Two-step immobilization and step-by-step separation method of RNA-protein compound

Abstract

The invention discloses a two-step immobilization and step-by-step separation method of an RNA-protein compound, and belongs to the field of analytical chemistry. The protein is crosslinked by utilizing the characteristic that N-hydroxysuccinimide ester (NHS) structures at two ends of a DSP (Digital Signal Processor) structure react with amino groups of the protein, and the complete RPC is enriched and captured from living cells by utilizing the characteristic that 254nm crosslinked RNA and a Psoralen probe capture RNA and are compatible with M-280 magnetic beads. And respectively eluting the RNA indirect binding protein and the RNA direct binding protein which form the RNA-protein compound by utilizing the property that the TCEP breaks the disulfide bond, so as to carry out proteomics mass spectrometry analysis on the RNA binding protein in the RNA-protein compound.

Description

TECHNICAL FIELD

[0001] The present application relates to a two-step fixation, stepwise separation method of RNA-protein complexes, more specifically to a method of using 254 nm ultraviolet cross-linking method and dithiobis succinimidyl propionate (DSP) protein cross-linking agent to fix the RNA-protein complexes (RPC) in two steps, and then using trichloroethyl phosphate (TCEP) to separate them step by step, and its subsequent application, belonging to the field of analytical chemistry. BACKGROUND

[0002] The structural formula of DSP (Lomant reagent) is as follows:

[0003]

[0004] DSP is a water-insoluble, homobifunctional N-hydroxysuccinimide ester (NHS ester) cross-linking agent, which has thiol cleavability and primary amine reactivity, and is suitable for various applications. DSP is usually used as a common protein cross-linking agent and has been widely used in the previous studies for protein cross-linking in cells and tissues, and there is no precedent for its use in RNA-protein complex enrichment.

[0005] With the development of proteomics, the research content of RPC is gradually expanding. First, the mRNA based on poly(A) sequence and complementary chain and its binding protein enrichment method was developed together with high-throughput sequencing technology. This method expands the identification scale of RBP from a single protein to a large-scale identification, but it cannot complete the RBP enrichment of all kinds of RNA because the enrichment handle depends on the poly(A) tail structure of RNA. In order to further expand the types of RNA binding proteins, researchers have developed an enrichment method based on alkyne uridine analog metabolic labeling tandem click chemistry reaction, a phase separation enrichment method based on the separation of RNA, protein and DNA with different densities, and a solid phase extraction method based on silicon affinity. The above three methods can enrich the RBP directly bound to RNA without bias. However, the metabolic labeling method has a long metabolic nucleotide analog labeling period and high experimental cost. The phase separation method still needs to be improved because the boundary is not clear when separating proteins and RNA. In addition, with the deepening of protein interaction research, more and more literature shows that genetic material such as RNA is not the only regulator of protein physiological activity, and protein-protein interaction also plays an important role in cell physiological activity and disease development. At the same time, studies have shown that the formation of RPC is achieved through multivalent RNA-RNA, RNA-protein and protein-protein interactions. Therefore, in addition to the RBP directly interacting with RNA, the RAP formed by protein-protein interaction also has significance for identification and research. Therefore, researchers further expand the identification boundary of RPC and develop R-Deep method based on density gradient centrifugation and RPC enrichment method based on immunopurification. Both methods treat these proteins interacting in RPC as a whole and enrich the RPC without distinguishing whether it is directly interacting with RNA or participating in the RPC through protein interaction, which lacks the accuracy and pertinence of RBP and RAP identification. SUMMARY

[0006] An object of the present application is to provide a method for broad-spectrum enrichment and identification of RNA-protein complexes under physiological conditions.

[0007] The present application aims to establish a two-step fixation and step-by-step separation enrichment method for RNA-protein complexes. The main principle is to crosslink RNA and protein, and protein and protein with 254 nm and DSP protein crosslinking agent respectively, and then separate them by their different chemical separation principles in the elution part. While expanding the RNA-protein interaction network, the accuracy of identifying RNA directly binding proteins is also considered. It can not only qualitatively identify large-scale and unbiased proteins in RNA-protein complexes in a broad sense, but also refine the identification data of RNA directly binding proteins.

[0008] The method for enriching RNA-protein complex under physiological conditions provided by the present application comprises the following steps:

[0009] 1) First, irradiate the cells or tissues with 254 nm ultraviolet light to form covalently cross-linked RNA-protein complex in the cells or tissues;

[0010] 2) Lyse the cells or tissues, and then add DSP reagent to the obtained supernatant for incubation, and then add psoralen-biotin probe to bind RNA under 365 nm ultraviolet light to facilitate the next step of enrichment;

[0011] 3) Incubate the complex sample with streptavidin-modified magnetic beads, collect the magnetic beads after the incubation, and clean them to obtain magnetic beads capturing the RNA-protein complex, thereby realizing specific enrichment of the RNA-protein complex.

[0012] In the step 1) of the above method, the power of the 254 nm ultraviolet irradiation is 40 W, and the irradiation time is 0.5-2 min;

[0013] In the step 2), the working concentration of the DSP reagent is 1 μM; the incubation time is 30 min, and the incubation temperature is room temperature; the power of the 365 nm ultraviolet irradiation is 150 W, and the irradiation time is 1-5 min;

[0014] In the step 3), the magnetic beads are collected by magnetic separation;

[0015] The cleaning method is as follows: sequentially clean once with 200 μL 0.2% (w / v) SDS PBS solution, 200 μL 8M urea PBS solution, and 200 μL PBS solution, respectively.

[0016] The application of the DSP reagent in the enrichment of RNA-protein complex and the mass spectrometric identification of RNA-binding proteins also belongs to the protection scope of the present application.

[0017] In the application, the RNA-protein complex comprises RNA direct-binding proteins (RNA-Binding Proteins, RBP) and RNA indirect-binding proteins (RNA-Associated Proteins, RAP);

[0018] Alternatively, the RNA-protein complex is the RNA-protein complex in a cell sample.

[0019] Another object of the present application is to provide a method for proteomic mass spectrometric analysis of RNA-binding proteins in the RNA-protein complex.

[0020] The method for proteomic mass spectrometry analysis of RNA-binding proteins in RNA-protein complexes provided by the present application comprises the following steps:

[0021] al) enriching the RNA-protein complexes in cells or tissues by using the above method to obtain magnetic beads capturing the RNA-protein complexes;

[0022] b1) adding a TCEP reducing agent to the magnetic beads of the RNA-protein complexes to break the disulfide bonds of DSP, so as to separate the RNA indirect binding proteins; then adding RNase to break the RNA and release the RNA direct binding proteins, so as to sequentially elute the RNA indirect binding proteins and the RNA direct binding proteins, and perform mass spectrometry analysis on them respectively.

[0023] The above method further comprises the following steps of setting a control group: after the enrichment in step a1) is completed, adding RNAse to the magnetic beads of the RNA-protein complexes in the washing step to remove the RNA-binding proteins, so that only the non-specifically adsorbed non-RNA-binding proteins are retained in the control group; the quantitative difference of the identified proteins in the experimental group and the control group is compared by mass spectrometry analysis, and after the non-specifically adsorbed non-RNA-binding proteins are deducted, the RNA-binding proteins with high confidence can be obtained.

[0024] The method provided by the present application has the following process (see Figure 1 ) and principles:

[0025] First, the interaction between RNA and direct binding proteins is fixed by 254 nm ultraviolet crosslinking; then, the protein-protein crosslinking agent disulfide bis-succinimidyl propionate (DSP) is innovatively introduced, and the RNA indirect binding proteins interacting with the RNA direct binding proteins are fixed by using the stable covalent bond formed by the reaction between the N-hydroxysuccinimide (NHS) end of DSP and the N-terminal and side chain amino groups of the proteins; then, the complete RNA-protein complexes are enriched from cells by using the covalent coupling between the psoralen end of the psoralen-biotin probe (PP probe) and the pyrimidine bases under the activation of 365 nm ultraviolet light, and the specific adsorption between the biotin end and the streptavidin magnetic beads; in the elution stage: first, the TCEP reducing agent is added to break the disulfide bonds of DSP, so as to separate the RNA indirect binding proteins; then, the RNase enzyme is added to break the RNA and release the RNA direct binding proteins, so as to achieve the purpose of stepwise separation of the RNA direct binding proteins and the RNA indirect binding proteins; finally, the two are subjected to proteomic analysis based on mass spectrometry, and the qualitative and quantitative information of the RNA direct binding proteins and the RNA indirect binding proteins is obtained.

[0026] The present application utilizes the characteristics of N-hydroxysuccinimide ester (NHS) structure at both ends of DSP structure and the reaction of amino group of protein to cross-link protein, and then utilizes 254nm cross-linking RNA and Psoralen probe to capture RNA and the characteristics of affinity with M-280 magnetic beads to enrich the captured complete RPC from living cells. Then, the properties of TCEP to break disulfide bond are utilized to elute the indirect binding protein and the direct binding protein of RNA in the RNA-protein complex, so as to perform proteomic mass spectrometry analysis on the RNA binding protein in the RNA-protein complex. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The flow chart of two-step fixation and step-by-step separation of RNA-protein complex in the present application.

[0028] Figure 2 In the figure, A is the silver staining diagram of DSP cross-linking whole protein in cells; B is the silver staining diagram of DSP cross-linking RNA-protein complex in cells; C is the silver staining diagram of RNA-protein complex separated by TCEP; and D is the silver staining diagram of RNA-protein separated by TCEP after DSP cross-linking.

[0029] Figure 3 In the figure, A is the scatter plot of data correlation in the data set of RNA direct binding protein; B is the scatter plot of data correlation in the data set of RNA indirect binding protein; C is the volcano plot of RNA direct binding protein (protein quantitative difference diagram of experimental group and control group); and D is the volcano plot of RNA indirect binding protein (protein quantitative difference diagram of experimental group and control group).

[0030] Figure 4 In the figure, A is the GO (Gene ontology) analysis diagram of RNA direct binding protein; B is the GO analysis diagram of RNA indirect binding protein; and C is the overlap Wayne diagram of RNA direct binding protein and known direct binding protein of human. DETAILED DESCRIPTION

[0031] The present application will be further described in detail below in combination with specific embodiments. The examples provided below are only for illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0032] In the following examples, the experimental methods are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0033] Example

[0034] After the HeLa cells cultured in 15 cm dish were grown, they were washed with 5 mL of cold PBS for three times, and then the liquid in the dish was completely absorbed. The dish was placed in a 254 nm UV crosslinking instrument (light energy was 0.25 J / cm2) on ice for crosslinking. Then the 100x DSP (40.44 mg / mL) was diluted with PBS to 1x DSP, and 3 mL of 1x DSP was added to each dish of cells for incubation at room temperature for 30 minutes. After the incubation was completed, 2 mL of cold PBS was added to each dish of cells, and the cells were collected with a cell scraper. The collected cells were placed in a 2 mL centrifuge tube, and the precipitate was obtained by centrifugation at 1000 G for 5 minutes. The solutions used thereafter were prepared using DEPC-treated water.

[0035] After the cell pellet was obtained, 250 μΐ of lysis buffer 1 (1x PBS, pH 7.4, final concentration of 0.5% SDS, RNAse inhibitor, proteinase inhibitor without EDTA) was added. The sample was homogenized by drawing up and down with a 1 mL syringe and incubated at 4°C with gentle rotation for 20 min. Then, 1 mL of lysis buffer 2 (1x PBS, pH 7.4, final concentration of 0.2% Triton; RNAse inhibitor, proteinase inhibitor without EDTA) was added. The sample was homogenized by drawing up and down with a 1 mL syringe and incubated at 4°C with gentle rotation for 20 min, followed by centrifugation at 14000G for 10 min. The supernatant was transferred to a six-well plate. The final concentration of 5 μΜ PP probe was added and incubated at 4°C with gentle shaking for 30 min in the dark. Then, the sample was cross-linked with UV light at 365 nm for 3 min on ice to complete the labeling of RNA with the PP probe. After the labeling was completed, the sample was centrifuged at 14000G and the supernatant was transferred to a 10 kDa ultrafiltration tube. The supernatant was washed with 1x PBS three times to remove excess PP probe. The volume of the supernatant in the ultrafiltration tube was adjusted to 500 μΐ with 2 M UA (1x PBS) and transferred to a 1.5 mL RNase-free centrifuge tube. Then, 50 μΐ of pre-treated streptavidin magnetic beads were added and incubated at 4°C with gentle rotation for 1 h. After the enrichment was completed, the control magnetic beads and the experimental magnetic beads were treated separately. First, the control magnetic beads were separated from the supernatant using a magnetic stand, and the supernatant was discarded. Then, the control magnetic beads were washed twice with 200 μΐ of 1x PBS and then 200 μΐ of pre-prepared RNase (0.01 μg / μΐ, prepared with pure water) was added. After shaking at 37°C in a metal bath for 1 h, the supernatant was discarded. The control magnetic beads were washed twice with 0.2% SDS, 2 M UA, 6 M UA, and 50 mM TEAB, respectively. The experimental magnetic beads were washed twice with 1x PBS, 0.2% SDS, 2 M UA, 6 M UA, and 50 mM TEAB, respectively, to remove non-specific adsorption of the magnetic beads (all the above reagents were prepared with RNase-free PBS solution). After the washing was completed, 25 μΐ of 100 mM TCEP (pH 8.5) was added to the experimental and control groups at the same time, and the sample was shaken at 37°C in a metal bath for 1 h. The RAP in the RPC was separated first, and the supernatant was collected. Then, 25 μΐ of pre-prepared RNase (0.01 μg / μΐ, prepared with 50 mM TEAB) was added to the magnetic beads and shaken at 37°C in a metal bath for 1 h to elute the RBP (the elution steps for the control and experimental groups were identical).

[0036] The experimental group RAP and the experimental group RBP were obtained according to the above steps. For the control group, the previous cell cross-linking, lysis, labeling, and enrichment steps were completely consistent with the experimental group, except that: after enrichment, the control group was first incubated with 0.1 μg / μL of RNase A solution at 37°C for 1 h, the purpose of which was to break the RNA chain to elute all RNA-related proteins, facilitating the differential screening with the experimental group later. After completing the SDS and UA washing synchronously with the experimental group, 20 μL of 100 mM TCEP solution was first added to the supernatant as the control of RAP; then 20 μL of 0.01 μg / μL RNase A solution was added to resuspend the magnetic beads, which served as the control of RBP. After that, the experimental group and the control group were added with 80 μL of 50 mM TEAB solution to 100 μL of the system, and then the proteomics sample preparation link was performed, with the specific steps as follows: after adding CAA at a final concentration of 40 mM and TCEP at a final concentration of 20 mM for reduction alkylation reaction in 37°C metal bath shaking for 1.5 h, an appropriate amount of trypsin solution was added at a ratio of 1:100, and the enzyme digestion was performed at 37°C for 12 h. After 12 h, the same amount of trypsin was added, and the enzyme digestion was continued at 37°C for 4 h. Subsequently, the magnetic beads were magnetically separated from the supernatant, and the supernatant was collected. The magnetic beads were washed with 50 μL of 0.2% TFA solution twice, and the washing liquid and the supernatant (200 μL in total) were collected into a 1.5 mL protein low-absorption centrifuge tube, and then vacuum rotary evaporator drying was performed at 45°C to obtain the final peptide fragments. Then, the peptide fragments were subjected to stable isotope dimethyl labeling: first, the peptide fragments were resuspended in 200 μL of 100 mM TEAB solution, 8 μL of 4% CH2O solution was added to the experimental group RBP and RAP, and 8 μL of 4% CD2O solution was added to the control group RAP and RBP. Meanwhile, 8 μL of 0.6 M NaBH3CN solution was added to each sample, which was vortexed and mixed, and then reacted at room temperature for 1 h. After the reaction was completed, 4 μL of 30% ammonia water solution was added to each sample to terminate the reaction. Finally, 4 μL of 10% TFA solution was added to each sample to acidify the sample, and then the light and heavy isotope-labeled experimental group and control group [RBP control group (heavy label) + experimental group (light label) / RAP control group (heavy label) + experimental group (light label)] were mixed. Then, StageTip C18 desalting was performed, and vacuum rotary evaporator drying was performed, and then the samples were stored at -80°C for mass spectrometry analysis.

[0037] The peptide segments obtained according to the above operation were redissolved in a 0.1% TFA solution, centrifuged at 14000G for 10 min, and the supernatant was loaded on a 2 cm self-packed pre-column (100 μM in diameter, packed with C18 filler with an inner diameter of 3 μM), and at the same time, a self-made reversed-phase analytical column (150 μM in diameter and 15 cm in length, packed with Ultimate XB-C18 filler with a particle size of 1.9 μM) was used for separation. An Easy-nLC1000 nanoliter liquid chromatography system was used, and the liquid phase gradient was 78 min (mobile phase A: 0.1% FA aqueous solution, mobile phase B: ACN solution containing 0.1% FA, flow rate: 600 nL / min), elution gradient: 0-8 min, 5%-8% B; 8-58 min, 8%-22% B; 58-70 min, 22%-32% B; 70-71 min, 32%-90% B; 71-78 min, 90% B. The eluted peptide segments entered the Orbitrap FusionTM TribidTM mass spectrometer in turn through the ion source. The spray voltage was 2 kV, the data acquisition mode was Data-Dependent Acquisition (DDA), the scanning method was positive ion scanning mode, the ion transmission tube temperature was set to 320°C, the full scan detection range was 300-1400 m / z, the scanning resolution was 120000, the maximum injection time was 100 ms, the automatic gain control (AGC) was set to 5e5, the parent ion was fragmented into secondary fragments by Higher-Energy Collisional Discussion (HCD) with a collision energy of 32%, and the secondary data acquisition was performed using the linear ion trap fast mode, the AGC was set to 5000, the maximum injection time was set to 35 ms, and the dynamic exclusion time was set to 18 s.

[0038] The raw files obtained by mass spectrometry were further analyzed using MaxQuant software (version 1.16.7.0), and the Uniprot Human (Release on 2022, 20198 entry) database was used for retrieval. The enzyme digestion mode was trypsin, the maximum allowed missed cleavage sites were set to 2, the minimum number of amino acids contained in each peptide was 6, cysteine carbamidomethylation was a fixed modification, methionine oxidation and N-terminal acylation were set as variable modifications. In terms of dimethylation labeling, the number of labels checked was 2, the light label checked DimethLys0 and DimethNter0; the heavy label checked DimethLys4 and DimethNter4, the maximum mass tolerance of parent ions and secondary fragment ions was 20 ppm and 0.5 Da, and the false positive rate (False Discovery Rate, FDR) of protein level and spectrum was less than or equal to 1%. According to the principle of normal distribution, the missing values were filled, and then the Persus software was used to calculate the enrichment fold (Fold Change, the ratio of signal intensity of the experimental group to the control group) of each protein and the P-Value value to represent the degree of T-test test calculation enrichment significance. The smaller the P-Value, the more significant the difference between the experimental group and the control group. The data obtained was considered to be a high-confidence RPC according to the following principles: 1. At least two proteins identified in three experiments; 2. The protein contains at least 2 unique peptides; 3. The enrichment fold of the experimental group / control group is not less than 2 times (Fold change) and P_Value is less than 0.01.

[0039] The identified RPCs were subjected to GO analysis using the UNIPROT_ACCESSIOM database through the online software DAVID (https: / / david.ncifcrf.gov / tools.gvp), and pathway analysis was performed through Reactome. The Origin software was used to draw the volcano plot of the data. The correlation analysis diagram was drawn through the online website BioLadder (https: / / www.bioladder.cn / ).

[0040] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A method for enriching RNA-protein complex under physiological conditions, comprising the following steps: 1) irradiating cells or tissues with 254 nm ultraviolet light to form covalently cross-linked RNA-protein complex in the cells or tissues; 2) lysing the cells or tissues and adding DSP reagent to the supernatant obtained to incubate, and then adding psoralen-biotin probe to bind to RNA under 365 nm ultraviolet light to facilitate the next step of enrichment; 3) incubating the complex sample with streptavidin-modified magnetic beads, collecting the magnetic beads after incubation and washing to obtain magnetic beads capturing RNA-protein complex, thereby realizing specific enrichment of RNA-protein complex.

2. The method of claim 1, wherein, In step 1), the power of the 254 nm ultraviolet irradiation is 40 W, and the irradiation time is 0.5-2 min; In step 2), the working concentration of the DSP reagent is 1 μM; the incubation time is 30 min, and the incubation temperature is room temperature; the power of the 365 nm ultraviolet irradiation is 150 W, and the irradiation time is 1-5 min; In step 3), the magnetic beads are collected by magnetic separation; The washing method is as follows: sequentially washing once with 200 μL of 0.2% (w / v) SDS in PBS, 200 μL of 8 M urea in PBS, and 200 μL of PBS, respectively.

3. Application of DSP reagent in RNA-protein complex enrichment and RNA-binding protein mass spectrometry identification.

4. Use according to claim 3, characterized in that, In the application, the RNA-protein complex includes RNA directly binding protein and RNA indirectly binding protein; Or, the RNA-protein complex is the RNA-protein complex in a cell sample.

5. A method for proteomic mass spectrometry analysis of RNA-binding protein in RNA-protein complex, comprising the following steps: al) enriching the RNA-protein complex in cells or tissues by using the above method to obtain magnetic beads capturing RNA-protein complex; b1) adding TCEP reducing agent to the magnetic beads of the RNA-protein complex to break the disulfide bond of DSP, thereby separating out RNA indirectly binding protein; then adding RNase enzyme to break RNA, thereby releasing RNA directly binding protein, so as to sequentially elute out RNA indirectly binding protein and RNA directly binding protein, and respectively performing mass spectrometry analysis.

6. The method of claim 5, wherein, The method further comprises the step of setting a control group, specifically as follows: after the enrichment in step a1) is completed, adding RNAse to the magnetic beads of the RNA-protein complex during the washing step to remove RNA-binding protein, so that only non-specifically adsorbed non-RNA-binding protein is retained in the control group; by comparing the quantitative difference of the identified proteins in the experimental group and the control group through mass spectrometry analysis, and deducting the non-specifically adsorbed non-RNA-binding protein, the RNA-binding protein with high confidence can be obtained.

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