CN110824168B

CN110824168B - Polypeptide probe and application thereof in identifying binding protein modified after translation

Info

Publication number: CN110824168B
Application number: CN201810912796.6A
Authority: CN
Inventors: 张锴; 翟贵金
Original assignee: Tianjin Medical University
Current assignee: Tianjin Medical University
Priority date: 2018-08-10
Filing date: 2018-08-10
Publication date: 2023-06-09
Anticipated expiration: 2038-08-10
Also published as: CN110824168A

Abstract

The invention provides a polypeptide probe based on photocrosslinking and nano metal particle self-assembly technology, wherein a polypeptide with protein post-translational modification is modified on the polypeptide probe, and the affinity recognition and enrichment capture process are integrated, so that the convenient and efficient recognition and identification of protein post-translational modification binding proteins or complexes thereof are realized. The photocrosslinking groups are introduced into the nano metal particles, so that weak acting force between the polypeptide and the binding protein is converted into strong covalent bond action formed by the photocrosslinking groups, stronger elution conditions can be born, and proteins with lower abundance and transient interaction can be captured; and the influence of photo-crosslinking group modification on the polypeptide skeleton is avoided, the original structure and the recognition characteristic of the polypeptide skeleton are well maintained, and the influence on the binding specificity of the corresponding binding protein is greatly reduced. The invention also provides a preparation method of the polypeptide probe and application of the polypeptide probe in identification of binding proteins modified after translation, identification of interactions between protein modifications and discovery of novel protein modified binding proteins.

Description

Polypeptide probe and application thereof in identifying binding protein modified after translation

Technical Field

The invention relates to the field of biotechnology, in particular to a polypeptide probe based on photocrosslinking and nano metal particle self-assembly technology, a preparation method thereof and application thereof in identification of binding proteins modified after translation, identification of interactions between protein modifications and discovery of novel protein modified binding proteins.

Background

Post-translational modification of a protein refers to the process of covalently modifying individual amino acid residues on a protein after mRNA is translated into the protein. More than 400 post-translational modifications of proteins have been found in eukaryotic animal cells, with ubiquitination, phosphorylation, glycosylation, lipidation, methylation, acetylation, and the like being the most common. Post-translational modification of proteins plays a very important role in life. It makes the structure of protein more complex, its function more perfect, its regulation more fine and its action more specific. The functions of many proteins in cells are regulated by dynamic post-translational modification of proteins (development of post-translational modification of proteins, hu, et al, science bulletin, volume 6, 11, page 1061-1072, 2005).

Methylation modification of proteins is methylation of lysine or arginine side chain amino groups under the catalysis of methyltransferases. In addition, there are forms in which the carboxyl group of the aspartic acid or glutamic acid side chain is methylated to form methyl ester. Lysine residues may be monomethylated (Kme), dimethylated (Kme) or trimethylated (Kme 3) while arginine residues may be monomethylated (Rme 1), symmetrical dimethylated (Rme 2 s) or asymmetrical dimethylated (Rme 2 a) modified. Histone lysine methylation can occur on H3-K4, H3-K9, H3-K27, H3-K36, H3-K79, H4-K20, and the like, as well as on the H1N-terminus. Methylation of H3-K9, H3-K27, H4-K20 is involved in the inactivation of chromosomes, whereas methylation of H4-K9 may be involved in the inhibition of a wide range of chromatin levels. Methylation at positions H3-K4, H3-K36, H3-K79 is involved in the process of activation of chromosome transcription, wherein single methylation modification of H3-K4 can be against gene suppression caused by methylation of H4-K9. Histone arginine methylation sites can occur on H3-R2, H3-R4, H3-R17, H3-R26.

Acetylation is also an important form of post-translational modification of intracellular proteins. Many proteins such as histones can undergo acetylation. Acetylation of histones is catalyzed by Histone Acetyltransferases (HATs) and deacetylation is catalyzed by histone deacetylases (histone deacetylases, HDs or HDACs).

Ubiquitin consists of 76 amino acids, is highly conserved and is ubiquitous in eukaryotic cells. Proteins covalently bound to ubiquitin are recognized and degraded by proteases, a common route for degradation of short-lived proteins and some abnormal proteins within cells. Ubiquitin-protease systems are regulatory systems present in all eukaryotic cells, and three enzymes are required to participate in the degradation process: ubiquitin activating enzyme (E1), ubiquitin binding enzyme (E2) and ubiquitin protein ligase (E3). Specific recognition of proteins during ubiquitination of proteins depends on E3. The ubiquitination process mediated by E2s and E3s can be reversed by Deubiquitinase (DUBs). Ubiquitination is also an important form of histone modification, and H2A and H2B of histones are multiple sites of ubiquitination. Histone H2B ubiquitinase has been found and a correlation between histone H2B ubiquitination and histone methylation has been found.

Phosphorylation is the process by which the phosphoryl group of ATP is transferred to a specific site on a protein by a protein phosphorylating kinase. Most cellular processes are actually regulated by reversible protein phosphorylation, with at least 30% of the protein being modified by phosphorylation. The phosphorylation site is Ser, thr, tyr residue on the protein. During the phosphorylation regulation process, both the morphology and function of the cells are altered. Reversible phosphorylation processes involve almost all physiological and pathological processes such as cell signaling, tumorigenesis, metabolism, neural activity, muscle contraction, and proliferation, development and differentiation of cells.

Glycosylation of proteins is the process by which oligosaccharides are covalently bound in the form of glycosides to specific amino acid residues on the protein. Protein glycosylation can be divided into four classes according to the manner in which amino acids and sugars are linked: o-glycosylation, N-glycosylation, C-mannosylation and GPI (glycophosphatidlyinositol) -anchored ligation.

Protein lipidation is the process by which long fatty chains are conjugated to proteins via O or S atoms to give protein conjugates, typically where the S bond of a cysteine residue in the protein molecule is acetylated by palmitoyl groups. Or alkylated by farnesyl. These two fatty chains generally modify the same protein molecule together, and the protein is immobilized on the cell membrane by good compatibility of the fatty chains with the bio-phospholipid membrane.

In vivo, various post-translational modification processes are not isolated and, in many cellular activities, require the co-action of various post-translational modified proteins. More than one post-modification process can be provided for the same protein, and various post-translational modification forms are mutually influenced and mutually coordinated. Phosphorylation and glycosylation share similarities in many ways. Histones can be modified simultaneously by methylation and acetylation, and the main site of action for acetylation and methylation on histones is the conserved lysine residues at the ends of histones H3 and H4. Histone acetylation modifications span the entire cell cycle, whereas methylation modifications occur mostly in the G2 phase and heterochromatin assembly process.

Taking histone post-translational modifications as an example: genomic DNA exists in eukaryotes in the form of chromatin, and nucleosomes are the basic units constituting chromatin. The formation of nucleosomes and higher chromatin structures effectively stores and protects the genetic information contained in the DNA sequence on the one hand; on the other hand, as a specific existence mode of genome DNA, the structure of chromatin also becomes a natural obstacle for various cellular processes (such as transcription, replication, damage repair, etc.) requiring contact with DNA, so that chromatin becomes an important genetic information expression control platform. Chromatin condensation, formation of a relaxed structure, and switching between open and closed states provide a regulatory mechanism that exceeds the DNA sequence itself, i.e., epigenetic regulation.

Epigenetic regulatory mechanisms are involved in several aspects, such as histone modification, histone variant, DNA methylation, non-coding RNA, chromatin remodeling, and the like. Among them are many types of post-translational modifications of histones (especially at the N-terminal tail), including arginine methylation (me, me 2), lysine methylation (me, me2, me 3), acetylation (ac), propionylation (pr), butyrylation (bt), crotonylation (cr), formylation (fo), ubiquitination (ub), SUMO, lysine beta-hydroxybutyrylation (3 ohib), 2-hydroxyisobutyrylation (hib), succinylation (Succ), malonyl, glutaryl, and phosphorylation (ph), ADP ribosylation and biotinylation (bio), and the like. The modifications can be generated on different variants of histones and different sites of histones, can be distributed in groups or clusters on different sections of chromatin, are considered to form a group of 'histone codes' exceeding gene sequences, control the tissue level of intracellular genetic information, play an important role in gene expression and cell differentiation and development regulation, and are closely related to the generation and development of major diseases such as tumors.

To accomplish precise epigenetic regulation, around the "histone code," cells evolved a series of modification enzyme systems to accomplish the establishment and elimination of specific histone modifications. Enzymes that produce specific histone modifications are known as "writers" (writers), such as Histone Acetylases (HAT), methylases (HKMT), and the like; enzymes that function inversely and specifically remove specific histone modifications are referred to as "erasers" or "degmodified enzymes", such as various Histone Deacetylases (HDACs), demethylases (hqdms), and the like; likewise, there is also a large class of proteins or domains known as "readers" within cells that specifically recognize a variety of different types of histone modifications, coupling specific histone modifications to specific biological functional consequences. The synergistic effect of the above histone modification regulatory factors controls important cell physiological processes such as transcriptional activation, transcriptional silencing, DNA replication and repair, mitosis and the like, and is an important biochemical and material basis for epigenetic regulation.

The "modification dialogue" between histone modifications (modification crosstalk) is also of great importance for understanding histone modification-mediated epigenetic regulation. The histone "reader" element can co-exist with the catalytic domain of the "eraser" or "writer", which lays the biochemical foundation of the "finishing dialogue". For example, histone lysine demethylase PHF8 and KIAA1718 each contain one PHD zinc finger and jmc domain. The PHD zinc fingers of both proteins recognize the modification H3K4me3 and this modification can enhance the activity of the demethylase.

In recent years, with the development of high resolution mass spectrometry and specific post-translational modification antibody technology, many novel histone modifications have been discovered successively. The histone reader protein regulates gene expression by recognizing histone specific modification, and the functional disorder is closely related to diseases such as tumor and the like. Histone modification-removing enzyme protein corrects and regulates histone specific modification, and dysfunction is also found to be closely related to the occurrence and development of some diseases. Therefore, the identification and discovery of new histone modification reader proteins and unmodified enzyme proteins is of great importance for understanding the mechanism of disease occurrence and for finding drug targets. However, the discovery of the reader proteins and the de-modified enzyme proteins is also relatively delayed compared to the study of more mature histone post-translational modifications, mainly due to the lack of reliable and highly specific and sensitive methods. The bottleneck is that post-translational modification mediated protein forces are very weak and often occur transiently, thus making identification difficult.

Based on the affinity of a polypeptide probe with a specific protein, an effective method for finding a binding protein is provided, a specific amino acid sequence (or a polypeptide with specific modification) of a target protein is selected, a biotin tag is introduced into a side chain or a C end of the polypeptide, the binding protein is identified by the polypeptide, then the binding protein is obtained through enrichment of microspheres modified by avidin, and finally the binding protein is subjected to qualitative and quantitative analysis by technical means such as mass spectrometry. However, the polypeptide probe is combined with the target protein mainly through weak interaction forces such as electrostatic interaction and structural complementation, and the combination is easy to break away, and the combination is instantaneously and dynamically changed. Thus, the identification and screening of histone modified reader proteins or unmodified enzyme proteins using this method is not ideal and represents a major bottleneck in research technology in this field.

Currently, the introduction of photochemical cross-linking agents (such as azidobenzenes, benzophenones, bisazides, etc.) converts the binding between the probe and the protein into covalent binding. By modifying and reforming the peptide chain skeleton of the polypeptide and introducing photocrosslinking groups and affinity enrichment tags (such as biotin), the labeling and enrichment of the polypeptide binding protein can be realized, and the enrichment efficiency of the target protein is effectively improved. However, the synthesis of the polypeptide probe is complicated, and the photo-crosslinking group is introduced into the middle of the peptide chain skeleton, so that the structure of the peptide chain can be changed, and the recognition and the combination between histone modification and a reader protein or a de-modification enzyme protein are affected, so that the application of the photo-crosslinking technology in the research of protein combination effect is limited.

Disclosure of Invention

Aiming at the problem that the protein post-translational modification (including histone post-translational modification and non-histone post-translational modification) binding proteins (including reader proteins and complexes thereof and de-modification enzyme proteins and complexes thereof) are difficult to identify and identify in the application of the existing polypeptide probes, a novel polypeptide probe is developed by combining a nano metal particle self-assembly technology and an affinity photocrosslinking technology, a preparation method of the probe is established, and an identification and analysis method of the protein post-translational modification binding proteins with high efficiency, high sensitivity and high selectivity is performed by utilizing the probe.

In a first aspect of the invention, a polypeptide probe is provided.

The polypeptide probe is characterized by comprising nano metal particles, polypeptides modified on the surfaces of the nano metal particles and photocrosslinking groups modified on the surfaces of the nano metal particles.

According to the present invention, the nano-metal particles may be selected from nano-gold particles, nano-silver particles, nano-copper particles, nano-iron particles, nano-nickel particles and/or nano-aluminum particles. Gold nanoparticles and/or silver nanoparticles are preferred. In one embodiment of the invention, the gold nanoparticles.

The particle size of the nano metal particles is 1-50nm. The larger the particle size of the nano-metal particles, the larger the surface area, and the greater the number of modifiable polypeptides and photocrosslinking groups. From the viewpoint of combining the polypeptide loading, particle sedimentation, and post-treatment operation, the particle diameter is preferably 1 to 20nm, more preferably 1 to 10nm, for example, 4nm,5nm,7nm,8nm,10nm, and the like.

According to the invention, the amino acid residues of the polypeptide are not post-translationally modified by the protein (i.e., the unmodified polypeptide), or one or more amino acid residues of the polypeptide have post-translationally modified by the protein (i.e., the modified polypeptide), the C-terminal end of the polypeptide being a cysteine residue.

The sequence of the polypeptide can be a naturally occurring polypeptide sequence or an artificially fit polypeptide sequence. The polypeptide can be synthesized by artificial polypeptide synthesis technology, or can be produced by bacterial or cell expression by genetic engineering technology.

Preferably the polypeptide contains 5-100 amino acid residues. In combination with the posttranslational modification of the domains of the binding proteins found by current research, and the ability of the polypeptide synthesis technology, the polypeptides preferably contain 7-50 amino acid residues, more preferably 10-30 amino acid residues.

Protein post-translational modifications may be made at any amino acid residue of the polypeptide, including but not limited to lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and the like.

Post-translational modification of a protein refers to covalent modification of individual amino acid residues on the protein after translation of the mRNA into the protein, including, but not limited to, methylation, formylation, acetylation, ubiquitination, propionylation, butyrylation, succinylation, crotonylation, malonyl, 2-hydroxybutyrylation, 3-hydroxybutyrylation, glutaryl, phosphorylation, glycosylation, lipidation, SUMO, biotinylation, and the like.

In one embodiment, the polypeptide contains one or more lysine residues, one or more of which have a post-protein modification.

The post-translational modifications of the protein on the lysine residues are independently selected from various natural and/or chemical lysine modifications such as monomethylation (me 1), dimethylation (me 2), trimethylation (me 3), acetylation (ac), propionylation (pr), butyration (bt), crotonylation (cr), formylation (fo), ubiquitination (ub), sumoylation, β -hydroxybutyrylation (3 ohib), 2-hydroxyisobutyrylation (hib), succinylation (Succ), malonyl, glutaryl, etc.

Preferably, the polypeptide is an amino acid sequence fragment of a histone comprising a lysine residue in which post-translational modification of the protein can occur, and 2-50 amino acid residues before the N-terminus of the lysine residue and 2-50 amino acid residues after the C-terminus of the lysine residue; preferably, comprises a lysine residue that is post-translationally modifiable in the protein, and 3-25 amino acid residues before the N-terminus of the lysine residue and 3-25 amino acid residues after the C-terminus of the lysine residue; more preferably, it comprises a lysine residue in which post-translational modification of the protein may occur, and 4-15 amino acid residues before the N-terminus of the lysine residue and 4-15 amino acid residues after the C-terminus of the lysine residue.

Preferably, the lysine residues on the histone protein that may undergo post-translational modification of the protein are selected from the group consisting of: lysine residues at the H3-K4, H3-K9, H3-K14, H3-K18, H3-K23, H3-K27, H3-K36, H3-K56, H3-K79, H4-K5, H4-K8, H4-K12, H4-K16, H4-K20, H4-K31, H4-K44, H4-K59, H4-K77, H4-K79, H4-K91, H2A-K5, H2A-K9, H2A-K13, H2A-K15, H2A-K63, H2A-K119, H2B-K5, H2B-K16, H2B-K17, H2B-K5, H2B-K34, H2B-K120, H1K26 and/or H1N-terminal.

In a specific embodiment of the invention, the polypeptide is selected from the group consisting of: ARTKQTARKSC, ARTKQTARKSTGGKAC, ARTK (me 3) QTARKSC (trimethylated on lysine four), ARTKQTARK (cr) STGGKAC (crotonylated on lysine nine), ART (ph) K (me 3) QTARKSC (phosphorylated on threonine three, trimethylated on lysine four).

In another embodiment, the polypeptide contains one or more arginine residues, one or more of which have a post-protein modification thereon.

The post-translational modifications of the protein on the arginine residues are independently selected from the group consisting of single methylation modifications (me 1), symmetrical dimethyl modifications (me 2 s) and asymmetrical dimethyl modifications (me 2 a).

The polypeptide probe carrying the polypeptide with the post-translational modification of the protein can be used for identifying the binding protein with the post-translational modification of the protein, and the polypeptide probe carrying the polypeptide without the post-translational modification can be used as a control probe in the identification and identification process.

The polypeptide can be modified to the surface of the nano metal particles through the self-assembly reaction of sulfhydryl-gold of a cysteine residue at the C end.

According to the present invention, the photocrosslinking groups are known in the art and can undergo crosslinking reactions under the action of light, including but not limited to: azidobenzenes, benzophenones and bisazides.

Preferably, the photocrosslinking group is modified to the surface of the nano-metal particles by a linker molecule.

The linking molecule may be a chain polymer molecule such as polyethylene glycol, polyethylene, polypropylene, or other chemically functional scaffold molecule, such as an alkane branched chain. Polyethylene glycol is preferred, with a molecular weight of 200 to 1000, for example: polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, and the like. The length of the connecting molecule can be adjusted (for example, polyethylene glycol, polyethylene, polypropylene and the like with different polymerization degrees are adopted to change the length of the connecting molecule), so that the distance and the position between the photocrosslinking group at the tail end of the connecting molecule and the binding protein can be optimized, and the photocrosslinking efficiency of the target protein can be improved.

One end of the connecting molecule is covalently connected with the photocrosslinking group, the other end of the connecting molecule is modified by sulfydryl to form a sulfydryl-connecting molecule-photocrosslinking group covalent compound, and the connecting molecule is connected to the surface of the nano metal particle through sulfydryl-gold self-assembly reaction, so that the photocrosslinking group is modified to the surface of the nano metal particle. The covalent compounds of the thiol-linker-photocrosslinking group can be prepared using chemical synthesis methods known in the art.

In some embodiments of the invention, the covalent compound of a thiol-linker-photocrosslinking group is a thiol-polyethylene glycol-photocrosslinking group, for example: mercapto-polyethylene glycol-azidobenzene, mercapto-polyethylene glycol-benzophenone, mercapto-polyethylene glycol-biazidine.

When the polypeptide probe is incubated with the protein mixed solution system to be researched, the connecting molecules, especially the connecting molecules with PEG as components, can effectively reduce the adsorption of non-specific binding proteins, thereby improving the identification accuracy of target proteins.

The molar ratio of the nano metal particles to the polypeptide modified on the surfaces of the nano metal particles to the photocrosslinking groups is 1 (20-2000): 10-1000, preferably 1 (50-1000): 20-500. For example, for a 5nm size nano-metal particle, the molar ratio of the three may be 1 (50-500): (20-300), or 1 (100-300): (50-150), etc.

In a second aspect, the present invention provides a method for preparing the polypeptide probe.

The preparation method of the polypeptide probe is characterized in that nano metal particles, polypeptide and photocrosslinking groups are reacted in a solution, so that the polypeptide and the photocrosslinking groups are modified on the surfaces of the nano metal particles.

Preferably the solution is a 0.1-5% citrate aqueous solution, more preferably a 0.5-1.5% citrate aqueous solution. The citrate salt may be a sodium salt of citric acid, a potassium salt of citric acid, an ammonium salt of citric acid, including but not limited to monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, monoammonium citrate, diammonium citrate, triammonium citrate. In one embodiment of the invention, the solution is a 1% aqueous solution of trisodium citrate.

According to the invention, the reaction is carried out at a temperature of 10 to 40 ℃, preferably 20 to 30 ℃, for example 25 ℃.

According to the invention, the reaction is carried out for 2 to 24 hours, preferably 8 to 16 hours, for example 12 hours.

According to the invention, the nano-metal particles added to the solution: polypeptide: the molar ratio of the photo-crosslinking groups is 1 (50-5000): 25-2500, which can be selected according to the particle size of the nano metal particles, and is preferably 1 (100-1000): 50-500. In one embodiment of the invention, for 5nm nano-metal particles, the nano-metal particles: polypeptide: the molar ratio of photocrosslinking groups is 1 (400-600): (200-300), for example 1:500:250.

According to the invention, the photocrosslinking group is covalently bound to one end of a linker molecule, the other end of which is modified with a thiol group, forming a covalent compound of thiol-linker-photocrosslinking group. The linking molecule may be a chain polymer molecule such as polyethylene glycol, polyethylene, polypropylene, or other chemically functional scaffold molecule, such as an alkane branched chain. Polyethylene glycol is preferred, with a molecular weight of 200 to 1000, for example: polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, and the like. The length of the connecting molecule can be adjusted (for example, polyethylene glycol, polyethylene, polypropylene and the like with different polymerization degrees are adopted to change the length of the connecting molecule), so that the distance and the position between the photocrosslinking group at the tail end of the connecting molecule and the binding protein can be optimized, and the photocrosslinking efficiency of the target protein can be improved. Covalent compounds for synthesizing thiol-linker-photocrosslinking groups, such as thiol-polyethylene glycol-coupled benzophenones in particular embodiments of the present invention, may be prepared using chemical synthesis methods conventional and known in the art.

According to the invention, the preparation method further comprises a post-treatment step of the polypeptide probe, wherein the step comprises washing the polypeptide probe and preserving the polypeptide probe in a liquid suspension. The liquid may be water or buffers commonly used in the art including, but not limited to, phosphate buffer, carbonate buffer, tris buffer, hepes buffer, PBS buffer, and the like.

FIG. 1 is a schematic flow chart of the preparation of the polypeptide probes of the present invention and the enrichment of capture proteins post-translational modification of binding proteins using the polypeptide probes of the present invention. The invention is described with reference to fig. 1. The preparation of the polypeptide with the post-translational modification of the protein and the photocrosslinking group functionalized nano metal polypeptide probe is realized by taking the polypeptide with the post-translational modification of the protein as an identification template, coupling a photocrosslinking group (covalent compound of the mercapto-connecting molecule-photocrosslinking group) with a mercapto-modified connecting molecule, taking nano metal particles as a carrier and self-assembling the polypeptide, the photocrosslinking group and the nano metal particles. Incubating the polypeptide probe with a complex protein system in which a binding protein or a complex thereof may exist, and binding the polypeptide on the polypeptide probe with the binding protein or the complex thereof having an interaction therewith, wherein the photocrosslinking group on the polypeptide probe is brought close to the target protein, and the photocrosslinking group reacts under photoinitiation to covalently link the polypeptide probe with the target protein. The subsequent elution of nonspecifically adsorbed proteins on the polypeptide probe can be performed by using an eluent, so that the interference of nonspecifically bound proteins on enrichment and purification of target proteins is reduced. The polypeptide probe crosslinked with the target protein can be gathered through means of centrifugal operation or magnetic adsorption and the like, so that the purpose of finding, enriching and separating the binding protein or the complex thereof from a complex protein system is realized. Finally, the binding protein or its complex crosslinked to the polypeptide probe may be replaced by a replacement means commonly used in the art, for example, by using a thiol-containing substance, and the purified binding protein or its complex may be obtained by purification or the like. The binding proteins or complexes thereof may be subsequently analyzed quantitatively or qualitatively for experimental and research purposes.

When the polypeptide which is not subjected to protein post-translational modification is used as a recognition template to prepare a polypeptide probe, the polypeptide probe can be used as a control probe for comparing and researching the interaction between the binding protein and the protein post-translational modification. As described in example 2 below.

Since at least one post-translational modification of a protein may occur in a polypeptide sequence, the modifications may affect the recognition and binding capacity of different binding proteins to the corresponding modifications, including enhancing the recognition and binding capacity and reducing the recognition and binding capacity. Thus, a polypeptide having one protein post-translational modification, a polypeptide having at least two protein post-translational modifications, respectively, may be prepared as corresponding polypeptide probes, and a comparative study and analysis of interactions between different protein post-translational modifications may be performed, as described in example 4 below.

Accordingly, a third aspect of the present invention provides a method for identifying or identifying a protein post-translationally modified binding protein or complex thereof, characterized in that a polypeptide probe of the present invention is used, the polypeptide probe is contacted with a binding protein or complex thereof, or a protein mixture which may contain said binding protein or complex thereof, in a solution suitable for binding of the polypeptide probe to the binding protein or complex thereof at 0 to 8 ℃ and incubated for 1 to 24 hours, followed by photocrosslinking and removal of the uncrosslinked protein.

According to the invention, the solution suitable for binding of the polypeptide probe to the binding protein or complex thereof comprises: 25-100mM Tris-HCl,100-400mM sodium chloride, 1-5mM magnesium chloride and 1-5mM potassium chloride; preferably, the composition comprises: 40-60mM Tris-HCl, pH 7-8, 150-250mM sodium chloride, 2-3mM magnesium chloride and 2-3mM potassium chloride. In one embodiment of the invention, the solution contains: 50mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride.

According to the present invention, the amount of the polypeptide probe in the solution is adjusted accordingly to the amount of the protein. In general, the total amount of polypeptide probe in solution, calculated as the mole number of the nano metal particles, is proportional to the total amount of protein contacted: 0.02pmol to 1 pmol/. Mu.g protein, and may be 0.025pmol to 0.5 pmol/. Mu.g protein. For example, in one embodiment of the invention, the polypeptide probe is used in an amount of 4pmol in solution for 10 μg of binding protein; for 600. Mu.g of the protein mixture, the amount of polypeptide probe in the solution was 16pmol.

According to the invention, the light crosslinking is carried out by selecting the wavelength of light used according to the chemical structure of the photocrosslinking groups employed, so that the photocrosslinking reaction of the corresponding photocrosslinking groups can take place. To avoid the influence of light and heat on the protein structure, the photocrosslinking reaction is preferably carried out in an environment of 0 to 8℃for example in an ice-water bath or on ice.

According to the present invention, the uncrosslinked proteins can be removed by various methods known in the art, including but not limited to washing with a suitable solution, including but not limited to various buffers commonly used in the art. In one embodiment of the invention, the buffer contains 25-100mM Tris-HCl,100-400mM sodium chloride, 1-5mM magnesium chloride, and 1-5mM potassium chloride; preferably, the composition comprises: 40-60mM Tris-HCl, pH 7-8, 150-250mM sodium chloride, 2-3mM magnesium chloride and 2-3mM potassium chloride. In one embodiment of the present invention, the buffer contains: 50mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride. In another embodiment of the invention, the buffer is a PBS buffer containing 1-10M urea, preferably a PBS buffer containing 3-5M urea, and in one embodiment of the invention is a 50mM PBS buffer containing 4M urea.

According to the present invention, the method further comprises the steps of obtaining a polypeptide probe by centrifugation or magnetic separation, and displacing the protein bound on the polypeptide probe from the nano-metal particles.

The substitution employs a thiol-bearing compound including, but not limited to, mercaptoethanol.

According to the present invention, the method further comprises the step of purifying the displaced binding protein or complex thereof.

According to the invention, the binding protein or complex thereof is a protein post-translationally modified reader protein or complex thereof, or a protein post-translationally modified unmodified enzyme protein or complex thereof.

According to the present invention, the obtained binding protein or its complex may be subjected to subsequent quantitative, structural or biological functional studies using various protein analysis methods in the art, including but not limited to SDS-PAGE, liquid chromatography-mass spectrometry, western-blot, bioantibody detection, biofunctional activity analysis, etc.

In one embodiment of the invention, the polypeptide in the polypeptide probe is ARTK (me 3) QTARKSC; the binding proteins or complexes thereof are proteins with domains WD40, PWWP, PHD, tudor and/or chomo, including but not limited to: WDR5, EED, PPWD1, WDR61, cha 1B, WDR, BRPF1, HDGF, PWWP2A, BRD1, PSIP1, ZMYND11, BPTF, ING1, CXXC1, ING2, DIDO1, ING4, ING5, KDM2B, KDM5A, KDM5B, KDM5C, KMT2A, PHF2, PHF8, PHF13, TAF3, ATRX, TRIM24, JADE3, RSF1, SGF29, KDM4C, SPIN1, SND1, SMNDC1, TDRD3, CHD1, CBX8, CDYL, CHD9, SUV39H1, ORC1, KDM18, L3MBTL2, FAM60A, PBRM1.

In one embodiment of the invention, the polypeptide in the polypeptide probe is ARTKQTARK (cr) STGGKAC; the binding protein or complex thereof is a protein with the domains DPF and/or YEATS, including but not limited to: DPF2, KAT6A, DPF1, KAT6B, KMT2A, MTF2, NSD2, AF9, ENL, YEATS4, YEATS2, ATAD2, KAT2A, KAT7, SMARCA4.

In a fourth aspect the present invention provides a method for determining the interaction between a post-translational modification of a protein and a binding protein or complex thereof, characterized in that in each of at least two separate reaction systems, a polypeptide probe according to the invention is used, the polypeptide probe is contacted with the binding protein or complex thereof, or a mixture of proteins possibly containing said binding protein or complex thereof, in a solution suitable for binding of the polypeptide probe to the binding protein or complex thereof at a temperature of 0-8 ℃ and incubated for 1-24 hours, followed by photocrosslinking, to remove uncrosslinked proteins.

The interaction between the protein post-translational modification and the binding protein or complex thereof is selected from the group consisting of: interactions between a single modification and a single binding protein or complex thereof; interactions between a single modification and multiple binding proteins or complexes thereof; interactions between multiple modifications and a single binding protein or complex thereof; interactions between various modifications and various binding proteins or complexes thereof.

The binding protein or complex thereof is a reader protein or complex thereof that is post-translationally modified by the protein, or a de-modified enzyme protein or complex thereof that is post-translationally modified by the protein.

According to the invention, in determining the interaction between a single modification and a single binding protein or complex thereof, one system in the independent reaction system employs a polypeptide probe carrying a polypeptide which is not post-translationally modified by the protein, and another system employs a polypeptide probe carrying the same polypeptide sequence and having post-translationally modified by the protein at one or more amino acids. In one embodiment of the invention, in determining the interaction between the H3K4me3 modification and its binding protein or its complex, one of the separate reaction systems employs a polypeptide probe carrying the polypeptide ARTKQTARKSC and the other system employs a polypeptide of the polypeptide probe being ARTK (me 3) QTARKSC; in determining the interaction between the H3K9cr modification and its binding protein or its complex, one system in the independent reaction system employs a polypeptide probe carrying the polypeptide ARTKQTARKSTGGKAC, and the other system employs a polypeptide probe of ARTKQTARK (cr) STGGKAC;

In determining the interaction between a single modification and a plurality of binding proteins or complexes thereof, according to the invention, the number of independent reaction systems may be equal to the number of binding proteins or complexes thereof, each reaction system employing the same polypeptide probe carrying a polypeptide having a post-translational modification of the protein. For comparison, a separate reaction system can be added as a control, wherein the polypeptide probes used in the system carry the corresponding polypeptides without post-translational modification of the proteins.

In determining the interaction between a plurality of modifications and a single binding protein or complex thereof, the number of independent reaction systems is equal to or greater than the number of modifications, the polypeptides on the polypeptide probes in each system carrying at least one post-translational modification of the protein. For example, in one embodiment of the invention, where the interaction of methylation and phosphorylation occurring on H3 with H3K4me3 modified binding protein or complex thereof is determined, one system in the independent reaction system employs a polypeptide probe carrying ARTK (me 3) QTARKSC and the other system employs a polypeptide probe carrying ART (ph) K (me 3) QTARKSC.

In determining the interaction between the plurality of modifications and the plurality of binding proteins or complexes thereof, the number of independent reaction systems is adjusted according to the number of modifications and the number of binding proteins or complexes thereof, the polypeptides on the polypeptide probes in each system carrying at least one posttranslational modification of the protein, according to similar principles.

According to the invention, the solution suitable for binding of the polypeptide probe to the binding protein comprises: 25-100mM Tris-HCl,100-400mM sodium chloride, 1-5mM magnesium chloride and 1-5mM potassium chloride; preferably, the composition comprises: 40-60mM Tris-HCl, pH 7-8, 150-250mM sodium chloride, 2-3mM magnesium chloride and 2-3mM potassium chloride. In one embodiment of the invention, the solution contains: 50mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride.

According to the present invention, various methods known in the art may be used to remove uncrosslinked proteins, including but not limited to washing with suitable solutions, including but not limited to: various buffers are commonly used in the art. In one embodiment of the invention, the buffer contains 25-100mM Tris-HCl,100-400mM sodium chloride, 1-5mM magnesium chloride, and 1-5mM potassium chloride; preferably, the composition comprises: 40-60mM Tris-HCl, pH 7-8, 150-250mM sodium chloride, 2-3mM magnesium chloride and 2-3mM potassium chloride. In one embodiment of the present invention, the buffer contains: 50mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride. In another embodiment of the invention, the buffer is a PBS buffer containing 1-10M urea, preferably a PBS buffer containing 3-5M urea, and in one embodiment of the invention is a 50mM PBS buffer containing 4M urea.

According to the present invention, the effect of increasing or decreasing modification on binding protein interactions can be appreciated by comparing the binding proteins obtained for each independent assay system, e.g., comparing the amount of binding protein obtained for each independent assay system.

In a fifth aspect, the invention provides the use of the polypeptide probe to identify or identify a protein post-translational modification binding protein or complex thereof, or to determine the interaction between a protein post-translational modification binding protein or complex thereof and a protein post-translational modification.

The binding protein or complex thereof is a reader protein or complex thereof that is post-translationally modified by the protein.

The binding protein or complex thereof is a protein post-translationally modified, de-modified enzyme protein or complex thereof.

The use is for non-diagnostic or therapeutic purposes.

The application is performed in vitro.

In a sixth aspect, the present invention provides a detection reagent for identifying or identifying a protein post-translational modification binding protein or complex thereof, or for determining the interaction between a protein post-translational modification binding protein or complex thereof and a protein post-translational modification, characterized in that it comprises at least one polypeptide probe according to the present invention, each of said polypeptide probes being present in a separate liquid system.

A test kit for identifying or identifying a protein post-translational modification binding protein or complex thereof, or for determining the interaction between a protein post-translational modification binding protein or complex thereof and a protein post-translational modification, comprising at least one polypeptide probe according to the invention, each of said polypeptide probes being present in a separate liquid system.

According to the present invention, the liquid may be water or buffers commonly used in the art, including but not limited to phosphate buffers, carbonate buffers, tris buffers, hepes buffers, PBS buffers, and the like.

According to the invention, the concentration of the polypeptide probe in the liquid is 10nM to 500nM, preferably 50nM to 150nM, more preferably 70 nM to 100nM, in terms of mole of nano-metal particles.

According to the invention, the detection kit further comprises a solution for identification and authentication, wherein the solution contains 25-100mM Tris-HCl,100-400mM sodium chloride, 1-5mM magnesium chloride and 1-5mM potassium chloride; preferably, the composition comprises: 40-60mM Tris-HCl, pH 7-8, 150-250mM sodium chloride, 2-3mM magnesium chloride and 2-3mM potassium chloride. In one embodiment of the invention, the solution contains: 50mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride.

According to the invention, the detection kit further comprises a buffer for eluting the uncrosslinked protein. In one embodiment of the invention, the buffer is the same as the solution used for the identification, characterization reaction, e.g., contains 25-100mM Tris-HCl,100-400mM sodium chloride, 1-5mM magnesium chloride, and 1-5mM potassium chloride; preferably, the composition comprises: 40-60mM Tris-HCl, pH 7-8, 150-250mM sodium chloride, 2-3mM magnesium chloride and 2-3mM potassium chloride. In one embodiment of the present invention, the buffer contains: 50mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride. In another embodiment of the invention, the buffer is a PBS buffer containing 1-10M urea, preferably a PBS buffer containing 3-5M urea, and in one embodiment of the invention is a 50mM PBS buffer containing 4M urea.

In the present invention, the general description of post-translational modifications of proteins is defined and described using methods known in the art. The corresponding modification is put in english lowercase abbreviation after the modified amino acid residue code, for example, "H3K4me3" represents a trimethylation at the fourth lysine residue of the H3 protein, and "(me 3)" in the polypeptide sequence of "ARTK (me 3) QTARKSC" represents a trimethylation at the immediately preceding "K" thereof. In addition, "modified H3K4me3" and "H3K4me3 modified" are the same meaning and each represents a trimethylation modification at the fourth lysine residue of the H3 protein; other modifications are explained in the same manner.

The invention has the beneficial effects that:

according to the polypeptide probe based on the photo-crosslinking technology and the nano metal self-assembly technology, the nano metal particles are adopted as the carrier, and the photo-crosslinking technology is used, so that the affinity recognition and enrichment capture process are integrated, and the recognition and identification operation of the binding protein is convenient and efficient.

The photocrosslinking group is introduced into the polypeptide probe, so that weak acting force between the polypeptide and the binding protein is converted into strong covalent bond action formed by the photocrosslinking group, and therefore, the polypeptide probe can bear stronger elution conditions, and simultaneously, the polypeptide probe can capture proteins with lower abundance and transient interaction.

Compared with the traditional method, the method has the advantages that the photocrosslinking group is connected to the connecting molecule (such as PEG) and then modified on the nano metal particles, so that the influence of the photocrosslinking group modification on the polypeptide skeleton is skillfully avoided, the original structure and the identification characteristic of the polypeptide skeleton are well maintained, and the influence on the binding specificity of the corresponding binding protein is greatly reduced. In addition, the length of the connecting molecule can be adjusted, so that the position and distance between the photocrosslinking group and the target binding protein can be optimized, and the crosslinking efficiency of the target protein can be increased. In addition, nanoparticles are used as carriers, so that a dense arrangement mode of the photocrosslinkers and the polypeptides on the nano metal is realized, the effect and crosslinking probability of the probes and the binding proteins are increased, and the high-efficiency and high-selectivity enrichment of the probes on the target proteins is improved.

In addition, the photocrosslinking group is modified on the nano metal particles instead of the polypeptide, after the polypeptide recognizes and binds the target protein, the covalent connection point formed by the photocrosslinking group on the target protein is positioned at a non-recognition site of the target protein, usually on the surface of the protein, so that on one hand, the subsequent replacement treatment of the photocrosslinking site is very easy to react, and on the other hand, the recognition site of the replaced target protein is not subjected to any chemical reaction and change, thereby being beneficial to the subsequent structural and functional study of the target protein.

In the preferred scheme of the invention, the adopted sulfhydryl-connecting molecule-photocrosslinking group covalent compound, in particular to a sulfhydryl polyethylene glycol coupling photocrosslinking group, has simple chemical structure and easy synthesis, and compared with other sulfhydrylation functional groups with sulfur atom structures in the field, the self-assembly reaction condition with nano metal particles is simple and mild, and the reaction efficiency is high.

Drawings

FIG. 1 is a flow chart of enrichment capture histone modification binding protein based on self-assembled multivalent photoaffinity polypeptide probes.

FIG. 2 shows a MALDI mass spectrum of the probe (2).

FIG. 3 is a transmission electron microscope characterization of probe (2).

FIG. 4 is a representation of the ultraviolet visible spectrum of probe (2).

FIG. 5, probe (2) liquid chromatogram and analysis thereof, wherein (A) probe (2) liquid chromatogram, wherein peak (1) is polypeptide (2) (denoted "H3K4me 3"), peak (2) is PEG600 peak; (B) a standard curve of PEG600 concentration versus chromatographic peak area; (C) Standard curve for polypeptide (2) concentration versus chromatographic peak area.

FIG. 6 results of mass spectrometry characterization of BPTF domain proteins. Wherein, (A) SDS-PAGE patterns of expressed proteins; (B) Sequence coverage of mass spectrometry identified BPTF domain proteins.

FIG. 7 efficient enrichment of target proteins by probes. Wherein (A) SDS-PAGE patterns of different probes after enrichment of target proteins; and (B) statistical calculations of the enrichment efficiency of different probes. In the figure, "H3K4me3" represents probe (2), and "H3K4" represents probe (1).

FIG. 8 shows selective enrichment of target proteins by the probe against BSA and cell lysates. Wherein, (A) the probe is in the background of Bovine Serum Albumin (BSA), and (B) the probe is in the background of cell lysate, so as to selectively enrich the target protein. In the figure, "H3K4me3" represents probe (2).

FIG. 9 recognition of protein multi-modification interactions with binding proteins by probes, exemplified by the effect of phosphorylation on methylation recognition proteins. Wherein (A) a pattern diagram of the effect of phosphorylation on methylation-recognizing proteins; (B) Enrichment comparison of target proteins by the two probes (2) and (5) (denoted by "H3K4me3" and "H3T3phK4me3", respectively).

FIG. 10 Western blot analysis of histone modification H3K4me3 binding protein-SND 1 in nuclear protein by probe.

Enrichment and characterization of histone modified H3K9cr binding proteins in nuclear proteins by the probe of fig. 11. Wherein (a) the probes enrich for binding proteins containing different domains; (B) Western blot analysis of the identified H3K9cr binding protein-KAT 6B; and (C) structural docking analysis of the binding protein KAT6B with the polypeptide (4).

Detailed Description

The invention is further described below with reference to examples.

The chemical reagents used in the examples below are all conventional and commercially available.

Example 1 preparation of polypeptide probes

Five polypeptides, the numbers and sequences of which are respectively: (1) the method comprises the following steps ARTKQTARKSC, (2): ARTK (me 3) QTARKSC, (3): ARTKQTARKSTGGKAC, (4): ARTKQTARK (cr) STGGKAC, (5): ART (ph) K (me 3) QTARKSC. The polypeptides were synthesized by Beijing-Asia-photobiotechnology Co. For convenience of description, the numbers of the polypeptide probes used in the following examples are consistent with the numbers of the polypeptides carried thereon, and the corresponding relationship of the numbers is followed.

Mercapto polyethylene glycol coupled diphenyl ketone with molecular formula of HS- (CH) ₂ CH ₂ O) ₁₃ -C ₆ H ₄ COC ₆ H ₅ Synthesized by Shanghai Tuo Biotechnology Co.

Gold nanoparticles (particle size 5 nm) were purchased from BBI company.

Preparation of polypeptide probes: mixing 2 mu L (1 mM) polypeptide and 1 mu L (1 mM) mercapto polyethylene glycol coupled benzophenone, adding into 50 mu L (80 nM) nano gold particle solution (1% trisodium citrate aqueous solution), shaking and mixing at room temperature for 12 hours; then, the functionalized and modified nano gold particles are washed twice by using 50 mu L of deionized water, and are stored at 4 ℃ for standby. The same preparation method is adopted to prepare and obtain the nano gold particle polypeptide probes respectively carrying the polypeptide sequences (1), (2), (3), (4) or (5), and the mercapto polyethylene glycol coupled benzophenone, and the nano gold particle probes only carrying the mercapto polyethylene glycol coupled benzophenone.

Characterization of the probes: characterization was performed using MALDI-TOF mass spectrometry, transmission electron microscopy, and uv-vis spectroscopy; and calculating the number of the modified polypeptide and the photocrosslinking groups on the probe by utilizing high performance liquid chromatography. Taking the polypeptide probe (2) carrying the sequence (2) as an example, the characterization result is shown in figures 2-5.

Example 2 identification and efficient enrichment of target proteins by polypeptide probes

The effect of the polypeptide probe of example 1 was verified by using BPTF (PHD domain, which is known to bind to modified H3K4me 3) as the target protein. BPTF domain proteins were self-expressed as described in reference (Angew Chem Int Ed Engl.2016Jul 4,55 (28): 7993-7) and the identified structure is shown in FIG. 6.

Experimental group: the target protein (10. Mu.g) was incubated with polypeptide probe (2) (4 pmol) in binding buffer (50 mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride) overnight at 4 ℃. The sample tube was then transferred to ice and irradiated under a 365nm uv lamp for 15min to initiate the photocrosslinking reaction. The uncrosslinked proteins are then washed using a binding buffer solution. Separating the captured target protein by centrifugation, and adding a loading buffer (250 mM Tris-HCl pH 6.8,10% SDS,0.5% bromophenol blue, 7.5% dithiothreitol) containing 12% mercaptoethanol to compete the target protein from the nano-gold particles; finally, the target protein is loaded after being heated for 30min at 95 ℃ and is characterized by SDS-PAGE.

At the same time 3 parallel control experiments were performed. Control test 1: the rest of the test methods and operations were the same as the test groups, except that no uv light irradiation operation was performed. Control test 2: the rest of the test method and operation were the same as those of the test group except that the polypeptide probe (1) was used. Control test 3: the rest of the test methods and operations are the same as the test groups except that the polypeptide probe uses a nano gold particle probe modified only with sulfhydryl polyethylene glycol coupled benzophenone.

For SDS-PAGE characterization, the results of the experimental group and 3 control experiments, respectively, and untreated equivalent amounts of the initial target protein were loaded and the gels were silver stained to observe the effect of probe enrichment, as shown in FIG. 7A. Lane 1 is the experimental group, lane 2 is control 1, lane 3 is control 2, lane 4 is control 3, and lane 5 is an untreated equivalent amount of the starting target protein. The amount of the target protein in lane 1 is significantly higher than that in

lanes

3 and 4, compared with

lanes

3 and 4, indicating that the polypeptide probe (2) specifically binds to the target protein. Compared with the lane 2, the amount of the target protein in the lane 1 is obviously higher than that in the lane 2, which indicates that the photocrosslinker overcomes the defect of weak acting force of the combination between proteins by carrying out covalent combination on the target protein and the polypeptide in the polypeptide probe after the light treatment, and can resist the subsequent washing and other operations. Compared with the lane 5, the lane 1 has the target protein content close to that of the lane 1, which indicates that the polypeptide probe can efficiently enrich the target protein. The calculation result of comparing the amounts of the target proteins in lanes 1 to 4 with those in lane 5 is shown in FIG. 7B.

As can be seen from fig. 7, the polypeptide probe (2) was subjected to the binding light treatment, compared with the control probes (probe (1)) and PEG, and the non-light condition, thereby realizing specific binding and efficient enrichment of the target protein.

Example 3: and under the background condition of cell lysate or BSA, the polypeptide probe is selectively enriched for target proteins.

The effect of the polypeptide probe (2) of example 1 was also verified by using BPTF as the target protein.

Experimental group: the target protein (10. Mu.g) was mixed with cell lysate (50. Mu.g) (or Bovine Serum Albumin (BSA), 50. Mu.g) in a certain ratio and then incubated with polypeptide probe (4 pmol) in buffer (50 mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride) overnight at 4 ℃. The sample tube was then transferred to ice and irradiated under a 365nm uv lamp for 15min to initiate the photocrosslinking reaction. The uncrosslinked proteins were then washed three times with PBS buffer (50 mM, pH 7.4) containing 4M urea. Separating the captured proteins by centrifugation and adding a loading buffer (250 mM Tris-HCl pH 6.8,10% SDS,0.5% bromophenol blue, 7.5% dithiothreitol) containing 12% mercaptoethanol to compete the target proteins from the gold nanoparticles; finally, the protein is loaded after heating at 95 ℃ for 30min, and is characterized by SDS-PAGE.

1 parallel control test was performed simultaneously: the rest of the test methods and operations were the same as the test groups, except that no uv light irradiation operation was performed.

In SDS-PAGE characterization, the mixture of the untreated target protein and BSA or cell lysate and the equivalent initial target protein are respectively loaded and detected, and the gel is subjected to silver staining and color development to observe the enrichment effect of the polypeptide probe on the target protein, and the result is shown in FIG. 8.

In FIG. 8A, with BSA as background, lane 1 is the experimental group, lane 2 is the control group, lane 3 is the mixture of untreated target protein and BSA, and lane 4 is the untreated equivalent amount of starting target protein. Lane 1 is closer to the amount of target protein in lane 1 than in lane 4; lane 1 shows a band only at the molecular weight of the target protein compared to lane 3, and the color of the band at the corresponding position in lane 3 is close to that of the polypeptide probe (2), indicating that the polypeptide probe (2) specifically binds to the target protein. Compared with the lane 2, the amount of the target protein in the lane 1 is obviously higher than that in the lane 2, which indicates that the photocrosslinker overcomes the defect of weak acting force of the combination between proteins by carrying out covalent combination on the target protein and the polypeptide in the polypeptide probe after the light treatment, and can resist the subsequent washing and other operations.

Similar results were also observed in experiments with cell lysates as background, and the results are shown in fig. 8B.

It follows that the polypeptide probe (2) has very good specificity and enrichment ability for the target protein, and that the polypeptide probe (2) maintains very high selectivity and specificity for the target protein even in a complex system.

Example 4: evaluation of protein multiple modification by polypeptide probes and binding protein interactions

The experimental conditions were the same as those of the experimental group in example 2. The probe used was the probe of (2) or (5), respectively.

As shown in FIG. 9, lane 1 is the probe (2) and BPTF _PHD Lane 2 is the incubation of probe (5) with BPTF _PHD Lane 3 is the input control. The same procedure as described in example 2 for incubation, elution, enrichment and SDS-PAGE identification was followed and the results are shown in FIG. 9: probe (5) and target protein BPTF _PHD Is weaker than the binding and enrichment of the probe (2) to the target protein BPTF _PHD Indicating that the target protein BPTF is affected when the phosphorylation modification of T3 on the H3K4 sequence is present _PHD The combination with the modification of H3K4me3 indicates the influence of T3ph on the identification process of the modification of K4me 3. This result is consistent with literature reports (Nucleic Acids Research 2016,44,6102-6112), demonstrating that the polypeptide probe of example 1 can also be used to analyze interactions between protein modifications.

Example 5: enrichment and characterization of histone H3K4me3 modified binding proteins in nuclear proteins by polypeptide probes.

Nucleoprotein was extracted from Hela cells using Hela cell lysate or nucleoprotein extraction kit (purchased from Shanghai Biotechnology Co., ltd.). The extracted nucleoprotein (0.6 mg) was incubated with polypeptide probe (2) (16 pmol) in buffer (50 mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride) overnight at 4 ℃. And performing subsequent ultraviolet irradiation and elution experiments, and performing SDS-PAGE analysis and liquid chromatography-mass spectrometry combined analysis (Nano-LC-Q-exact mass spectrometry) and identification on the enriched protein. The specific conditions are as follows: the liquid phase gradient is 60 minutes, the flow rate is 300nL/min, the primary mass spectrum resolution is set to 70000, the scanning range is 350-1750m/z, the normalized energy is 27%, and the secondary mass spectrum resolution is set to 17500. The scanning window was 1.6Da. The results of identifying all the enriched proteins obtained are shown in table 1 below:

TABLE 1 identification of probes for H3K4m3 binding proteins

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The identified proteins were classified for biological functions using a bioinformatics database, and it was found that of the 48 proteins enriched, 11.9% had chromatin binding activity, 23.2% had transcriptional activity, 32.1% had DNA binding activity, 15.8% had transcription factor binding activity, 5.8% had helicase activity, 1.2% had demethylase activity, 1.9% had methyltransferase activity, 2.3% had acetyltransferase activity, and 5.8% had atpase activity.

The result of western blot verification of the binding protein SND1 and the polypeptide (2) is shown in FIG. 10.

In the implementation, the polypeptide probe (2) is enriched to 23 out of 35H 3K4me3 binding proteins reported in Hela cells, which indicates that the probe has strong capability of enriching histone modified binding proteins. In addition, other proteins are enriched, and the proteins possibly have a domain for recognizing H3K4me3 and have corresponding biological functions, so that subsequent structural and functional researches on the proteins are beneficial to finding new functional proteins and biological functions thereof. The probe has important application value in discovering new unknown protein modified binding protein.

Example 6: enrichment and characterization of histone H3K9cr modified binding proteins in nuclear proteins by polypeptide probes.

Using probe (4), the binding protein of H3K9cr was obtained (Table 2) in the same manner as in example 5, and all the binding proteins known to date recognizing the modification were found in the enriched and identified proteins, including: AF9, ENL and YEATS4 (Mol Cell 2016,62,181-193;Nat Chem Biol 2016,12,396-398;Structure 2017,25,571-573.). A series of proteins with recognition domains not reported are also found.

FIG. 11B is a western blot verification of binding protein KAT6B and polypeptide (4), and FIG. 11C is a further confirmation of the binding protein using computer docking analysis.

TABLE 2 identification of probes for H3K9cr binding proteins

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Claims

1. The polypeptide probe is characterized by comprising nano metal particles, polypeptides modified on the surfaces of the nano metal particles and photocrosslinking groups modified on the surfaces of the nano metal particles;

the amino acid residues of the polypeptide are not subjected to protein post-translational modification, or one or more amino acid residues of the polypeptide are provided with protein post-translational modification, and the C end of the polypeptide is a cysteine residue; the polypeptide is modified to the surface of the nano metal particles through a C-terminal cysteine residue;

the photocrosslinking group is modified to the surface of the nano metal particle through a connecting molecule, one end of the connecting molecule is covalently connected with the photocrosslinking group, the other end of the connecting molecule is modified by a sulfhydryl group to form a sulfhydryl-connecting molecule-photocrosslinking group covalent compound, and the sulfhydryl group is modified to the surface of the nano metal particle; the connecting molecule is polyethylene glycol with the molecular weight of 200-1000;

the polypeptide contains 7-50 amino acid residues;

the nano metal particles are selected from nano gold particles, nano silver particles, nano copper particles, nano iron particles, nano nickel particles and/or nano aluminum particles; the particle size of the nano metal particles is 1-10nm;

The molar ratio of the nano metal particles to the polypeptide modified on the surfaces of the nano metal particles to the photocrosslinking groups is 1 (50-1000) (20-500).

2. The polypeptide probe of claim 1, wherein the polypeptide comprises 10-30 amino acid residues.

3. The polypeptide probe of any one of claims 1-2, wherein the post-translational modification of the protein is a covalent modification of an amino acid residue, independently selected from methylation, formylation, acetylation, ubiquitination, propionylation, butyrylation, succinylation, crotonylation, malonyl, 2-hydroxybutyrylation, 3-hydroxybutyrylation, glutaryl-ylation, phosphorylation, glycosylation, lipidation, SUMO-ylation, and/or biotinylation.

4. The polypeptide probe of any one of claims 1-2, wherein the polypeptide comprises one or more lysine residues, one or more of which has a post-protein translational modification.

5. The polypeptide probe of claim 4, wherein the post-translational modifications of the protein on lysine residues are independently selected from the group consisting of monomethylation, dimethylation, trimethylation, acetylation, propionylation, butyrylation, crotonylation, formylation, ubiquitination, sumoylation, β -hydroxybutyrylation, 2-hydroxyisobutyrylation, succinylation, malonyl and/or glutaryl ation of lysine residues.

6. The polypeptide probe of claim 5, wherein the polypeptide is an amino acid sequence fragment of a histone protein, comprising a lysine residue that can undergo post-translational modification of the protein, and 3-25 amino acid residues before the N-terminus of the lysine residue and 3-25 amino acid residues after the C-terminus of the lysine residue.

7. The polypeptide probe of claim 6, comprising a lysine residue that is post-translationally modified with the protein, and 4-15 amino acid residues before the N-terminus of the lysine residue and 4-15 amino acid residues after the C-terminus of the lysine residue.

8. The polypeptide probe of claim 6, wherein the lysine residue on the histone that is subject to post-translational modification of the protein is selected from the group consisting of: lysine residues at the H3-K4, H3-K9, H3-K14, H3-K18, H3-K23, H3-K27, H3-K36, H3-K56, H3-K79, H4-K5, H4-K8, H4-K12, H4-K16, H4-K20, H4-K31, H4-K44, H4-K59, H4-K77, H4-K79, H4-K91, H2A-K5, H2A-K9, H2A-K13, H2A-K15, H2A-K63, H2A-K119, H2B-K5, H2B-K16, H2B-K17, H2B-K5, H2B-K34, H2B-K120, H1K26 and/or H1N-terminal.

9. The polypeptide probe of any one of claims 1-2, wherein the polypeptide is selected from the group consisting of: ARTKQTARKSC, ARTKQTARKSTGGKAC, ARTK (me 3) QTARKSC, ARTKQTARK (cr) STGGKAC, or ART (ph) K (me 3) QTARKSC.

10. The polypeptide probe of any one of claims 1-2, wherein the polypeptide comprises one or more arginine residues, one or more of which has a post-protein translational modification.

11. The polypeptide probe of claim 10, wherein the post-translational modifications of the protein on the arginine residue are independently selected from the group consisting of a single methylation modification, a symmetrical dimethyl modification, and an asymmetrical dimethyl modification.

12. The polypeptide probe of any one of claims 1-2, wherein the nano-metal particles are selected from nano-gold particles or/and nano-silver particles.

13. The polypeptide probe of any one of claims 1-2, wherein the photocrosslinking group is selected from the group consisting of azidobenzenes, benzophenones, and/or biazides.

14. The method for preparing the polypeptide probe according to any one of claims 1 to 13, wherein the nano metal particles, the polypeptide and the photocrosslinking group are reacted in a solution to modify the polypeptide and the photocrosslinking group to the surface of the nano metal particles;

nano-metal particles added to the solution: polypeptide: the molar ratio of the photo-crosslinking groups is 1 (100-1000): 50-500;

the solution is 0.1-5% citrate aqueous solution, and the citrate is sodium salt of citric acid, potassium salt of citric acid or ammonium salt of citric acid;

The reaction is carried out at 10-40 ℃.

15. The method of claim 14, wherein the solution is a 0.5-1.5% aqueous citrate solution.

16. The method of claim 14, wherein the solution is a 1% aqueous solution of trisodium citrate.

17. The method of claim 14, wherein the reaction is carried out at 20-30 ℃.

18. The method of any one of claims 14 to 17, further comprising a post-treatment step of the polypeptide probe, said step comprising washing the polypeptide probe and preserving it in liquid suspension.

19. A method for identifying or identifying a protein post-translationally modified binding protein or complex thereof, characterized in that a polypeptide probe according to any one of claims 1-13 is used, the polypeptide probe is contacted with the binding protein or complex thereof, or a protein mixture possibly containing said binding protein or complex thereof, in a solution suitable for binding the polypeptide probe to the binding protein or complex thereof at 0-8 ℃ and incubated for 1-24 hours, followed by photocrosslinking and removal of the uncrosslinked protein;

20. The method of claim 19, wherein the polypeptide in the polypeptide probe is ARTK (me 3) QTARKSC; the binding protein or complex thereof is a protein with domains WD40, PWWP, PHD, tudor and/or chomo.

21. The method of claim 20, wherein the binding protein or complex thereof is selected from WDR5, EED, PPWD1, WDR61, chap 1B, WDR, BRPF1, HDGF, PWWP2A, BRD1, PSIP1, ZMYND11, BPTF, ING1, CXXC1, ING2, DIDO1, ING4, ING5, KDM2B, KDM5A, KDM5B, KDM5C, KMT2A, PHF2, PHF8, PHF13, TAF3, ATRX, TRIM24, jace 3, RSF1, SGF29, KDM4C, SPIN1, SND1, SMNDC1, TDRD3, CHD1, CBX8, CDYL, CHD9, SUV39H1, ORC1, KDM18, L3MBTL2, FAM60A, PBRM1.

22. The method of claim 19, wherein the polypeptide in the polypeptide probe is ARTKQTARK (cr) STGGKAC; the binding protein or complex thereof is a protein with the domains DPF and/or YEATS.

23. The method of claim 22, wherein the binding protein or complex thereof is selected from the group consisting of: DPF2, KAT6A, DPF1, KAT6B, KMT2A, MTF2, NSD2, AF9, ENL, YEATS4, YEATS2, ATAD2, KAT2A, KAT7, SMARCA4.

24. A method for determining the interaction between a post-translational modification of a protein and a binding protein or complex thereof, characterized in that in each of at least two separate reaction systems, a polypeptide probe according to any one of claims 1-13 is used, the polypeptide probe is contacted with the binding protein or complex thereof, or a mixture of proteins possibly containing said binding protein or complex thereof, in a solution suitable for binding of the polypeptide probe to the binding protein or complex thereof, at 0-8 ℃, and incubated for 1-24 hours, followed by photocrosslinking, removing the uncrosslinked proteins;

25. The method of claim 24, wherein in determining the interaction between the H3K4me3 modification and its binding protein or its complex, one of the separate reaction systems employs a polypeptide on the polypeptide probe of ARTKQTARKSC and the other system employs a polypeptide on the polypeptide probe of ARTK (me 3) QTARKSC.

26. The method of claim 24, wherein in determining the interaction between the H3K9cr modification and its binding protein or its complex, one of the separate reaction systems employs a polypeptide probe having a polypeptide of ARTKQTARKSTGGKAC and the other system employs a polypeptide probe having a polypeptide of ARTKQTARK (cr) STGGKAC.

27. The method of claim 24, wherein in determining the interaction of methylation and phosphorylation occurring at H3 with the H3K4me3 modified binding protein or complex thereof, one of the separate reaction systems employs a polypeptide probe whose polypeptide is ART (me 3) QTARKSC and the other system employs a polypeptide probe whose polypeptide is ART (ph) K (me 3) QTARKSC.

28. The method of any one of claims 19 to 27, wherein the solution suitable for binding of the polypeptide probe to the binding protein or complex thereof comprises: 25-100 mM Tris-HCl,100-400 mM sodium chloride, 1-5 mM magnesium chloride and 1-5 mM potassium chloride;

the total dosage of the polypeptide probe in the solution is calculated by the mole number of the nano metal particles, and the proportion relation between the total dosage and the total amount of the contacted protein is as follows: 0.02 pmol-1 pmol/mug protein;

the illumination crosslinking reaction is carried out at 0-8 ℃;

The uncrosslinked proteins were removed by washing with a buffer containing 25-100 mM Tris-HCl,100-400 mM sodium chloride, 1-5 mM magnesium chloride and 1-5 mM potassium chloride, or alternatively, a PBS buffer containing 1-10M urea.

29. The method of claim 28, wherein the solution suitable for binding of the polypeptide probe to the binding protein or complex thereof comprises: 40-60 mM Tris-HCl,150-250 mM sodium chloride, 2-3 mM magnesium chloride, 2-3 mM potassium chloride, pH 7-8.

30. The method of claim 29, wherein the solution suitable for binding of the polypeptide probe to the binding protein or complex thereof comprises: 50 mM Tris-HCl, pH 7.8, 200 mM sodium chloride, 2.5 mM magnesium chloride and 2.5 mM potassium chloride.

31. The method of claim 28, wherein the uncrosslinked proteins are removed by washing with a buffer containing 40-60 mM Tris-HCl, pH 7-8, 150-250 mM sodium chloride, 2-3 mM magnesium chloride and 2-3 mM potassium chloride.

32. The method of claim 31, wherein the uncrosslinked protein is removed by washing with a buffer comprising: 50 mM Tris-HCl, pH 7.8, 200 mM sodium chloride, 2.5 mM magnesium chloride and 2.5 mM potassium chloride.

33. The method of claim 28, wherein the uncrosslinked proteins are removed by washing with a buffer of PBS buffer containing 3-5M urea.

34. The method of claim 33, wherein the uncrosslinked proteins are removed using a buffer wash of 50mM PBS buffer containing 4M urea.

35. The method of any one of claims 19 to 27, further comprising the steps of obtaining a polypeptide probe by centrifugation or magnetic separation and displacing the protein bound to the polypeptide probe from the nano-metal particles, said displacement using a thiol-bearing compound.

36. The method of claim 35, further comprising the step of purifying the displaced binding protein or complex thereof.

37. Use of the polypeptide probe of any one of claims 1-13 to identify or determine the interaction between a protein post-translational modification binding protein or complex thereof and a protein post-translational modification;

the binding protein or complex thereof is a reader protein or complex thereof that is post-translationally modified by the protein; alternatively, the binding protein or complex thereof is a protein post-translationally modified, de-modified enzyme protein or complex thereof.

38. A test kit for identifying or identifying a protein post-translationally modified binding protein or complex thereof, or for determining the interaction between a protein post-translationally modified binding protein or complex thereof and a protein post-translational modification, comprising at least one polypeptide probe according to any one of claims 1-13, each polypeptide probe being present in a separate liquid system;

the liquid is selected from water, carbonate buffer, tris buffer, hepes buffer or PBS buffer;

the concentration of the polypeptide probe in the liquid is 10 nM-500 nM in terms of the mole number of the nano metal particles.

39. The assay kit of claim 38, wherein the concentration of the polypeptide probe in the liquid is 50nm to 150nm, based on moles of nano-metal particles.

40. The test kit of any one of claims 38-39, further comprising a solution for the identification, characterization reaction, the solution comprising 25-100 mM Tris-HCl,100-400 mM sodium chloride, 1-5 mM magnesium chloride, and 1-5 mM potassium chloride.

41. The test kit of claim 40, wherein the solution for the identification and authentication reaction comprises: 40-60 mM Tris-HCl, pH 7-8, 150-250 mM sodium chloride, 2-3 mM magnesium chloride and 2-3 mM potassium chloride.

42. The test kit of claim 41, wherein the solution for the identification and authentication reaction comprises: 50 mM Tris-HCl, pH 7.8, 200 mM sodium chloride, 2.5 mM magnesium chloride and 2.5 mM potassium chloride.

43. The test kit of claim 40, further comprising a buffer for eluting the uncrosslinked protein, wherein the buffer is the same as the solution used for the identification and characterization reaction, or wherein the buffer is a PBS buffer containing 1-10M urea.

44. The test kit of claim 43, wherein the buffer is a PBS buffer containing 3-5M urea.