Trace protein extraction method for tear Schirmer test paper

Abstract

The invention discloses a trace protein extraction method for tear Schirmer test paper, and relates to the technical field of biologication.The extraction method comprises the steps that wet part tear Schirmer test paper is taken, a Triton X-100 solution is added, and low-temperature uniform mixing and oscillation are conducted; centrifuging, taking supernate, adding a precooled acetone solution, uniformly mixing, standing at low temperature, centrifuging, and collecting protein precipitate; adding a RapiGest SF solution to resuspend protein, adding a DTT solution, carrying out a heating reaction, adding an IAA solution, and carrying out a reaction at room temperature in a dark place; adding trypsin, and heating for reaction; and adding formic acid until the final concentration of the solution is 1%, terminating pancreatin reaction, desalting by a solid-phase extraction column, and drying in vacuum. According to the invention, the in-depth proteomics research on the tear Schirmer test paper sample is facilitated.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a trace protein extraction reagent and an extraction method for tear Schirmer test paper. Background Art

[0002] Tears are watery liquids secreted by the lacrimal glands, conjunctival goblet cells, cornea, and blood vessels. The tear film covers the cornea and conjunctival epithelium, preventing water evaporation while also providing lubrication, nutrition, and protection against pathogens. Tears contain thousands of molecules, including water, proteins, lipids, electrolytes, and various small molecule metabolites, of which water accounts for 98%-99%. The various components in tears play an important role in maintaining eye health, and tear proteins are a potential source of pathophysiological biomarkers for diagnosing and monitoring eye diseases and even some systemic diseases.

[0003] Proteomics was proposed by Australian scientist Marc Wilkins in 1994. It mainly explores the composition and change patterns of proteins in cells, tissues and organisms. It has been used for the determination of tumor markers for a long time and is now also used in the study of eye diseases. Its process mainly includes the extraction, separation, analysis and identification of proteins, and the research techniques are diversified, including multidimensional chromatography, multidimensional electrophoresis, amino acid composition analysis, protein chip technology and mass spectrometry. In recent years, with the advent of high-throughput and high-sensitivity mass spectrometers, mass spectrometry-based proteomics technology has developed to a new level, which can complete the qualitative and quantitative analysis of thousands of proteins in biological samples within tens of minutes, and has a positive impact on the diagnosis, monitoring and pathogenic mechanism research of eye diseases. As early as 2006, foreign researchers used in-gel enzymatic hydrolysis combined with mass spectrometry to characterize 491 proteins in human tears, and newly discovered 18 antioxidant enzymes that can be used as important features to maintain the ocular microenvironment. In 2012, the coverage of tear proteome identification increased significantly, reaching more than 1,500 species. Tear proteomic analysis is very promising in clinical health monitoring and disease treatment.

[0004] Clinical tear testing has many advantages, such as convenient and fast collection, easy operation, and low invasiveness. Unlike other ocular fluids (such as aqueous humor and vitreous), tears are easier to obtain from healthy subjects, providing sufficient conditions for omics studies that require a control group. At present, there are two main ways to collect tear specimens in clinical practice, namely glass microcapillary collection and Schirmer test strip collection. The former requires high technical experience for the processing personnel. In comparison, the Schirmer test strip collection method is safer and faster, has good patient tolerance, and is more suitable for routine examinations. However, the Schirmer test strip collection method poses challenges to subsequent sample processing and protein extraction methods. In the subsequent processing steps, samples need to be extracted from the test strip material and prepared for transfer, which will involve protein loss and contamination problems, increasing the variability of the results. The extraction strategies currently described in the literature have many steps, low protein recovery rates, and insufficient identification depth to support the needs of deep tear proteomics analysis.

[0005] Therefore, technicians in this field are committed to developing a new method for extracting trace proteins from tear test strips to ensure the depth, accuracy and reliability of tear analysis. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a new type of tear test paper trace protein extraction reagent and an accurate and efficient extraction method.

[0007] To achieve the above object, the present invention provides a trace protein extraction reagent, comprising: Triton X-100, ammonium bicarbonate solution, trypsin solution, formic acid, RapiGest SF, iodoacetamide solution and dithiothreitol solution.

[0008] Furthermore, the concentration of Triton X-100 is 1%, the solvent is an ammonium bicarbonate solution, and the concentration of the ammonium bicarbonate solution is 100 mM.

[0009] Furthermore, trypsin is of mass spectrometry grade and is prepared into a trypsin solution, and formic acid is of mass spectrometry grade.

[0010] Furthermore, the concentration of the iodoacetamide solution is 50 mM, and the concentration of the dithiothreitol solution is 50 mM.

[0011] Furthermore, the concentration of the trypsin solution is 0.2 ug / ul.

[0012] The present invention also provides a method for extracting trace protein from tear Schirmer test paper using a trace protein extraction reagent, comprising the following steps:

[0013] Step 1, take the Schirmer test paper of the wet tear fluid, transfer it to the first low-adsorption centrifuge tube, add Triton X-100 solution, mix and shake at 4°C; centrifuge for the first time, take the supernatant, transfer it to the second low-adsorption centrifuge tube, add 4 times the volume of pre-cooled acetone solution, mix, let it stand at -20°C, centrifuge for the second time, and collect the protein precipitate;

[0014] Step 2: Add RapiGest SF solution to the protein precipitate obtained in step 1 to resuspend the protein, add dithiothreitol (DTT) solution, heat for the first reaction, then add iodoacetamide (IAA) solution, react at room temperature in the dark; add trypsin, heat for the second reaction; add formic acid to a final concentration of 1% to terminate the trypsin reaction and obtain a peptide solution;

[0015] Step 3: Desalt the peptide obtained in step 2 using a MonoSpin C18 solid phase extraction column, and dry it in a vacuum dryer to obtain a dried sample.

[0016] Furthermore, step 1 also includes: taking 20 mm wet tear Schirmer test paper and cutting it into 2 mm*2 mm small pieces; the concentration of Triton X-100 solution is 1%; the oscillation time is 1 hour; and the standing time is 2 hours.

[0017] Furthermore, in step 2, the concentration of RapiGest SF solution is 0.1% resuspended protein, the concentration of 50mM DTT solution is 50mM, the temperature of the first heating reaction is 55°C, the time of the first heating reaction is 30 minutes, the concentration of IAA solution is 50mM, and the reaction time at room temperature in the dark is 30 minutes.

[0018] Furthermore, in step 2, the concentration of trypsin is 0.2 ug / ul, the temperature of the second heating reaction is 37° C., and the time of the second heating reaction is 2 hours.

[0019] Furthermore, the method further comprises detecting the dried sample using an LCMS liquid spectrometer.

[0020] In preferred embodiment 1 of the present invention, the process of extracting protein using a trace protein extraction reagent of tear Schirmer test paper is described in detail.

[0021] The beneficial technical effects of the present invention are as follows:

[0022] The composition of the extraction reagent disclosed in the present invention is different from that of the prior art. It is based on Triton X-100 reagent and combined with acetone protein precipitation method. It has better solubility and stronger elution ability for proteins on tear Schirmer test paper. It can deeply analyze more than 4,000 proteins in tear samples, with rich protein types, which is twice as high as the prior art.

[0023] The extraction method disclosed in the present invention has simple steps, does not require multi-step elution and transfer, has low protein loss, and greatly improves the sample recovery rate. Compared with the existing method (16-18 hours), it takes less time (7 hours) and has high protein extraction efficiency, and can extract 250-300ug protein from 20mm tear Schirmer test paper.

[0024] 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

[0025] Figure 1 is a mass spectrometry test result diagram of a tear sample 1 according to a preferred embodiment 1 of the present invention;

[0026] Figure 2 is a mass spectrometry test result diagram of a tear sample 1 according to a preferred embodiment 1 of the present invention;

[0027] Figure 3 This is a GO functional annotation classification bar chart of a preferred embodiment 1 of the present invention. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1 Trace protein extraction reagent and extraction method for tear Schirmer test paper

[0030] The materials and reagents required for this example are shown in Table 1:

[0031] Table 1: Materials and Reagents

[0032]

[0033]

[0034] The extraction steps are as follows:

[0035] 1. Preparation of extraction reagents

[0036] 1) Prepare 100 mM ammonium bicarbonate solution with water (mass spectrometry grade) and dilute Triton X-100 to 1% concentration with this solution;

[0037] 2) Prepare 100 mM ammonium bicarbonate solution with water (mass spectrometry grade) and dilute RapiGest SF to 0.1% concentration with this solution;

[0038] 3) Prepare 50 mM DTT solution and 50 mM IAA solution with water (mass spectrometry grade);

[0039] 4) Prepare 0.2ug / ul trypsin solution with water (mass spectrometry grade).

[0040] 2. Tear Schirmer test paper sample extraction

[0041] 1) Take a 20mm wet tear Schirmer test paper, cut it into 2mm*2mm small pieces, and transfer it to a 1.5ml low-absorption centrifuge tube;

[0042] 2) Add 1% Triton X-100 solution to the sample centrifuge tube, mix and shake at 4°C for 1 hour (this step reacts at low temperature to ensure that the protein on the test paper is fully eluted and extracted completely without protein degradation);

[0043] 3) Centrifuge and take the supernatant, and transfer it to a new 1.5 ml low-absorption centrifuge tube;

[0044] 4) Add 4 times the volume of pre-cooled acetone solution, mix well, and let stand at -20°C for 2 hours (this step can fully precipitate the protein, and the extraction reagent Triton X-100 is removed in the supernatant by centrifugation, which does not interfere with mass spectrometry detection, and does not require multiple washing steps, reducing the processing time.)

[0045] 5) Collect protein precipitate by centrifugation, add 0.1% RapiGest SF solution to resuspend the protein, add 50 mM DTT solution, heat at 55°C for 30 minutes, add 50 mM IAA solution, and react at room temperature in the dark for 30 minutes;

[0046] 6) Add 0.2ug / ul trypsin and heat at 37°C for 2 hours;

[0047] 7) Add formic acid to a final concentration of 1% to terminate the trypsin reaction;

[0048] 8) The peptide fragments obtained in the previous step were desalted using a MonoSpinC18 solid phase extraction column, and the samples were dried using a vacuum dryer for detection using an LCMS liquid spectrometer.

[0049] The results are as follows:

[0050] The extraction method was applied and combined with liquid chromatography-mass spectrometry to achieve a high-depth proteomic analysis of tear samples collected by Schirmer test strips. 4120 and 4032 proteins were identified in the two tear samples, respectively. The identification results are shown in Table 2. The mass spectrometry results of tear sample 1 are shown in Figure 1 As shown, the mass spectrometry results of tear sample 2 are as follows Figure 2 As shown in the figure, the false positive rate is less than 1%, and the sample mass spectrometry detection signal intensity is high, indicating that the sample extraction effect of this method is good and the protein recovery rate is high. The test results of the two samples are comparable, indicating that the method has good repeatability and the results are accurate and reliable. The GO (Gene Ontology) protein function annotation of the 4,000 proteins identified in the tear samples is further performed. The results are as follows Figure 3 As shown, these proteins are involved in a variety of biological processes (Biological Process, BP), including signal transduction, transcriptional regulation, innate immune response, etc. Their cellular localization (Cellular Component, CC) is mainly in the cytoplasm and cytoplasm, and their molecular functions (Molecular Function, MF) mainly involve protein binding, RNA binding, etc.

[0051] Table 2 Statistics of proteomic identification results

[0052]

[0053] 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 trace protein extraction reagent, characterized in that: The reagents include: Triton X-100, ammonium bicarbonate solution, trypsin solution, formic acid, RapiGest SF, iodoacetamide solution and dithiothreitol solution.

2. The reagent according to claim 1, characterized in that The concentration of Triton X-100 is 1%, the solvent is ammonium bicarbonate solution, and the concentration of the ammonium bicarbonate solution is 100 mM.

3. The reagent according to claim 1, characterized in that The trypsin solution is prepared with mass spectrometry grade trypsin, and the formic acid is of mass spectrometry grade.

4. The reagent according to claim 1, characterized in that The concentration of the iodoacetamide solution is 50 mM, and the concentration of the dithiothreitol solution is 50 mM.

5. The reagent according to claim 3, characterized in that The concentration of the trypsin solution is 0.2 ug / ul.

6. A method for extracting trace protein from tear fluid using a trace protein extraction reagent as described in any one of claims 1 to 5 using a Schirmer test paper, characterized in that: The method comprises the following steps: Step 1, take the Schirmer test paper of the wet tear fluid, transfer it to the first low-adsorption centrifuge tube, add Triton X-100 solution, mix and shake at 4°C; centrifuge for the first time, take the supernatant, transfer it to the second low-adsorption centrifuge tube, add 4 times the volume of pre-cooled acetone solution, mix, let it stand at -20°C, centrifuge for the second time, and collect the protein precipitate; Step 2: Add RapiGest SF solution to the protein precipitate obtained in step 1 to resuspend the protein, add DTT solution, heat for the first reaction, then add IAA solution, and react at room temperature in the dark; add trypsin, heat for the second reaction; add formic acid to a final concentration of 1% to terminate the trypsin reaction and obtain a peptide solution; Step 3: Desalt the peptide obtained in step 2 using a MonoSpin C18 solid phase extraction column, and dry it in a vacuum dryer to obtain a dried sample.

7. The method according to claim 6, characterized in that The step 1 also includes: taking 20 mm wet tear Schirmer test paper and cutting it into 2 mm×2 mm small pieces; the concentration of the Triton X-100 solution is 1%; the oscillation time is 1 hour; and the standing time is 2 hours.

8. The method according to claim 6, characterized in that The concentration of the RapiGest SF solution in step 2 is 0.1% resuspended protein, the concentration of the 50mM DTT solution is 50mM, the temperature of the first heating reaction is 55°C, the time of the first heating reaction is 30 minutes, the concentration of the IAA solution is 50mM, and the time of the room temperature light-protected reaction is 30 minutes.

9. The method according to claim 6, characterized in that The concentration of trypsin in step 2 is 0.2 ug / ul, the temperature of the second heating reaction is 37° C., and the time of the second heating reaction is 2 hours.

10. The method according to claim 6, characterized in that The method also includes detecting the dried sample using an LCMS liquid spectrometer.

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