Methods of screening or identifying ligand binding proteins

Abstract

The present application belongs to the technical field of protein interaction or proteomics, and particularly relates to a method for screening or identifying ligand-binding proteins, which uses a biosensor to enrich proteins, separates strong-binding proteins and weak-binding proteins through a two-step trypsin digestion method, and finally identifies and quantitatively analyzes through mass spectrometry. The method determines the target protein and binding region of the ligand based on the change in local stability of the protein after the biosensor (such as a biological layer interference technology) detects the ligand binding. The present application also provides a device for screening or identifying ligand-binding proteins, which comprises a protein sample to be tested module, a biosensor module, a first trypsin digestion module, an elution module, a second trypsin digestion module, and an identification module.

Description

Technical Field

[0001] This invention belongs to the technical field of protein interaction or proteomics, specifically relating to a method or apparatus for screening or identifying ligand-binding proteins. The method utilizes a biosensor to enrich proteins, then separates strong-binding and weak-binding proteins using a two-step enzymatic digestion method, and finally identifies and quantifies them using mass spectrometry. The method is based on a biosensor (e.g., biolayer interferometry) to detect changes in the local stability of proteins after ligand binding, thereby determining the target protein and binding region of the ligand. Background Technology

[0002] Molecular fishing, originating from the study of intermolecular interactions, is a technical system that achieves specific molecule capture based on experimental methods such as ligand fishing, surface plasmon resonance (SPR), biolayer interferometry (BLI), and pull-down. Molecular fishing involves immobilizing a known biomolecule (usually called a ligand) on a solid support via coupling. A complex sample solution containing the potential target molecule is then brought into contact with the solid support. During this contact, the target molecule in the sample solution binds to the ligand molecule on the solid support. Finally, unbound or weakly bound components are washed away with a washing solution, and the bound ligand molecules are eluted from the solid phase using an elution buffer for subsequent analysis and identification.

[0003] Drug affinity responsive target stability (DARTS) is a new technology developed based on the principle that the binding of small molecule drugs to their target proteins leads to a decrease in the sensitivity of the target proteins to protease degradation. It is currently mainly used to identify drug targets. The basic steps of DARTS are as follows: (1) preparation of protein library; (2) incubation of protein and small molecule; (3) protease hydrolysis; (4) detection of protein differences between the control group and the drug-treated group by Coomassie brilliant blue, silver staining, immunoblotting and other methods; (5) collection of target protein gel bands; (6) identification of target protein by mass spectrometry and other methods; (7) verification of the target by parallel experiments. However, the DARTS technique has limitations, including: Its sensitivity is limited, relying on SDS-PAGE gel staining for visualization, making it difficult to detect low-abundance target proteins; high-abundance proteins may mask the signal of low-abundance targets. Furthermore, DARTS is susceptible to specific interference during detection; some proteins in their native state have low sensitivity to proteases, potentially affecting the accuracy of results. Simultaneously, drug binding may alter the enzymatic sensitivity of non-target proteins, increasing the risk of false positives, and the aggregation of non-specific proteins can also interfere with the signal. In vivo application is challenging, currently mainly limited to in vitro cell lysis systems, making it difficult to simulate the interactions of the in vivo physiological environment. Additionally, the DARTS method cannot distinguish the binding strength of interacting factors.

[0004] Limited proteolysis-coupled mass spectrometry (LiP-MS) is a compound target screening technique based on limited protease digestion. Its unique feature is that it can screen for specific compound-binding peptides without requiring modification of the compound. The entire process requires two rounds of protease digestion: first, proteinase K digests the peptides, followed by trypsin digestion. This technique has the following drawbacks: low sensitivity; the two-step digestion process leads to dilution of peptides reflecting conformational changes by a large number of non-specific peptides, resulting in low identification efficiency and insufficient detection capability for low-abundance proteins; complex data interpretation; binding site identification depends on peptide sequence coverage, requiring sufficient coverage of key target protein regions, and dynamic changes in protein conformation may interfere with binding site localization, increasing the difficulty of result interpretation; and strict control of experimental conditions; parameters such as the digestion time and enzyme concentration need precise optimization, otherwise over- or under-digestion can easily occur, affecting the reproducibility of results. Furthermore, LiP-MS lacks an enrichment and separation process; it directly digests the entire protein mixture with enzymes, and then uses mass spectrometry to identify differences in protein abundance to determine interacting proteins. This method places high demands on subsequent data analysis and processing, and it is prone to failing to detect some weak signals. LiP-MS cannot distinguish the binding strength of interacting factors.

[0005] Peptide-centric Local Stability Assay (PELSA) is used to identify ligand-binding proteins and their binding sites. This technique determines the target protein and binding region of the ligand by detecting changes in the local stability of the protein after ligand binding. The PELSA method does not require chemical modification of the ligand and is independent of affinity. It can directly detect proteins that undergo local stability changes after ligand binding in complex samples such as cell lysates, thereby achieving a systematic analysis of ligand-binding proteins, binding sites, and local affinity. This technique has the following disadvantages: (1) It involves the processing and display of a large amount of peptide quantitative data, and the data processing is relatively complex; (2) Because it involves enzymatic digestion of total protein and then quantitative analysis of all peptides produced after digestion; since the peptides obtained by digestion of total protein are numerous, finding differential proteins is like finding a needle in a haystack, which may lead to some weak differences being masked and not detected, resulting in a large limitation of the detection targets and a strong dependence on protein abundance and conformational changes; (3) It can only play an identification role and cannot distinguish between strong and weak binding.

[0006] Therefore, new methods for screening and identifying ligand-binding proteins are still needed to distinguish between strong and weak binding proteins in interacting proteins, and / or to improve the sensitivity of screening for protein interactions. Summary of the Invention

[0007] This invention provides a method for screening and identifying ligand-binding proteins. It determines the target protein and binding region of the ligand by detecting changes in the local stability of the protein after ligand binding. Notably, this method uses a two-step enzymatic digestion method to distinguish between strong-binding and weak-binding proteins.

[0008] Therefore, in a first aspect, the present invention provides a method for screening or identifying target proteins that bind to ligands, the method comprising: (1) preparing a protein sample to be tested, the protein sample containing the target protein; (2) ligand enrichment: enriching the ligand onto the surface of a biosensor, wherein the ligand is capable of binding to the target protein; (3) interacting protein binding: contacting the protein sample to be tested with a biosensor on which the ligand is enriched; (4) first trypsin digestion: placing the biosensor from step (3) in a first working solution containing trypsin for enzymatic digestion and collecting the first digestion product; (5) protein elution: eluting the biosensor after enzymatic digestion in step (4) with an elution buffer and collecting the eluted protein; (6) second trypsin digestion: subjecting the eluted protein collected in step (5) to trypsin digestion to obtain a second digestion product; and (7) identification: identifying the first digestion product to obtain a first target protein that weakly binds to the ligand, and / or identifying the second digestion product to obtain a second target protein that strongly binds to the ligand.

[0009] In some implementations, the first trypsin digestion in step (4) serves to "remove weak binding and retain strong binding." In some implementations, after the ligand binds to the protein, a first trypsin digestion is performed. At this time, the weakly binding protein (or the loosely bound region of the protein) is digested by trypsin and removed from the biosensor due to its weak protective effect, and enters the first digestion product. The strong binding protein (or the tightly bound region of the protein) is retained on the biosensor due to its stable conformation.

[0010] In some embodiments, in step (4), the concentration of the trypsin is 0.1 μg / ml to 2 μg / ml, for example 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, or 2.0 μg / ml. In some embodiments, in step (4), the concentration of the trypsin is 1.0 μg / ml.

[0011] In some embodiments, in step (4), the digestion time of the trypsin is 100s to 5000s, for example, 100s, 500s, 1000s, 1500s, 2000s, 2500s, 3000s, 3500s, 4000s, 4500s, or 5000s. In some embodiments, in step (4), the digestion time of the trypsin is 3600s.

[0012] In some implementations, in step (4), the concentration of the trypsin is 1 μg / ml, and / or the digestion time is 3600 s.

[0013] In some implementations, in step (4), the enzymatic digestion temperature of the trypsin is 25℃~37℃, for example 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃.

[0014] In some embodiments, the second trypsin digestion in step (6) serves to "capture the strong binding protein". In some embodiments, after eluting the strong binding protein that was not cleaved in the first digestion from the sensor with an elution buffer, the eluted protein is subjected to a second trypsin digestion to obtain a second enzymatic hydrolysate.

[0015] In some implementations, in step (6), the enzymatic digestion temperature of the trypsin is 25℃~37℃, for example 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃.

[0016] In some implementations, in step (6), the trypsin digestion time is 7h to 12h, for example 7h, 8h, 9h, 10h, 11h, 12h.

[0017] In some embodiments, in step (6), the concentration of the trypsin is 0.1 μg / ml to 2 μg / ml, for example, 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, or 2.0 μg / ml. In some embodiments, in step (6), the concentration of the trypsin is 1.0 μg / ml.

[0018] In some implementations, in step (6), the trypsin digestion temperature is 37°C, and / or the digestion time is 12 h.

[0019] As used herein, the term "trypsin (EC 3.4.4.4)" refers to a serine proteolytic enzyme extracted from the pancreas of cattle, sheep, or pigs. Trypsin is an endopeptidase that cleaves the carboxyl side of lysine and arginine residues in polypeptide chains. In this invention, the trypsin is used to digest protein-protein linkages, rather than for complete degradation.

[0020] In some implementations, the eluent in step (5) is selected from urea solution.

[0021] In some embodiments, the concentration of the urea solution in step (5) is 0.1M to 10M, for example, 0.1M, 0.5M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, or 10M. In some preferred embodiments, the concentration of the urea solution is 1M.

[0022] In some embodiments, the treatment time of the urea solution in step (5) is 100s to 1000s, for example, 100s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, or 1000s. In some preferred embodiments, the treatment time of the urea solution is 600s.

[0023] In some implementations, the concentration of the urea solution in step (5) is 1M, and / or the treatment time of the urea solution is 600s.

[0024] In some implementations, step (3) is followed by washing the biosensor with a washing buffer to remove unbound contaminating proteins.

[0025] In some implementations, step (4) is followed by: washing the enzyme-digested biosensor with a washing buffer.

[0026] In some embodiments, the washing buffer is selected from phosphate buffer.

[0027] In some implementations, in step (2), a binding agent is fixed on the surface of the biosensor, and the ligand binds to the binding agent through covalent or non-covalent linkage, thereby enriching the biosensor surface.

[0028] In some implementations, the non-covalent interaction is selected from electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, π-π stacking interactions, and coordination bonds.

[0029] In some embodiments, the binder is a negatively charged binder. In some embodiments, the negatively charged binder is aminopropylsilane (APS).

[0030] In some embodiments, the ligand is selected from nanoplastics, proteins, DNA, RNA, or small molecule compounds.

[0031] In some embodiments, the biosensor is selected from biolayer interferometry (BLI) sensors and surface plasmon resonance (SPR) biosensors.

[0032] In some embodiments, the biosensor is selected from biolayer interferometry (BLI) sensors, which are used to determine the presence and / or amount of target molecules that bind to the ligand.

[0033] In some implementations, the identification methods in step (7) include, but are not limited to, mass spectrometry identification, immunological identification, and chromatographic identification.

[0034] In some implementations, the immunological identification includes, but is not limited to, Western blotting identification.

[0035] In some implementations, the chromatographic identification includes, but is not limited to, high-performance liquid chromatography, ion exchange chromatography, and affinity chromatography.

[0036] In some embodiments, the protein sample to be tested is a single protein solution containing one protein or a mixed protein solution containing two or more proteins.

[0037] In some implementations, the protein sample to be tested is a protein extracted from a biological sample.

[0038] In some implementations, the biological sample is derived from a human, animal, plant, or microorganism, or from an extract of a human, animal, plant, or microorganism's cell or tissue.

[0039] In some implementations, the protein sample to be tested is total protein extracted from Arabidopsis thaliana seeds.

[0040] In a second aspect, the present invention provides an apparatus for screening or identifying target proteins that bind to ligands, the apparatus comprising: (1) a test protein sample module, the test protein sample module comprising a test protein sample having a target protein; (2) a biosensor module having a surface capable of enriching ligands and being capable of binding to the target protein in the test protein sample module through the ligands; (3) a first trypsin digestion module, the first trypsin working solution contained therein capable of digesting the target protein bound in the biosensor module and generating a first digestion product; (4) an elution module, the elution buffer contained therein capable of eluting the biosensor after digestion in module (3) and generating eluted protein; (5) a second trypsin digestion module, the trypsin contained therein capable of digesting the eluted protein generated in module (4) and generating a second digestion product; (6) an identification module, capable of identifying the first digestion product as a first target protein weakly bound to the ligand, and / or capable of identifying the second digestion product as a second target protein strongly bound to the ligand.

[0041] In some embodiments, the concentration of the trypsin in module (3) is 0.1 μg / ml to 2 μg / ml, for example 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, and 2.0 μg / ml. In some embodiments, the concentration of the trypsin in module (3) is 1.0 μg / ml.

[0042] In some embodiments, the digestion time of the trypsin in module (3) is 100s to 5000s, for example, 100s, 500s, 1000s, 1500s, 2000s, 2500s, 3000s, 3500s, 4000s, 4500s, and 5000s. In some embodiments, the digestion time of the trypsin in module (3) is 3600s.

[0043] In some implementations, in module (3), the concentration of the trypsin is 1 μg / ml, and / or the digestion time is 3600 s.

[0044] In some implementations, the enzyme digestion temperature of the trypsin in module (3) is 25℃~37℃, for example 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃.

[0045] In some implementations, the enzyme digestion temperature of the trypsin in module (5) is 25℃~37℃, for example 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃.

[0046] In some implementations, in module (5), the digestion time of the trypsin is 7h to 12h, for example 7h, 8h, 9h, 10h, 11h, 12h.

[0047] In some embodiments, the concentration of the trypsin in module (5) is 0.1 μg / ml to 2 μg / ml, for example 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.5 μg / ml, and 2.0 μg / ml. In some embodiments, the concentration of the trypsin in module (5) is 1.0 μg / ml.

[0048] In some implementations, in module (5), the digestion temperature of the trypsin is 37°C, and / or the digestion time is 12h.

[0049] In some implementations, the eluent in module (4) is selected from urea solution.

[0050] In some embodiments, the concentration of the urea solution is 0.1M to 10M, for example, 0.1M, 0.5M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, or 10M. In some preferred embodiments, the concentration of the urea solution is 1M.

[0051] In some embodiments, the treatment time of the urea solution is 100s to 1000s, for example, 100s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, or 1000s. In some preferred embodiments, the treatment time of the urea solution is 600s.

[0052] In some preferred embodiments, the concentration of the urea solution is 1M, and / or the treatment time of the urea solution is 600s.

[0053] In some implementations, the surface of the biosensor in module (2) is immobilized with a binding agent, and the ligand binds to the binding agent through covalent or non-covalent linkage, thereby enriching the biosensor surface.

[0054] In some implementations, the non-covalent interaction is selected from electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, π-π stacking interactions, and coordination bonds.

[0055] In some embodiments, the binder is a negatively charged binder. In some embodiments, the negatively charged binder is aminopropylsilane (APS).

[0056] In some embodiments, the ligand is selected from nanoplastics, proteins, DNA, RNA, or small molecule compounds.

[0057] In some implementations, the biosensor is selected from biosensors used in biolayer interferometry (BLI).

[0058] In some embodiments, the device is capable of using biolayer interferometry (BLI) to determine the presence and / or amount of target molecules bound to the ligand.

[0059] In some embodiments, the device also includes components for measuring signals from biosensors using biolayer interferometry.

[0060] In some implementation schemes, the identification method in module (6) is selected from mass spectrometry identification, immunological identification, and chromatographic identification.

[0061] In some implementations, the immunological identification includes, but is not limited to, Western blotting identification.

[0062] In some implementations, the chromatographic identification includes, but is not limited to, high-performance liquid chromatography, ion exchange chromatography, and affinity chromatography.

[0063] In some embodiments, the protein sample to be tested is a single protein solution containing one protein or a mixed protein solution containing two or more proteins.

[0064] In some implementations, the protein sample to be tested is a protein extracted from a biological sample.

[0065] In some implementations, the biological sample is derived from a human, animal, plant, or microorganism, or from an extract of a human, animal, plant, or microorganism's cell or tissue.

[0066] In some implementations, the protein sample to be tested is total protein extracted from Arabidopsis thaliana seeds.

[0067] In some embodiments, the method or apparatus of this invention can be used to evaluate efficacy and side effects in drug screening. Through two trypsin digestions, on the one hand, the strongly binding proteins screened typically indicate that the drug binds tightly to the expected target, representing the basis of potential efficacy; on the other hand, the weakly binding proteins screened exhibit weak "off-target" interactions with other proteins in the body. These weak interactions may not be apparent in the short term, but long-term accumulation may lead to toxic side effects, thus providing a theoretical basis for subsequent drug side effect evaluation.

[0068] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular biology, and immunology laboratory procedures used herein are all conventional procedures widely used in the field. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0069] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0070] As used herein, the term "Bio-Layer Interferometry (BLI)" is a label-free, real-time analytical technique based on the principle of optical interference, widely used for determining the affinity of interactions such as protein-protein, protein-small molecule, and nucleic acid-protein. Its core component is a biosensor, typically consisting of an optical fiber probe and a biomolecular coating at its end. When the biosensor is immersed in a solution containing the target molecule, the biomolecule specifically binds to molecules on the coating, causing a change in the thickness of the biolayer on the biosensor surface. This thickness change interferes with the light waves in the optical fiber, resulting in a detectable change in the interference pattern. By analyzing the changes in the interference pattern, the kinetic parameters of biomolecule binding can be precisely measured, including the binding rate constant (kon), dissociation rate constant (koff), and affinity constant (KD).

[0071] Beneficial effects of the invention: This invention provides a method for screening or identifying ligand-binding proteins. The method involves a two-step enzymatic proteolysis treatment of the protein sample to effectively distinguish between strong and weak binding proteins. Compared to existing methods for screening interacting proteins, such as the peptide-centric Local Stability Assay (PELSA), the method of this invention can effectively distinguish between strong and weak binding proteins in the sample, thereby significantly improving the sensitivity and / or specificity of screening interacting proteins.

[0072] In some implementations, the method for screening or identifying target proteins that bind to ligands according to the present invention transforms the dynamic information of the "binding strength" between the ligand and the target protein into static information of "different samples" through two trypsin digestion steps. This allows the target protein to be found, its binding strength to be assessed, and the binding site or binding region to be located in subsequent identification.

[0073] The method described in this invention, through two trypsin digestions, can effectively distinguish between "transient binding" and "stable binding" between the partition body and the target protein.

[0074] The method described in this invention can improve the accuracy of target screening and eliminate "background noise". Specifically, it can effectively screen for interacting proteins of interest that bind directly to ligands and have high specificity and affinity.

[0075] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0076] Figure 1 Schematic diagram of a method for screening and identifying ligand-binding proteins based on biolayer interferometry (BLI).

[0077] Figure 2 Characterization image of 25nm polystyrene plastic under transmission electron microscopy.

[0078] Figure 3 The size distribution of nanoplastics was identified by dynamic light scattering (DLS).

[0079] Figure 4 Enrichment results of interacting proteins in PS25 nanoplastic based on BLI. The control group was obtained without ligands. Detailed Implementation

[0080] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention.

[0081] The flowchart of the present invention for screening and identifying ligand-binding proteins based on the BLI method is shown below. Figure 1 As shown. Experimental steps: 1. Ligand synthesis or preparation.

[0082] 2. Ligand enrichment: Ligands (e.g., 25nm polystyrene nanoplastic (PS25)) are bound to the sensor.

[0083] 3. Total protein extraction.

[0084] 4. Rolling interaction protein binding: After the ligand binds to the sensor, the sensor is washed with phosphate-buffered saline (PBS). The total protein and other proteins are repeatedly rolled around and incubated with the ligand-bound sensor, which allows the interaction protein to repeatedly bind to the ligand, thereby enriching the target protein and amplifying the signal.

[0085] 5. First trypsin digestion: After washing with PBS, the sensor containing the bound protein was placed in trypsin working solution for enzymatic digestion. The digestion products were then identified by mass spectrometry. The identified proteins were a set of weakly binding proteins to the ligand. These weakly binding protein sets mainly included peripheral proteins that did not directly bind to the ligand.

[0086] 6. Protein elution: After washing with PBS, the bound proteins on the sensor are eluted with a strong elution buffer (such as urea solution).

[0087] 7. Second trypsin digestion: The eluted protein is then subjected to trypsin digestion.

[0088] 8. Mass spectrometry identification: The products after the above enzyme digestion are identified by mass spectrometry. The proteins identified in this step are a collection of proteins that strongly bind to the ligand. These strongly binding proteins are proteins that are in direct contact with the ligand.

[0089] Example: Identification of target proteins interacting with nanoplastics in plants and isolation and identification of interacting epitopes. In situ molecular fishing is a technique for screening target proteins and binding epitopes. Its basic principle is to extract receptor molecules that can bind to ligands from a molecular library (such as total plant protein or cell lysate). Typically, target protein screening is achieved through a stepwise process of solid-phase immobilization, incubation, trypsin digestion, and mass spectrometry identification. Based on biolayer interferometry (BLI), we can observe the specific steps of binding, enzymatic digestion, and elution in real time.

[0090] 1. Extraction of total plant protein a) Place Arabidopsis ecotype Col-0 seeds in a 1.5ml EP tube, add 1ml of 15% sodium hypochlorite solution and soak for 10min, then rinse 5 times with sterile water.

[0091] b) Suspend the sterilized seeds in sterile water and sow them evenly on MS medium using a pipette.

[0092] c) Place the seeds sown on MS medium in a 4°C refrigerator for 3 days to ensure uniform germination.

[0093] d) After growing the culture plate in a white light box for 7 days, collect the material.

[0094] e) Total protein extraction from Arabidopsis thaliana seedlings. 1 g of Arabidopsis thaliana material was thoroughly ground into powder using liquid nitrogen and transferred to a 5 ml centrifuge tube. 1 ml of non-denaturing protein extraction buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 10% Glycerol, 1% (V / V) protease inhibitor) was added, and the mixture was vortexed to mix. The mixture was then placed on ice for 20 min. Centrifuged at 4 ℃ and 13000 rpm for 10 min, and the supernatant was collected (repeated twice). The clarified total protein extract was then analyzed using the Bradford method to determine protein concentration.

[0095] 2. Synthesis and Characterization of Nanoplastics a) The 25nm polystyrene plastic used in this study was purchased from Zhongke Leiming (Beijing) Technology Co., Ltd.

[0096] b) The purchased nanoplastics were characterized using transmission electron microscopy and dynamic light scattering (DLS) techniques.

[0097] The results of transmission electron microscopy characterization of 25 nm polystyrene plastic are as follows: Figure 2 As shown.

[0098] The results of DLS identification of the size distribution of nanoplastics are as follows: Figure 3 As shown.

[0099] The DLS patterns of nanoplastics are shown in Table 1.

[0100] Table 1: DLS mode of nanoplastics

[0101] 3. Preparation of biosensors a) Select a suitable sensor based on the ligand type. In this study, the ligand is 25nm polystyrene plastic with a negative charge. Therefore, it is immobilized on the probe surface of the biosensor via non-covalent adsorption.

[0102] b) Sensor activation. Remove the aminopropylsilane (APS) biosensor probe and immediately place it in PBS buffer. Let it stand at room temperature for 10 minutes for pre-hydration to prevent the probe surface from drying out and causing the active sites to become ineffective. This step must be completed before instrument debugging to ensure that the probe is fully hydrated.

[0103] c) Instrument baseline calibration (activation verification): After pre-hydration, remove the probe and debug the BLI detector according to the original operation (1000 rpm, 30℃). Install the probe in the instrument calibration position, add the buffer solution used in the experiment, start the baseline recording program, and continue to calibrate until the signal is stable and there is no drift (baseline fluctuation ≤ ±0.05nm). Confirm that the probe activation is qualified.

[0104] d) Post-activation processing: Calibrated probes (activated) must be used immediately in subsequent ligand fixation steps. Throughout the process, avoid contact between the probe surface and impurities, drying, or contact with non-sterile containers. If the probe is not used immediately after activation, it must be placed back into the pre-hydration buffer for storage, and the storage time shall not exceed 30 minutes. If the storage time exceeds this period, it must be reactivated.

[0105] 4. Immobilization of nanoplastics into biosensors a) Ligand pretreatment: The synthesized nanoplastics (50 mg / ml) were diluted 1000 times with ddH2O, shaken to mix, and then left to stand for use.

[0106] b) Immobilization of nanoplastics onto the biosensor surface: Nanoplastic immobilization is achieved through non-covalent adsorption. Loading time was 100 s, rotation speed was 1000 rpm, and temperature was set at 30℃. After loading, the BLI signal curve was observed to confirm successful immobilization of the ligand-nanoplastic complex on the probe surface, with a stable signal and no significant drift (if the signal is abnormal, the probe needs to be replaced and the operation repeated). A control group without ligands (i.e., an empty sensor) was also set up.

[0107] 5. Enrichment and identification of interacting proteins a) Gradient enrichment of interacting proteins ("snowball" model enrichment): The extracted total plant protein solution is added to the sample chamber, and the APS sensor is used to allow it to interact with immobilized ligand molecules, thereby enriching the interacting proteins. The BLI real-time monitoring mode is activated, and a cyclic loading-binding program is started with the following parameters: single loading 100s → equilibration 60s (maintaining a rotation speed of 1000 rpm and a temperature of 30℃) → binding reaction 100s. The BLI system monitors and records the binding signal with the ligand molecules in real time. This loading 100s-equilibration 60s cycle is repeated 20 times to form a "snowball" enrichment model. Throughout the process, the protein binding is observed in real time using the BLI signal curve to ensure that the affinity exhibits a gradient distribution—the stronger the binding, the deeper the position occupied; the weaker the binding, the greater the relative distance.

[0108] b) Step 1: Trypsin digestion and separation of the protein corona – “soft corona” component (weakly bound, located on the periphery of the protein corona): Carefully remove the BLI probe and gently rinse the probe surface three times with PBS to remove unbound contaminating proteins and avoid interference with subsequent experiments. Place the biosensor containing the bound protein in 1 μg / ml trypsin for a “snow-melting” process for 3600 s to ensure complete digestion. Perform mass spectrometry analysis on the obtained polypeptide fragments to identify the protein corona – “soft corona” component. During enzymatic digestion, the digestion process can be monitored with the help of BLI. When the signal curve tends to be stable, it indicates that the digestion is basically complete and small molecule protein / peptide fragments are obtained.

[0109] c) Elution of peptide / protein residues: After washing the biosensor treated with trypsin three times with PBS solution, place it in 1M urea for 600 s to elute all remaining strongly interacting proteins bound to the APS sensor. After elution, carefully collect the eluent and store it on ice to prevent peptide fragment degradation; at the same time, observe the BLI signal curve to confirm that there are no protein residues on the probe surface (signal returns to baseline).

[0110] d) Second round of trypsin digestion to obtain the protein crown – the “hard crown” component (strongly bound, located in the inner circle of the protein tube): The urea-eluting protein was subjected to a second round of trypsin treatment at a concentration of 1 μg / ml, digested overnight at 37°C. The digested peptides were analyzed by mass spectrometry to determine the sequence of the separated fragments, and library search analysis was used to identify the types of proteins.

[0111] e) Data analysis and post-validation: The identified sequences are compared to find identical or adjacent sequences, enabling both protein identification and epitope analysis.

[0112] The results of BLI-based enrichment of interacting proteins in step a) are as follows: Figure 4 As shown.

[0113] Table 2 shows the identification results of COP1, an exemplary protein that strongly interacts with the PS25 ligand, as screened by this method.

[0114] Table 2: Results of analysis of identified strongly interacting proteins .

Claims

1. A method for screening or identifying target proteins that bind to ligands, the method comprising: (1) Prepare a protein sample to be tested, wherein the protein sample to be tested contains a target protein; The protein sample to be tested is a protein extracted from a plant sample or an extract from plant cells or tissues; the plant is Arabidopsis thaliana. (2) Ligand enrichment: Ligands are enriched onto the surface of the biosensor, wherein the ligands are capable of binding to the target protein; the ligands are 25 nm polystyrene plastic; (3) Interacting protein binding: The protein sample to be tested is brought into contact with a biosensor whose surface is enriched with ligands; (4) First trypsin digestion: The biosensor from step (3) is placed in a first working solution containing trypsin for digestion and the first digestion product is collected; wherein the concentration of the trypsin is 1 μg / ml and the digestion time is 3600s; (5) Protein elution: The biosensor after enzymatic hydrolysis in step (4) is eluted with urea solution and the eluted protein is collected; wherein the concentration of the urea solution is 1M and the treatment time of the urea solution is 600s; (6) Second trypsin digestion: The eluted protein collected in step (5) is subjected to trypsin digestion to obtain the second digestion product; wherein the concentration of the trypsin is 1 μg / ml, the digestion time is 12 h, and the digestion temperature is 37℃. as well as, (7) Identification: The first enzymatic hydrolysis product is identified to obtain a first target protein that weakly binds to the ligand, and the second enzymatic hydrolysis product is identified to obtain a second target protein that strongly binds to the ligand, wherein the second target protein is COP1.

2. The method of claim 1, wherein, The method includes any one of the following: (i) Step (3) is followed by: washing the biosensor with a washing buffer to remove unbound contaminating proteins; (ii) Step (4) is followed by: washing the enzyme-digested biosensor with a washing buffer; or (iii) The washing buffer is selected from phosphate buffer.

3. The method of claim 1, wherein step (2) has a feature selected from any one of the following: (i) A binding agent is immobilized on the surface of the biosensor, and the ligand binds to the binding agent through covalent or non-covalent linkage, thereby accumulating on the surface of the biosensor; (ii) The non-covalent mechanism is selected from electrostatic interaction, hydrogen bonding, hydrophobic interaction, van der Waals force, π-π stacking interaction, and coordination bond; (iii) The binder is a negatively charged binder; or (iv) The negatively charged binder is aminopropylsilane.

4. The method of claim 1, wherein, The method has any of the following characteristics: (i) The biosensor is selected from biosensors used in biolayer interferometry; or (ii) The method includes using biolayer interferometry to determine the presence and / or amount of target molecules that bind to the ligand.

5. The method of claim 1, wherein, Step (7) has any of the following characteristics: (i) The identification methods described are selected from mass spectrometry, immunology, and chromatography. (ii) The immunological identification is selected from Western blotting; or (iii) The chromatographic identification method is selected from high performance liquid chromatography, ion exchange chromatography, and affinity chromatography.

6. The method of claim 1, wherein, The protein sample to be tested is selected from any of the following: (i) The protein sample to be tested is a single protein solution containing one protein or a mixed protein solution containing two or more proteins; or (ii) The protein sample to be tested is total protein extracted from Arabidopsis thaliana seeds.

7. An apparatus for screening or identifying target proteins that bind to a ligand, the apparatus comprising: (1) A test protein sample module, wherein the test protein sample module contains a test protein sample containing a target protein; The protein sample to be tested is a protein extracted from a plant sample or an extract from plant cells or tissues; the plant is Arabidopsis thaliana. (2) A biosensor module whose surface is capable of accumulating ligands and can bind to target proteins in the protein sample module to be tested through the ligands; the ligands are 25nm polystyrene plastic; (3) The first trypsin digestion module contains a first working solution of trypsin that can digest the target protein bound in the biosensor module and produce the first digestion product; wherein the concentration of the trypsin is 1 μg / ml and the digestion time is 3600s. (4) Elution module, which contains an eluent that can elute the enzymatically hydrolyzed biosensor in module (3) and generate eluted protein; wherein the eluent is a urea solution with a concentration of 1M and a treatment time of 600s. (5) A second trypsin digestion module, which contains trypsin that can digest the eluted protein produced in module (4) and produce a second digestion product; wherein the concentration of the trypsin is 1 μg / ml, the digestion time is 12 h, and the digestion temperature is 37 °C. (6) An identification module, which is capable of identifying the first enzymatic hydrolysis product as a first target protein that is weakly bound to the ligand, and is capable of identifying the second enzymatic hydrolysis product as a second target protein that is strongly bound to the ligand, wherein the second target protein is COP1.

8. The apparatus of claim 7, wherein, Module (2) has a feature selected from any of the following: (i) A binding agent is immobilized on the surface of the biosensor, and the ligand binds to the binding agent through covalent or non-covalent linkage, thereby accumulating on the surface of the biosensor; (ii) The non-covalent mechanism is selected from electrostatic interaction, hydrogen bonding, hydrophobic interaction, van der Waals force, π-π stacking interaction, and coordination bond; (iii) The binder is a negatively charged binder; or (iv) The negatively charged binder is aminopropylsilane.

9. The apparatus of claim 7, wherein, The device has any of the following features: (i) The biosensor is selected from biosensors used in biolayer interferometry; (ii) The device is capable of determining the presence and / or amount of target molecules bound to the ligand using biolayer interferometry; or (iii) The device also includes components for measuring signals from biosensors of biolayer interferometry.

10. The apparatus of claim 7, wherein, Module (6) has any of the following characteristics: (i) The identification methods described are selected from mass spectrometry, immunology, and chromatography. (ii) The immunological identification is selected from Western blotting; or (iii) The chromatographic identification method is selected from high performance liquid chromatography, ion exchange chromatography, and affinity chromatography.

11. The apparatus of claim 7, wherein, The protein sample module to be tested has any of the following characteristics: (i) The protein sample to be tested is a single protein solution containing one protein or a mixed protein solution containing two or more proteins; or (ii) The protein sample to be tested is total protein extracted from Arabidopsis thaliana seeds.

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