Proteome targeted enrichment method

Abstract

The invention discloses a targeted enrichment method for proteome. According to the cell nucleus targeted RNA binding protein enrichment reagent, one end is provided with a furocoumarin group, the other end is provided with a cell nucleus targeted group, and the other end is provided with biotin; the enrichment or identification of the RNA binding protein in the cell nucleus can be carried out. The method for enriching the nucleus-targeted RNPs by adopting the nucleus-targeted RNA binding protein enrichment reagent comprises the following steps: firstly, adding the nucleus-targeted RNA binding protein enrichment reagent into cells for co-incubation, then, irradiating a cell incubation system by adopting ultraviolet light with the wavelength of 254nm, and then, irradiating the cell incubation system by adopting ultraviolet light with the wavelength of 365nm, then carrying out cracking to obtain a cracking solution; incubating the lysis solution and streptavidin magnetic beads, and finally collecting the magnetic beads enriched with the cell nucleus RNPs, so that the cell nucleus targeted RNPs are enriched. According to the method, the cell nucleus positioning of the reagent can be efficiently realized, the RBPs enrichment selectivity and the negative electrode efficiency are improved, and the varieties of RNPs obtained through enrichment are more comprehensive.

Description

Technical Field

[0001] The present invention relates to a proteome targeted enrichment method, and belongs to the field of analytical chemistry. Background Art

[0002] Interactions between RNA and proteins play a wide range of crucial roles in cellular processes. RNA-protein complexes (RNPs) formed by these interactions regulate various aspects of RNA metabolism, including physiological conditions and pathological states, and participate in the pathogenesis of numerous human diseases. Large-scale analysis of the types and intracellular localization of RNA-binding proteins (RBPs) within RNPs is of great significance for physiological and pathological research. However, due to the low abundance and poor homogeneity of endogenous RBPs, the precise enrichment and identification of RBPs located in the cell nucleus makes the already difficult task of RBP enrichment even more challenging. In recent years, orthogonal organic phase separation (OOPS) has become a widely adopted method for enriching intracellular RBPs in cell lysates. However, this method has inherent drawbacks due to glycoprotein contamination and is significantly limited by its inability to specifically enrich and identify RBPs in the nucleus. To address these issues, a method has recently been developed that first crosslinks RNA-RBPs using 254nm ultraviolet irradiation, then uses oligo(dT) to bind to the poly(A) residues at the ends of mRNAs to capture RNPs for enrichment and identification. This method addresses the difficulties in enriching and purifying RBPs to some extent, but it still has the limitation of only enriching mRNA-RBP complexes within RNPs and the technical bottleneck of being unable to enrich and identify RBPs localized in the cell nucleus. Summary of the Invention

[0003] The purpose of the present invention is to provide a proteome targeted enrichment method, specifically to provide a nucleus-targeted RNA-binding protein enrichment reagent and its preparation method, and applications: a proteome targeted enrichment method, a method for sequencing and analyzing RNA in nucleus RNPs.

[0004] The present invention provides a compound as a nucleus-targeted RNA-binding protein enrichment reagent (BNlsTP reagent for short). The compound comprises three parts: a furanocoumarin group, a nucleus-targeting group, and a biotin group. The furanocoumarin group can specifically bind to uracil on RNA and achieve covalent cross-linking under 365nm ultraviolet light. The nucleus-targeting group can introduce a probe into the nucleus of a living cell. The biotin group can capture the reagent by binding to streptavidin magnetic beads. The compound can enrich or identify RNA-binding proteins in the nucleus.

[0005] The present invention provides a compound, the structural formula of which is shown in Formula 1 below:

[0006]

[0007] The present invention also provides a method for preparing the above-mentioned compound, comprising the steps of: reacting the compound represented by formula 2 with

[0008] The compound represented by Formula 3 is subjected to a cycloaddition reaction to obtain the compound represented by Formula 1;

[0009]

[0010] In the above method, the molar ratio of the compound represented by Formula 2 to the compound represented by Formula 3 may be 1:1-2, specifically 1:1.5;

[0011] The cycloaddition reaction is carried out in an organic solvent, which is acetonitrile, methanol, dichloromethane or DMF;

[0012] The conditions of the cycloaddition reaction are as follows: the temperature can be 4-30°C, specifically room temperature (10-30°C, such as 25°C), and the time can be 2-48h, specifically 4h, 2-48h, 2-4h, 4-48h, 3-20h or 2-30h.

[0013] In the above method, the compound represented by Formula 2 is prepared by a method comprising the following steps: using the peptide segment CPKKKRKVYGRKKRRQRRR represented by Formula 4 as a starting material and performing an amidation reaction with biotin to prepare the compound represented by Formula 2;

[0014]

[0015]

[0016] The amidation reaction is carried out in an organic solvent, which is acetonitrile, methanol, dichloromethane or DMF, preferably DMF.

[0017] In the present invention, the molar ratio of the peptide segment represented by formula 4 to the biotin is 1:1 to 1:10, specifically 1:3;

[0018] The conditions for the amidation reaction between the peptide segment represented by Formula 4 and the biotin can be conventional conditions in the art; the specific operation of the amidation reaction is: first, mix the peptide segment represented by Formula 4 and biotin, and shake at room temperature overnight to react.

[0019] In the above method, the compound represented by Formula 3 is prepared by a method comprising the following steps: amidating a maleimide tetraethylene glycol amino group represented by Formula 5 with a 4-furanocoumarin n-butyric acid ether represented by Formula 6 to prepare the compound represented by Formula 3;

[0020]

[0021] The amidation reaction is carried out in the presence of saturated sodium bicarbonate;

[0022] The amidation reaction is carried out in an organic solvent, which is acetonitrile, methanol, dichloromethane or DMF, preferably DMF.

[0023] In the present invention, the molar ratio of the maleimide tetraethylene glycol amino group represented by Formula 5 to the 4-furocoumarin n-butyric acid ether represented by Formula 6 can be 1:1 to 5:1, specifically 2:1;

[0024] The conditions for the amidation reaction of the maleimide tetraethylene glycol amino group represented by Formula 5 and the 4-furocoumarin n-butyric acid ether represented by Formula 6 are conventional conditions in the art; the specific operation of the amidation reaction is: dissolving the compound represented by Formula 5 in saturated sodium bicarbonate, dissolving the compound represented by Formula 6 in DMF and then adding dropwise to the reaction solution, reacting at room temperature (specifically 25° C.) overnight (specifically 12 hours), extracting the product with dichloromethane, and then drying and concentrating to obtain a crude product, and separating on a preparation plate to obtain the final product.

[0025] The present invention also provides a nucleus-targeted RNA-binding protein enrichment reagent, which comprises the compound represented by the above formula 1.

[0026] The compound represented by Formula 1 above is composed of a furanocoumarin group, a cell nucleus targeting group, and a biotin group, and can be used as an RNA-binding protein enrichment reagent (referred to as BNlsTP reagent). This reagent achieves the enrichment and mass spectrometry identification of RNA-binding proteins in the cell nucleus through the cell membrane penetration of the cell nucleus targeting group and the specific recognition of importin α and importin β on the cell nuclear membrane, the photocrosslinking reaction of the furanocoumarin group with uracil in RNA, and the binding of biotin to streptavidin magnetic beads.

[0027] Use of the nucleus-targeted RNA-binding protein enrichment reagent of the present invention in any of the following 1)-3):

[0028] 1) Enrichment of nucleus-targeted RNPs;

[0029] 2) Proteomic mass spectrometry analysis of RBPs in nuclear RNPs;

[0030] 3) Sequencing and analysis of RNA in nuclear RNPs,

[0031] The present invention also provides a proteome targeted enrichment method, comprising the following steps: first, adding the nucleus-targeted RNA-binding protein enrichment reagent to cells for co-incubation, then irradiating the cell incubation system with 254nm wavelength ultraviolet light, then irradiating the cell incubation system with 365nm wavelength ultraviolet light, and then lysing to obtain a lysate; incubating the lysate with streptavidin magnetic beads, and finally collecting the magnetic beads enriched with nucleus RNPs to enrich the nucleus-targeted RNPs.

[0032] In the present invention, the streptavidin magnetic beads are prepared by directly mixing streptavidin and magnetic beads; wherein the magnetic beads are commercially available to those skilled in the art.

[0033] In the above enrichment method, the number of cells can be 10 6 ~10 8 indivual;

[0034] The co-incubation conditions of the cells and the cell nucleus-targeted RNA-binding protein enrichment reagent are as follows: the incubation time can be 10 to 300 minutes; the incubation temperature can be 4 to 37° C.; the working concentration of the cell nucleus-targeted RNA-binding protein enrichment reagent can be 1 to 100 μM, specifically 5 μM, 1 to 5 μM, 5 to 100 μM, 1 to 50 μM or 1 to 80 μM;

[0035] The power of the 254nm wavelength ultraviolet light irradiation can be 40W, and the irradiation time can be 0.5 to 5 minutes, specifically 1.5 minutes, 0.5 to 1.5 minutes, 1.5 to 5 minutes or 1 to 3.5 minutes;

[0036] The power of the 365nm wavelength ultraviolet light irradiation can be 150W, and the irradiation time can be 0.5 to 5 minutes, specifically 1.5 minutes, 0.5 to 1.5 minutes, 1.5 to 5 minutes or 1 to 3.5 minutes;

[0037] The working concentration of the streptavidin magnetic beads can be 0.01-10 mg / mL, specifically 1 mg / mL, 0.01-1 mg / mL, 1-10 mg / mL, 0.05-5 mg / mL or 0.05-8.5 mg / mL.

[0038] The present invention also provides a method for proteomic mass spectrometry analysis of RBPs in nuclear RNPs, comprising the following steps: first, adding the nuclear-targeted RNA-binding protein enrichment reagent to cells for co-incubation, then irradiating the cell incubation system with 254 nm ultraviolet light, then irradiating the cell incubation system with 365 nm ultraviolet light, and then lysing to obtain a lysate; incubating the lysate with streptavidin magnetic beads, and finally collecting the magnetic beads enriched with nuclear RNPs;

[0039] RNAse is added to the magnetic beads enriched with nuclear RNPs to release the RBPs in the RNPs and perform proteomic mass spectrometry analysis.

[0040] In the above method, the working concentration of the RNase is specifically 0.1-100 ng / L.

[0041] The present invention further provides a method for sequencing and analyzing RNA in nuclear RNPs, comprising the following steps: first, adding the nuclear-targeted RNA-binding protein enrichment reagent to cells for co-incubation, then irradiating the cell incubation system with 254 nm ultraviolet light, then irradiating the cell incubation system with 365 nm ultraviolet light, and then lysing to obtain a lysate; incubating the lysate with streptavidin magnetic beads, and finally collecting the magnetic beads enriched with nuclear RNPs;

[0042] A competitive elution solution is added to the magnetic beads enriched with nuclear RNPs to elute the RNPs as a whole, and then proteinase K is added to degrade RBPs, and then RNA sequencing analysis is performed.

[0043] In the above method, the competitive eluent is a biotin eluent;

[0044] The biotin eluent is composed of the following components: 12.5 mM biotin, 75 mM sodium chloride, 7.5 mM Tris-HCl, 1.5 mM EDTA, 0.15% SDS (sodium dodecyl sulfate), 0.075% sodium lauryl sarcosinate and 0.02% sodium deoxycholate; the above components are all in the same system, and % are w / v.

[0045] In the above method, the working concentration of proteinase K can be 10-100 ng / μL, specifically 50 ng / μL.

[0046] The present invention further provides a compound that can be used as a nucleus-targeted RNA-binding protein enrichment reagent (BNlsTP reagent for short), which includes the following three parts: a furanocoumarin group, a nucleus-targeting group, and a biotin group.

[0047] In the above-mentioned compounds, the cell nucleus targeting group can penetrate the cell membrane and can specifically recognize importin α and / or importin β on the cell nuclear membrane.

[0048] In the above compounds, tetraethylene glycol serves as a flexible connecting arm between the furanocoumarin group and the biotin group.

[0049] The present invention has the following advantages:

[0050] 1. Compared with the physical method of enriching RBPs through OOPS at the cell lysate level, this method can not only directly label and enrich RBPs in cells, maintain the intracellular environment to the greatest extent, and reproduce the localization and physiological state of RBPs in cells, but also specifically bind to RBPs through photochemical cross-linking, thereby improving the selectivity of RBPs enrichment.

[0051] 2. Compared with the method of enriching and identifying RBPs by capturing RNPs through the pairing and binding of oligo(dT) to the poly(A) at the end of mRNA at the cell lysate level, this method can achieve unbiased direct labeling and enrichment of RNPs in cells, making the types of enriched RNPs more comprehensive, especially the enrichment and identification of non-coding RNPs, which is of great significance in the field of epigenetic research.

[0052] 3. The BNlsTP reagent has the dual functions of penetrating the cell membrane and targeting the cell nucleus, breaking through the difficulty of existing reagents in penetrating the membrane and being able to efficiently achieve the cell nuclear localization of the reagent, which is of great significance for the discovery of RBPs with new functions in the cell nucleus.

[0053] 4. The use of tetraethylene glycol as a flexible linker between furanocoumarin and biotin in the BNlsTP reagent greatly reduces steric hindrance, facilitates the function of each functional group, and improves the enrichment efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Flowchart of the method for specific enrichment of RBPs in the cell nucleus.

[0055] Figure 2 The figure is an SDS-polyacrylamide gel electrophoresis diagram of the RNPs enrichment product in HeLa cells using the method of the present invention.

[0056] Figure 3 (A) is a volcano plot of RBPs in HeLa cells identified by the method of the present invention, and (B) is the cellular component analysis of the GO analysis of the RBPs identified that meet the chi-value criteria, and the 10 most enriched entries are obtained. The horizontal axis is the -log P value.

[0057] Figure 4 This is a distribution diagram of RNA species obtained by RNA enrichment and sequencing analysis of RNPs in HeLa cell nuclei using the method of the present invention. DETAILED DESCRIPTION

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0059] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0060] The BNlsTP reagent in the following examples has a structural formula as shown in Formula 1:

[0061]

[0062] The preparation method of the BNlsTP reagent comprises the following steps: in acetonitrile, a compound represented by Formula 2 and a compound represented by Formula 3 at a molar ratio of 1:1.5 are subjected to a cycloaddition reaction at room temperature (25° C.) in the absence of light for 4 hours to obtain a compound represented by Formula 1;

[0063]

[0064]

[0065] Furthermore, the compound represented by formula 2 is prepared by a method comprising the following steps: using the peptide segment CPKKKRKVYGRKKRRQRRR represented by formula 4 at a molar ratio of 1:3 as a starting material and performing an amidation reaction with biotin (shaking at room temperature 25°C overnight for 12 hours) to prepare the compound represented by formula 2;

[0066]

[0067] Furthermore, the compound represented by Formula 3 is prepared by a method comprising the following steps: dissolving the maleimide tetraethylene glycol amino group represented by Formula 5 in saturated sodium bicarbonate, dissolving the 4-furanocoumarin n-butyric acid ether represented by Formula 6 (the molar ratio of the maleimide tetraethylene glycol amino group represented by Formula 5 to the 4-furanocoumarin n-butyric acid ether represented by Formula 6 is 2:1) in DMF, and then adding dropwise to the reaction solution. After reacting at room temperature (specifically 25° C.) overnight (specifically 12 hours), the product is extracted with dichloromethane, and then dried and concentrated to obtain a crude product to prepare the compound represented by Formula 3;

[0068]

[0069] Example 1. Evaluation of the effect of BNlsTP reagent in enriching RNPs in Hela cells

[0070] When the HeLa cell confluence rate in a 15 cm culture dish reaches approximately 80%, remove the culture medium from the culture dish and rinse the cells three times with 10 mL of PBS each time. Add 2 mL of 0.05% (w / v) Trypsin-EDTA to the culture dish and incubate in a 37°C incubator for 2 minutes. Add fresh culture medium to stop the digestion and collect the cells in a 15 mL RNase-free centrifuge tube. Centrifuge at 1000 g for 3 minutes and discard the supernatant. Add 2 mL of culture medium containing 5 μM BNlsTP reagent (the solvent is an aqueous solution containing 75% acetonitrile) to allow approximately 10 7 After the cells are completely dispersed, the cell suspension is placed in an RNase-free six-well plate and incubated in a 37°C incubator for 10 minutes. After incubation, the suspension is centrifuged at 1000g for 3 minutes, the supernatant discarded, and 2 mL of ice-cold PBS is added to redisperse the cells in an RNase-free six-well plate. The six-well plate is placed on ice and cross-linked under UV light at 254 nm and 40 W for 1.5 minutes. The cells are then collected in a 2 mL RNase-free centrifuge tube and centrifuged at 1000g for 3 minutes, the supernatant discarded. The cells are then redispersed in an RNase-free six-well plate with 2 mL of ice-cold PBS. The six-well plate is placed on ice and cross-linked under UV light at 365 nm and 150 W for 1.5 minutes. The cells are then collected in a 2 mL RNase-free centrifuge tube and centrifuged at 1000g for 3 minutes, the supernatant discarded. After adding 250 μL of whole-cell lysis buffer (0.5% w / v SDS), the cells were thoroughly homogenized using a syringe with a fine needle (0.7 mm). After adding 1 mL of diluent and homogenizing again, the cells were centrifuged at 16,000 g for 15 min. The supernatant was transferred to a 2 mL RNase-free centrifuge tube, and streptavidin magnetic beads were added to a final concentration of 0.5 μg / μL. The cells were incubated at 4°C for 1 h. After magnetic separation and discarding the supernatant, the beads were washed 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. After magnetic separation and discarding the supernatant, 20 μL of 0.01 μg / μL RNase A in PBS was added and the cells were incubated at 37°C for 1 h. After magnetic separation, the supernatant was removed, 5 μL of 5× loading buffer was added, and the cells were denatured at 95°C for 10 min. Finally, SDS-polyacrylamide gel electrophoresis was performed.

[0071] like Figure 2As shown, the three groups that did not use 254nm for RNA-RBP crosslinking, did not use 365nm for BNlsTP reagent-RNA crosslinking, and did not use BNlsTP reagent did not show obvious protein bands, while the experimental group showed a large number of protein bands. This shows that the BNlsTP reagent of the present invention has good selectivity for RNPs and a significant enrichment effect.

[0072] Example 2: Proteomic Mass Spectrometry Analysis of RBPs Enriched in Hela Cell Nuclei Using BNlsTP Reagent

[0073] In Example 1 of the present invention, after magnetic separation and removal of the supernatant, trypsin hydrolysis and mass spectrometry analysis can be performed. The specific steps are as follows: add 10mM TCEP and 50mM CAA and incubate at room temperature for 40min for reductive alkylation treatment, take the denatured protein and add 1μg trypsin, place it in a 37°C constant temperature box and incubate for 16 hours to obtain the enzymatic hydrolysis product peptide segment. The enzymatic hydrolysis product peptide segment is desalted and eluted by a C18Zip-Tips desalting column and then freeze-dried for use. In order to obtain more accurate and reliable RBPs identification results, the control group samples were subjected to exactly the same experimental conditions except that the BNlsTP reagent was not added.

[0074] Mass spectrometry analysis: Nanoliter liquid chromatography (EASY-nLC 1000) was coupled with a biomass spectrometer (Orbitrap FusionTribrid) to perform mass spectrometry analysis on the enzymatic peptide fragments. The liquid chromatography used a column packed with C18 reverse chromatographic filler (filler diameter 1.9 μm, chromatographic column inner diameter 75 μm, column length 30 cm), and the sample was separated at a flow rate of 300 nL / min. The scanning range of the primary mass spectrometry analysis was set to 300-1400 m / z, with a resolution of 120K. The secondary mass spectrometry analysis selected the data-dependent scanning mode, and the energy of the high-energy collision dissociation (HCD) fragmentation mode was set to 32%.

[0075] Mass spectrometry data analysis: The raw files of mass spectrometry data were searched and parsed using MaxQuant software. The protease cleavage mode was set to trypsin, and each peptide was allowed to contain a maximum of 2 missed cleavage sites and a minimum of 6 amino acid residues. Carbamidomethyl-modified cysteine ​​was set as a fixed modification, and oxidized methionine and N-terminal acetyl modification were set as variable modifications. For protein identification, the FDR upper limit was set to 0.01, and each protein needed to be identified with at least two unique peptides to be considered a reliable identification result. The data were screened and the proteins that were identified at least twice in three repeated experiments were quantitatively compared with the corresponding proteins in the control group for differential analysis. The software used for differential analysis was Perseus, which achieved differential protein screening by calculating the average Log2 (enrichment ratio) and P value of proteins in the experimental group and the control group. Proteins with a P value less than 0.01 and an enrichment ratio greater than or equal to 2 were considered to be high-confidence RBPs. Figure 3 As shown, the method provided by the present invention identified 1119 high-confidence RBPs, and the identified RBPs had top-ranked P values ​​among the cell nucleus-related entries.

[0076] Example 3: Sequencing analysis of RNA enriched in RNPs in Hela cell nuclei using BNlsTP reagent

[0077] Using the same experimental conditions as in Example 1 of the present invention, after incubation with streptavidin magnetic beads, multiple washes, and magnetic separation with the supernatant discarded, 400 μL of biotin eluent (12.5 mM biotin, 75 mM sodium chloride, 7.5 mM Tris-HCl, 1.5 mM EDTA, 0.15% SDS, 0.075% sodium lauryl sarcosinate, and 0.02% sodium deoxycholate, all percentages are w / v) was added. The mixture was incubated at room temperature (25°C) with shaking (800 rpm) for 20 minutes, followed by vortexing at 65°C for 10 minutes (800 rpm). After magnetic separation, the supernatant was retained, and the magnetic beads were re-added with the eluent, and the above process was repeated. The resulting supernatant was combined with the retained supernatant. 2 mg / mL proteinase K was added, and the mixture was incubated at 55°C for 1 hour. After complete degradation of the proteins in the complex, RNA was further extracted using TRIzol solution. Each experiment was performed with three independent biological replicates, using 1.5 μg of purified RNA for each replicate. cDNA libraries were constructed using the NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB). Libraries were purified using AMPure XP magnetic beads (Beckman) and quality checked using the Bioanalyzer 2100 system (Agilent). Libraries were sequenced on the Illumina HiSeq 4000 platform using 150bp paired-end sequencing.

[0078] like Figure 4 As shown, the method provided by the present invention can enrich various types of RNA. This shows that compared with the method of enriching and identifying RBPs by pairing oligo(dT) with the poly(A) residue at the end of mRNA to capture RNPs, this method can achieve unbiased direct labeling and enrichment of intracellular RNPs, making the enriched RNP types more comprehensive. In particular, the enrichment and identification of non-coding RNPs is of great significance in the field of epigenetic research.

Claims

1. A compound, characterized in that The structural formula of the compound is shown in Formula 1 below:

2. The method for preparing the compound according to claim 1, characterized in that The method comprises the following steps: performing a cycloaddition reaction on the compound represented by Formula 2 and the compound represented by Formula 3 to obtain the compound represented by Formula 1; 3. The method according to claim 2, wherein: The molar ratio of the compound represented by formula 2 to the compound represented by formula 3 is 1:1-2; The cycloaddition reaction is carried out in an organic solvent, which is acetonitrile, methanol, dichloromethane or DMF; The conditions of the cycloaddition reaction are as follows: temperature of 4 to 30° C., time of 2 to 48 h; and / or, The compound of formula 2 is prepared by a method comprising the following steps: using the peptide CPKKKRKVYGRKKRRQRRR of formula 4 as a starting material and performing an amidation reaction with biotin to prepare the compound of formula 2; The amidation reaction is carried out in an organic solvent, which is acetonitrile, methanol, dichloromethane or DMF; The compound of formula 3 is prepared by a method comprising the following steps: subjecting maleimide tetraethylene glycol amino group of formula 5 to amidation reaction with 4-furanocoumarin n-butyric acid ether of formula 6 to prepare the compound of formula 3; The amidation reaction is carried out in the presence of saturated sodium bicarbonate; The amidation reaction is carried out in an organic solvent, which is acetonitrile, methanol, dichloromethane or DMF.

4. A nucleus-targeted RNA-binding protein enrichment reagent, characterized in that: It is composed of the compound represented by formula 1 according to claim 1.

5. Use of the compound of formula 1 according to claim 1 or the nucleus-targeted RNA-binding protein enrichment reagent according to claim 4 in any of the following 1)-3): 1) Enrichment of nucleus-targeted RNPs; 2) Proteomic mass spectrometry analysis of RBPs in nuclear RNPs; 3) Sequencing and analysis of RNA in nuclear RNPs.

6. A method for targeted proteome enrichment, comprising the following steps: first, adding the nucleus-targeted RNA-binding protein enrichment reagent of claim 4 to cells for co-incubation, then irradiating the cell incubation system with 254 nm ultraviolet light, then irradiating the cell incubation system with 365 nm ultraviolet light, and then lysing to obtain a lysate; incubating the lysate with streptavidin magnetic beads, and finally collecting the magnetic beads enriched with nuclear RNPs to enrich the nucleus-targeted RNPs.

7. The enrichment method according to claim 6, characterized in that: The number of cells is 10 6 ~10 8 indivual; The co-incubation conditions of the cells and the cell nucleus-targeted RNA-binding protein enrichment reagent are as follows: incubation time is 10 to 300 minutes; incubation temperature is 4 to 37° C.; the working concentration of the cell nucleus-targeted RNA-binding protein enrichment reagent is 1 to 100 μM; The power of the 254nm wavelength ultraviolet light irradiation is 40W, and the irradiation time is 0.5 to 5 minutes; The power of the 365nm wavelength ultraviolet light irradiation is 150W, and the irradiation time is 0.5 to 5 minutes; The working concentration of the streptavidin magnetic beads is 0.01-10 mg / mL.

8. A method for performing proteomic mass spectrometry analysis on RBPs in nuclear RNPs, comprising the following steps: using the nuclear-targeted RNA-binding protein enrichment reagent of claim 4 to obtain magnetic beads enriched with nuclear RNPs using the enrichment method of claim 6 or 7; adding RNase to the magnetic beads enriched with nuclear RNPs to release the RBPs in the RNPs, and performing proteomic mass spectrometry analysis; The working concentration of the RNase is specifically 0.1-100 ng / L.

9. A method for sequencing and analyzing RNA in nuclear RNPs, comprising the following steps: using the nuclear-targeted RNA-binding protein enrichment reagent of claim 4 to obtain magnetic beads enriched with nuclear RNPs using the enrichment method of claim 6 or 7; adding a competitive eluent to the magnetic beads enriched with nuclear RNPs to elute the RNPs as a whole; then adding proteinase K to degrade RBPs; and then performing RNA sequencing analysis.

10. A compound characterized by: The compound includes the following three parts: a furanocoumarin group, a cell nucleus targeting group and a biotin group; and / or, The cell nucleus targeting group can penetrate the cell membrane and can specifically recognize importin α and / or importin β on the cell nuclear membrane; and / or, Tetraethylene glycol serves as a flexible linker between the furanocoumarin group and the biotin group.

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