60-Q, 50mg, or Sepax packing materials are recommended. Alternatively, columns packed with Monomix HC60-Q-II, Polar MC60-Q9, or similar packing materials can also be used. These packing materials are all based on porous hydrophilic polymethyl methacrylate, and the matrix contains -N... + (CH3)3 functional group.

[0082] Activation: 3.0 mL ultrapure water

[0083] Equilibration: 1.5 mL Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0)

[0084] Sample loading: 3g of eluent after the first solid-phase extraction

[0085] Wash: 1.5 mL Tris-HCl buffer D (20 mM Tris-HCl, 50 mM NaCl, pH=8.0)

[0086] Elution: 0.15 mL Tris-HCl buffer B (20 mM Tris-HCl, 500 mM NaCl, pH=8.0), collect the eluent as the injected sample 4.

[0087] Washing: 3.0 mL Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0)

[0088] Flow rate: not exceeding 1 d / s

[0089] Column temperature: room temperature.

[0090] The target protein was concentrated tenfold through a second solid-phase extraction.

[0091] 4) High-performance liquid chromatography analysis

[0092] Configure mobile phase E:

[0093] Weigh 2.4228 g of tris(hydroxymethyl)aminomethane (Tris), dissolve it in 1 L of ultrapure water, adjust the pH to 8.0 with hydrochloric acid (HCl), and then filter it through a 0.45 μm hydrophilic membrane to obtain mobile phase E.

[0094] Configure mobile phase F:

[0095] Weigh 2.4228 g of tris(hydroxymethyl)aminomethane (Tris) and 29.2214 g of sodium chloride (NaCl), dissolve them in 1 L of ultrapure water, adjust the pH to 8.0 with hydrochloric acid (HCl), and then filter through a 0.45 μm hydrophilic membrane to obtain mobile phase F.

[0096] Column selection: Proteomix SAX NP5, 4.6 × 50 mm, Sepax, anion exchange analytical column.

[0097] Mobile phase E: 20 mM Tris-HCl, pH=8.0

[0098] Mobile phase F: 20 mM Tris-HCl, 500 mM NaCl, pH=8.0

[0099] Elution gradient:

[0100] 0-0.5min 0%F

[0101] 0.5-7.0 min 0-100% F

[0102] 7.0-7.1 min 100-0%F

[0103] 7.1-15min 0%F

[0104] Detector: Ultraviolet detection wavelengths 210 and 280 nm

[0105] Column temperature: room temperature

[0106] Flow rate: 0.5 mL / min

[0107] Injection volume: 20 μL for sample 4.

[0108] The original urine sample 1, after filtration and two solid-phase extractions, was analyzed using high-performance anion exchange liquid chromatography (HPLC) as described in step 4 above. The analytical results are as follows: Figure 5-6 As shown in Table 1, qualitative analysis was completed by comparing the peak times of each single-labeled protein with those in Comparative Example 1, and quantitative analysis was completed by comparing the peak areas of each protein.

[0109] Table 1

[0110]

[0111] Comparative analysis revealed that only the peak area of ​​β2-microglobulin B2M in the original urine sample 1 was greater than the upper limit of 12.2 for β2-microglobulin B2M in normal human urine, indicating that the B2M content exceeded the upper limit of the normal reference range. The contents of the other four proteins were all less than the upper limit of the normal reference range. Elevated β2-microglobulin B2M in urine generally indicates renal tubular dysfunction or increased filtration load.

[0112] Example 2

[0113] The difference between Example 2 and Example 1 is the source of the original urine. Example 2 uses original urine sample 2. All other steps are the same as in Example 1, and the analysis results are as follows: Figure 7-9 As shown.

[0114] Qualitative analysis was completed by comparing the peak times of each single-labeled protein with those in Comparative Example 1, and quantitative analysis was completed by comparing the peak area of ​​each protein. The results are shown in Table 2.

[0115] Table 2

[0116]

[0117] In the original urine sample 2, the peak area of ​​B2M was 51.7 (280 nm), the peak area of ​​TRF was 56.0 (280 nm), the peak area of ​​RBP4 was 31.4 (280 nm), the peak area of ​​A1M was 94.1 (280 nm), and the peak area of ​​HSA was 322.1 (280 nm). Comparative analysis revealed that the levels of these five proteins all exceeded the upper limit of the normal reference range, indicating that the patient's renal function may have significant and serious damage.

[0118] In step 4), the Proteomix SAX NP5 anion exchange analysis column uses rigid, spherical, highly cross-linked non-porous polystyrene / divinylbenzene (PS / DVB) resin particles with particle sizes of 1.7 μm, 3 μm, 5 μm, and 10 μm, preferably 5 μm. The resin surface is bonded with a highly hydrophilic nanoscale-thickness neutral polymer layer. The hydrophobic PS / DVB resin surface is completely covered by this hydrophilic material, thereby eliminating the irreversible adsorption of biomolecules by PS / DVB and ensuring high separation efficiency and biological sample recovery rate. The polymer layer surface is densely and uniformly chemically bonded with strong anion exchange functional groups (quaternary ammonium groups). This stationary phase packing has three characteristics: First, the nanoscale-thick hydrophilic layer completely eliminates non-specific interactions between the carrier and the biological sample; second, the non-porous particle structure minimizes lateral diffusion of the sample while inhibiting its diffusion into the packing particles; third, using Saifen's unique chemical bonding technology, three-dimensional strong anion exchange groups are bonded to the hydrophilic layer, providing the best resolution and separation efficiency for proteins, oligonucleotides, carbohydrates, and peptides.

[0119] Because the stationary phase packing material is positively charged, and the isoelectric points (pI) of albumin, transferrin, retinol-binding protein, β2-microglobulin, and α1-microglobulin in urine are all less than 8, these proteins release protons and become negatively charged at pH 8.0. Therefore, there is an electrostatic binding force between the stationary phase and the proteins. Since the five proteins have different isoelectric points, the magnitude of the electrostatic force between the stationary phase and the five proteins also varies. During gradient elution, the proportion of mobile phase F is continuously increased, and the Cl in mobile phase F... - Ions compete with the five proteins for binding to the stationary phase, causing the proteins to elute sequentially in order of increasing binding strength, thus completing the separation. Typically, the proteins elute in order of decreasing isoelectric point.

[0120] Table 3

[0121]

[0122] Comparative Example 1

[0123] Configure each protein sample:

[0124] Using a pipette, 0.6 μL of 1.0 mg / mL β2-microglobulin (B2M), 1.4 μL of 1.0 mg / mL retinol-binding protein (RBP4), 2.0 μL of 2.0 mg / mL transferrin (TRF), 20 μL of 2.0 mg / mL albumin (HSA), and 12 μL of 2.0 mg / mL α1-microglobulin (A1M) were respectively diluted to 200 μL using mobile phase E as the diluent, thus preparing five single-label samples of proteins.

[0125] The single-label concentrations of the five proteins were as follows: β2-microglobulin (B2M) concentration was 3.0 μg / mL, corresponding to the upper limit of the normal reference range for urine (0.3 μg / mL); transferrin (TRF) concentration was 20.0 μg / mL, corresponding to the upper limit of the normal reference range for urine (2.0 μg / mL); retinol-binding protein (RBP4) concentration was 7.0 μg / mL, corresponding to the upper limit of the normal reference range for urine (0.7 μg / mL); albumin (HSA) concentration was 200.0 μg / mL, corresponding to the upper limit of the normal reference range for urine (20.0 μg / mL); and α1-microglobulin (A1M) concentration was 120.0 μg / mL, corresponding to the upper limit of the normal reference range for urine (12.0 μg / mL).

[0126] First, analyze the five protein single-label samples using the high-performance anion exchange liquid chromatography (HPLC) method described in step 4). The analytical results are as follows: Figure 10-14 The peak times for β2-microglobulin (B2M) were 3.736 min, with peak areas of 201 (210 nm) and 12.2 (280 nm); for transferrin (TRF), the peak times were 4.182 min, with peak areas of 871.4 (210 nm) and 46.7 (280 nm); for retinol-binding protein (RBP4), the peak times were 4.681 min, with peak areas of 314.9 (210 nm) and 20.9 (280 nm); for α1-microglobulin (A1M), the peak times were 4.932 min, with peak areas of 1035 (210 nm) and 78.4 (280 nm); and for albumin (HSA), the peak times were 5.493 min, with peak areas of 11068.5 (210 nm) and 292.8 (280 nm).

[0127] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An assay method for simultaneously detecting a plurality of proteins in urine, characterized by, Comprising the following steps: 1) Filtration: filtering urine with hydrophilic MCE filter membrane to obtain filtrate 1; 2) First solid phase extraction: treating the filtrate 1 with a dextran-containing size exclusion gel column to obtain filtrate 2; 3) Second solid phase extraction: the filtrate 2 is passed through a porous hydrophilic polymethacrylate with -N + (CH3)3 functional groups as a matrix anion exchange column, obtaining filtrate 3; 4) High performance anion exchange liquid chromatography: filtrate 3 was analyzed on an anion exchange column packed with non-porous resin beads functionalized with -N + (CH3)3 functional groups, eluted with a gradient of Tris containing Cl - ions; The proteins are albumin, transferrin, retinol binding protein, beta 2 microglobulin and alpha 1 microglobulin; The non-porous resin is polystyrene / divinylbenzene (PS / DVB) resin particles with a particle size of 5 μm, with a surface bonded hydrophilic polymer nanosheet, with the -N + (CH3)3 functional groups; The high-performance anion exchange liquid chromatography column is Proteomix SAX NP5, 4.6x50 mm, Sepax; The specific method of high-performance anion exchange liquid chromatography analysis is: Mobile phase: buffer A: 20 mM Tris-HCl, pH=8.0, buffer B: 20 mM Tris-HCl, 500 mM NaCl, pH=8.0 Loading: the filtrate 3 Elution gradient: 0-0.5min 0%B 0.5-7.0min 0-100%B 7.0-7.1min 100-0%B 7.1-15min 0%B Detector: ultraviolet detection wavelength 210, 280nm Column temperature: room temperature Flow rate: 0.5 mL / min.

2. The method according to claim 1, wherein the method is for simultaneous detection of multiple proteins in urine. The pore size of the hydrophilic MCE filter membrane is 0.22 microns.

3. The method according to claim 1, wherein the method is characterized by, The size exclusion gel column is HitrapTM Desalting, 5 mL, cytiva.

4. The method according to claim 3, wherein the method is for simultaneous detection of multiple proteins in urine. The specific method of the first solid phase extraction is: Replacement: completely replace the ethanol solution in the size exclusion gel column with 25 mL of the buffer A Equilibrium: 5.0 mL Tris-HCl buffer C: 20 mM Tris-HCl, 25 mM NaCl, pH=8.0 Loading: 1.5 mL of the filtrate 1 Elution: 2.0 mL of the Tris-HCl buffer C, and the eluate is the filtrate 2 Flow rate: 1.0-10.0 mL / min Column temperature: room temperature.

5. The method according to claim 1, wherein the method is for simultaneous detection of multiple proteins in urine. The anion exchange column of the second solid phase extraction is one of Monomix Mab60-Q, Monomix HC60-Q-II and PolarMC60-Q9.

6. The method according to claim 5, wherein the method is for simultaneous detection of multiple proteins in urine. The specific method of the second solid phase extraction is: Activation: 3.0 mL ultrapure water Equilibrium: 1.5 mL of the buffer A Loading: the filtrate 2 Elution: 1.5 mL Tris-HCl buffer D: 20 mM Tris-HCl, 50 mM NaCl, pH=8.0 Elution: 0.15 mL Tris-HCl buffer B: 20 mM Tris-HCl, 500 mM NaCl, pH=8.0, and the eluate is the filtrate 3 Flow rate: not more than 1 d / s Column temperature: room temperature.

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