Sample pretreatment method for aqueous humor trace protein detection and application of sample pretreatment method

Abstract

The invention discloses a sample pretreatment method for aqueous humor trace protein detection and application of the sample pretreatment method, and relates to the field of protein detection.The sample pretreatment method comprises the steps of preparing an aqueous humor extraction reagent and extracting an aqueous humor sample through the prepared reagent, specifically, sample treatment is conducted by innovatively extracting reagent sets of dodecyl-beta-D-maltoside and RapiGest SF, and the detection result is accurate. The pretreatment method disclosed by the invention is combined with a liquid chromatography-mass spectrometry technology for proteomic analysis, and the aqueous humor sample protein detection can be further carried out with high sensitivity, high flux, accurate result, good reproducibility, short time consumption, simple operation and low sample consumption.

Description

Technical Field

[0001] The present invention relates to the field of protein detection, and in particular to a sample pretreatment method for aqueous humor trace protein detection and application thereof. Background Art

[0002] Aqueous humor is a type of eye fluid that is produced by the ciliary epithelium and provides nutrition to the cells and tissues of the anterior chamber. The aqueous humor contains a large amount of proteins that are unique to ocular tissues, making it very suitable as a clinical screening specimen for eye diseases. In recent years, many studies have shown that plasma proteins can enter the aqueous humor through the blood-aqueous humor barrier formed by the ciliary epithelium, so proteins expressed in other organs have also been found to appear in the aqueous humor. [1-3] Analyzing proteins in aqueous humor is of great significance for understanding the physiological and pathological changes of ophthalmic and even extraocular diseases.

[0003] In 2010, foreign scientists used mass spectrometry to analyze proteins in human aqueous humor for the first time. They identified 355 proteins from aqueous humor samples, most of which were enzymes with biological catalytic functions. [4] In 2019, Kaur et al. found that there were significant differences in the content of 246 proteins in the aqueous humor of glaucoma patients and cataract patients through mass spectrometry proteomics analysis. [5] Continuously establishing and improving the human aqueous humor proteome database will provide new insights into understanding the mechanisms of ocular diseases and exploring potential prognostic biomarkers.

[0004] However, the single collection volume of aqueous humor is only 50-100ul, and the protein concentration is about 0.1-0.6ug / ml. Such a low protein content makes aqueous humor protein analysis extremely challenging. Combined with the development of mass spectrometry technology, updating the aqueous humor proteome sample pre-processing workflow is expected to have a profound impact on the analysis of such clinical samples.

[0005] Therefore, technicians in this field are committed to developing a method for detecting protein in aqueous humor samples with high sensitivity, high throughput, accurate results, good reproducibility, short time consumption, simple operation, and low sample consumption.

[0006] References:

[0007] 1.Coca-Prados M.The blood-aqueous barrier in health and disease.JGlaucoma.2014Oct-Nov;23(8Suppl 1):S36-8.

[0008] 2. Yavrum F, Elgin U, Kocer ZA, Fidanci V, Sen E. Evaluation of aqueous humor and serum clusterin levels in patients with glaucoma. BMC Ophthalmol. 2021 Jan 9;21(1):25.

[0009] 3. Wolf J, Rasmussen DK, Sun YJ, Vu JT, Wang E, Espinosa C, Bigini F, Chang RT, Montague AA, Tang PH, Mruthyunjaya P, Aghaeepour N, Dufour A, Bassuk AG, Mahajan VB. Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo. Cell. 2023 Oct 26;186(22):4868-4884.e12.

[0010] 4. Chowdhury UR, Madden BJ, Charlesworth MC, Fautsch MP. Proteome analysis of human aqueous humor. Invest Ophthalmol Vis Sci. 2010 Oct;51(10):4921-31.

[0011] 5. Kaur I, Kaur J, Sooraj K, Goswami S, Saxena R, Chauhan VS, Sihota R. Comparative evaluation of the aqueous humor proteome of primary angle closure and primary open angle glaucomas and age-related cataract eyes. Int Ophthalmol. 2019 Jan;39(1):69-104. Summary of the Invention

[0012] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a method for detecting protein in aqueous humor samples with high sensitivity, high throughput, accurate results, good reproducibility, short time consumption, simple operation and low sample consumption. To achieve the above-mentioned purpose, the present invention provides a sample pretreatment method for detecting trace protein in aqueous humor, characterized in that the method comprises the following steps:

[0013] Step 1, preparing aqueous humor extraction reagent;

[0014] Step 2: Use the reagent prepared in step 1 to extract the aqueous humor sample.

[0015] In a preferred embodiment of the present invention, the aqueous humor extraction reagent in step 1 includes Tris hydrochloride, RapiGest SF, dodecyl-β-D-maltoside, i.e. DDM, tris(2-carboxyethyl)phosphine hydrochloride, i.e. TCEP solution, chloroacetamide, i.e. CAA solution, and trypsin solution, wherein the RapiGest SF is sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate.

[0016] In another preferred embodiment of the present invention, the step 1 further comprises:

[0017] Step 1.1, prepare 10 mM Tris hydrochloride solution with water, and use the solution to dilute RapiGest SF to a W / V of 0.1%-0.2%;

[0018] Step 1.2, prepare 0.1%-0.5% W / V DDM with 10 mM Tris hydrochloride;

[0019] Step 1.3, prepare 5 mM TCEP solution and 10 mM CAA solution with 10 mM Tris hydrochloride;

[0020] Step 1.4: Prepare 0.2ug / ul trypsin solution with 10mM Tris hydrochloride.

[0021] In another preferred embodiment of the present invention, the water and trypsin in step 1 are both of mass spectrometry grade.

[0022] In another preferred embodiment of the present invention, the step 2 further comprises:

[0023] Step 2.1, take 5-10ul aqueous humor sample and transfer it to a low-adsorption PCR tube;

[0024] Step 2.2, add 10ul of diluted RapiGest SF and DDM mixture to the sample PCR tube in step 2.1, and mix and shake at low temperature for 20-30 minutes;

[0025] Step 2.3, determine the sample protein concentration by BCA method;

[0026] Step 2.4, add 3-5ul of a mixture of 5mM TCEP solution and 10mM CAA solution, and heat at 55°C for 30 minutes;

[0027] Step 2.5: After returning to room temperature, add 0.2ug / ul trypsin solution according to the protein concentration and heat at 37°C for 2-3 hours;

[0028] Step 2.6, add formic acid to a final concentration of 1% to terminate the trypsin reaction;

[0029] Step 2.7: Desalt the peptide fragments obtained in step 2.6, and dry the sample in a vacuum dryer.

[0030] In another preferred embodiment of the present invention, the volume ratio of the diluted RapiGest SF and DDM in step 2.2 is 2:1-1:1, and the volume ratio of the TCEP solution and the CAA solution in step 2.4 is 1:1.

[0031] In another preferred embodiment of the present invention, the amount of trypsin added in step 2.5 is enzyme: protein W / W of 1:20-1:50.

[0032] In another preferred embodiment of the present invention, the formic acid in step 2.6 is of mass spectrometry grade.

[0033] In another preferred embodiment of the present invention, in step 2.7, a Ziptip solid phase extraction column is used to perform peptide desalting treatment.

[0034] The present invention also provides an application of a sample pretreatment method for detecting trace proteins in aqueous humor, characterized in that the pretreated sample is subjected to proteomic analysis of trace aqueous humor samples by liquid chromatography-mass spectrometry to identify proteins in the aqueous humor samples.

[0035] Technical Effects

[0036] 1. The abundance of proteins in aqueous humor samples is low, so a new extraction method needs to be established to improve the depth of aqueous humor sample detection. This method has an innovative extraction reagent composition (dodecyl-β-D-maltoside and RapiGest SF). Dodecyl-β-D-maltoside is a non-ionic detergent with good extraction and solubilization effects on membrane proteins. Combined with RapiGest SF, it promotes the enzymatic hydrolysis of difficult-to-hydrolyze proteins. The two work together to enhance protein extraction and digestion effects. Both are highly compatible with mass spectrometry and can be directly tested on a mass spectrometer. This method can deeply analyze more than 700 proteins in aqueous humor samples. The protein types are rich and the detection sensitivity is high. More than 700 proteins in aqueous humor samples can be identified, creating favorable conditions for the discovery of eye disease markers.

[0037] 2. The existing method has a complex processing flow and is time-consuming. The main processes include protein extraction and quantification (1 hour), ultrafiltration tube washing (2 hours), proteolysis digestion and desalting (16-18 hours). This method is simple to operate and takes less time. The main processes include protein extraction and quantification (1 hour), proteolysis digestion and desalting (2-3 hours). This method has simple operating steps, which reduces more than ten hours compared to traditional methods. It has good reproducibility, does not require transfer and multi-step elution during the process, takes less time, has less protein loss, and has a high sample recovery rate, which can significantly improve patient tolerance.

[0038] 3. The existing methods consume a lot of samples, and one experiment requires 50-100ul. This method uses less sample, only 10ul. The operation steps of this method are simple, and no transfer and multi-step elution are required during the process, so the sample consumption is low. This method reduces the sample consumption by 5-10 times compared with the traditional method. This method can achieve targeted and non-targeted protein qualitative and quantitative detection of 5-10ul aqueous humor.

[0039] 4. This method has good reproducibility and the results are accurate and reliable.

[0040] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a schematic diagram of the consistency of protein types identified in two aqueous humor samples in a preferred embodiment of the present invention;

[0042] Figure 2 It is a graph of raw data of protein detection identified in aqueous humor samples according to a preferred embodiment of the present invention;

[0043] Figure 3 It is a raw data diagram of protein detection identified in another aqueous humor sample according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0044] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0045] The materials and reagents required for the present invention are shown in Table 1

[0046] Table 1: Materials and Reagents

[0047]

[0048] Example 1. Preparation of extraction reagents

[0049] 1) Prepare 10 mM Tris hydrochloride solution with water (mass spectrometry grade) and dilute RapiGest SF to 0.1%-0.2% (W / V) with this solution;

[0050] 2) Prepare 0.1%-0.5% (W / V) DDM with 10 mM Tris hydrochloride;

[0051] 3) Prepare 5 mM TCEP solution and 10 mM CAA solution with 10 mM Tris hydrochloride;

[0052] 4) Prepare 0.2ug / ul trypsin solution with 10mM Tris hydrochloride.

[0053] Example 2: Aqueous humor sample extraction

[0054] 1) Take 5-10ul aqueous humor sample and transfer it to a low-adsorption PCR tube;

[0055] 2) Add 10ul of RapiGest SF and DDM mixture (2:1-1:1, V / V) to the above sample tube, and mix and shake at low temperature for 20-30 minutes (method principle: the two extraction reagents have good mass spectrometry compatibility while extracting aqueous humor protein, and the subsequent treatment does not require elution and removal steps, and can be directly used for mass spectrometry detection without interference with sample signals);

[0056] 3) Determination of sample protein concentration by BCA method;

[0057] 4) Add 3-5ul of 5mM TCEP solution and 10mM CAA mixture (1:1, V / V) and heat at 55°C for 30 minutes;

[0058] 5) After returning to room temperature, add 0.2ug / ul trypsin (enzyme: protein, 1:20-1:50, W / W) according to the protein concentration, and heat the reaction at 37°C for 2-3 hours (method principle: increase the enzyme concentration to accelerate the enzymatic reaction, while reducing the enzymatic reaction time to prevent high concentration trypsin from self-degradation);

[0059] 6) Add formic acid to a final concentration of 1% to terminate the trypsin reaction;

[0060] 7) Desalting the peptide fragments obtained in the previous step using a Ziptip solid phase extraction column, drying the sample using a vacuum dryer, and then testing it using an LCMS liquid chromatography-mass spectrometer.

[0061] Example 3: Proteomic Analysis

[0062] The pretreatment method combined with liquid chromatography-mass spectrometry was used to achieve a high-depth proteomic analysis of trace aqueous humor samples. A total of 751 proteins were identified in the two aqueous humor samples, of which 78% (581 proteins) were detected in both samples ( Figure 1 ), the protein detection coverage between samples is high, indicating that this method has good reproducibility. The 20 proteins with the strongest mass spectrometry detection signals of aqueous humor samples are shown in Table 2, of which albumin accounts for about 39%. Apolipoprotein APOA1, immunoglobulin IGHG1, etc., which are high-abundance proteins in aqueous humor, are also detected accordingly. The results are similar to those reported in foreign aqueous humor protein databases, indicating that this method is accurate and reliable. The original data of the detection of two aqueous humor samples are detailed in Figure 2 , Figure 3 .

[0063] Table 2 Mass spectrometry signal abundance TOP20 proteins

[0064]

[0065]

[0066] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A sample pretreatment method for detecting trace protein in aqueous humor, characterized in that: The method comprises the following steps: Step 1, preparing aqueous humor extraction reagent; Step 2: Use the reagent prepared in step 1 to extract the aqueous humor sample.

2. The method according to claim 1, characterized in that The aqueous humor extraction reagent in step 1 includes Tris hydrochloride, RapiGest SF, dodecyl-β-D-maltoside (DDM), tris(2-carboxyethyl)phosphine hydrochloride (TCEP solution), chloroacetamide (CAA solution), and trypsin solution, wherein the RapiGest SF is sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate.

3. The method according to claim 2, characterized in that The step 1 also includes: Step 1.1, prepare 10 mM Tris hydrochloride solution with water, and use the solution to dilute RapiGest SF to a W / V of 0.1%-0.2%; Step 1.2, prepare 0.1%-0.5% W / V DDM with 10 mM Tris hydrochloride; Step 1.3, prepare 5 mM TCEP solution and 10 mM CAA solution with 10 mM Tris hydrochloride; Step 1.4: Prepare 0.2ug / ul trypsin solution with 10mM Tris hydrochloride.

4. The method according to claim 3, characterized in that The water and trypsin in step 1 are both of mass spectrometry grade.

5. The method according to claim 3, characterized in that The step 2 also includes: Step 2.1, take 5-10ul aqueous humor sample and transfer it to a low-adsorption PCR tube; Step 2.2, add 10ul of diluted RapiGest SF and DDM mixture to the sample PCR tube in step 2.1, and mix and shake at low temperature for 20-30 minutes; Step 2.3, determine the sample protein concentration by BCA method; Step 2.4, add 3-5ul of a mixture of 5mM TCEP solution and 10mM CAA solution, and heat at 55°C for 30 minutes; Step 2.5: After returning to room temperature, add 0.2ug / ul trypsin solution according to the protein concentration and heat at 37°C for 2-3 hours; Step 2.6, add formic acid to a final concentration of 1% to terminate the trypsin reaction; Step 2.7: Desalt the peptide fragments obtained in step 2.6, and dry the sample in a vacuum dryer.

6. The method according to claim 5, characterized in that The volume ratio of the diluted RapiGest SF and DDM in step 2.2 is 2:1-1:1, and the volume ratio of the TCEP solution and the CAA solution in step 2.4 is 1:

1.

7. The method according to claim 5, characterized in that The amount of trypsin added in step 2.5 is enzyme: protein W / W 1:20-1:

50.

8. The method according to claim 5, characterized in that The formic acid in step 2.6 is of mass spectrometry grade.

9. The method according to claim 5, characterized in that In step 2.7, a Ziptip solid phase extraction column is used to perform peptide desalting.

10. Use of the sample pretreatment method for aqueous humor trace protein detection according to any one of claims 1 to 9, characterized in that: The pre-treated samples were subjected to proteomic analysis of trace aqueous humor samples using liquid chromatography-mass spectrometry to identify the proteins in the aqueous humor samples.

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