WO2021000946A1 - Sonde et procédé d'analyse d'un complexe protéique l'utilisant - Google Patents

Sonde et procédé d'analyse d'un complexe protéique l'utilisant Download PDF

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WO2021000946A1
WO2021000946A1 PCT/CN2020/100173 CN2020100173W WO2021000946A1 WO 2021000946 A1 WO2021000946 A1 WO 2021000946A1 CN 2020100173 W CN2020100173 W CN 2020100173W WO 2021000946 A1 WO2021000946 A1 WO 2021000946A1
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protein
probe
domain
proteins
enrichment
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PCT/CN2020/100173
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English (en)
Chinese (zh)
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田瑞军
李鹏飞
初碧珠
何岸
郑振东
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南方科技大学
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Priority claimed from CN201910599017.6A external-priority patent/CN110231490B/zh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers

Definitions

  • This application belongs to the field of biotechnology, and relates to a probe and its application, in particular to a probe containing three functional groups and its application in high-throughput detection of protein-protein interactions.
  • Proteins are the main components of living organisms. In living organisms, hundreds of thousands of protein-protein interactions occur at all times. These huge numbers of interactions assemble proteins into a wide variety of protein complexes with different functions. It executes and regulates almost all life processes and functions, including protein translation, cell cycle control, protein synthesis and degradation, etc. Protein complexes form a high-dimensional and complex cell signal transduction network through series and parallel interactions with each other; through exogenous signal stimulation and precise positioning based on time and subcellular organelles, the macroscopic and precise control of cell functions is achieved. The interactions between these proteins are usually found in the study of post-translational modifications of proteins, such as phosphorylation, acetylation, methylation, and ubiquitination.
  • SH2 domain Src homology 2 (SH2) domain is a very representative example of protein interaction domain.
  • SH2 domain can specifically recognize tyrosine phosphorylated proteins in many signal transduction pathways.
  • Currently, more than 120 different SH2 domains have been found in the human proteome.
  • Kinase or phosphatase can dynamically regulate the activity of tyrosine phosphorylated proteins. Therefore, the SH2 domain can be used to enrich and identify these dynamic protein complexes.
  • Tyrosine phosphorylated protein complex bound to SH2 domain plays an important role in signal transduction regulation.
  • these protein complexes usually have dynamically changing natural properties, weak binding (from micromolar to nanomolar binding constant), and exist near the insoluble cell membrane.
  • the existing protein extraction technology has limited ability to extract membrane proteins, and often destroys weak and transient interactions between proteins.
  • US20150177258A1 discloses a method for replacing threonine at position 138 in SH2 domain with valine and 188th on the basis of wild-type Src SH2 domain. The cysteine at position was replaced with alanine, and the lysine at position 206 was replaced with leucine.
  • the mutant Src SH2 domain significantly improves the affinity for tyrosine phosphorylated protein, and its affinity constant reaches the nanomolar level.
  • this method can only enrich proteins that undergo tyrosine phosphorylation, and cannot identify tyrosine phosphorylated protein complexes; inserting exogenous photoreactive groups in the SH2 domain realizes instantaneous interaction between proteins. Characterization of the effect (A. Uezu et al., Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells, PNAS, 1, E2929, 2012; Y.
  • US005532379A discloses a Sulfo-SBED probe which includes a biotin group, an NHS group and an aryl azide photoreactive group, and the aryl azide group has obvious non-specific label It cannot be directly applied to the study of protein-protein interactions.
  • US20140011212A1 discloses a trifunctional probe for studying the interaction between glycoprotein receptors and their ligands, but it cannot be used for the study of interactions between other proteins. Therefore, it is urgent to develop a method that can enrich these dynamically changing tyrosine phosphorylated protein complexes.
  • Protein array technology includes reverse phase protein assay (RPPA) and forward phase protein assay (FPPA).
  • RPPA reverse phase protein assay
  • FPPA forward phase protein assay
  • cell lysates or phosphorylated peptides are immobilized on a solid support, and a variety of soluble SH2 domains are probed as probes to characterize the binding specificity of SH2 domain-tyrosine phosphorylated proteins and analyze specific SH2 The domain recognizes the level of tyrosine phosphorylated protein.
  • the Ye research team proposed a high-throughput quantitative analysis method for tyrosine phosphorylation based on RPPA (Anal.Chem.2017 Feb 21; 89(4): 2304-2311. Sensitive Approaches for the Assay of the Global Protein Tyrosine Phosphorylation in Complex Samples Using a Mutated SH2 Domain), the SH2 superparent has the advantages of high affinity and specificity with tyrosine phosphorylated protein, and realizes the quantification of tyrosine phosphorylated protein with lower detection sensitivity than traditional antibody methods analysis. However, this method cannot detect protein complexes regulated by tyrosine phosphorylated proteins, nor can it be qualitatively analyzed.
  • FPPA uses a set of SH2 domains as immobilization baits to capture tyrosine phosphorylated proteins and their mediated protein complexes from cell lysates.
  • Liu et al. systematically described the interaction of 78 SH2 domains and 194 human immunoreceptor tyrosine phosphorylated protein peptides by further combining RPPA and FPPA (Liu, H.; Li, L.; Voss, C. ;Wang,F.;Liu,J.;Li,SS-C.Mol.Cell.Proteomics 2015,14,1846-1858.).
  • Affinity enrichment combined with mass spectrometry is widely used to capture and identify tyrosine phosphorylated protein complexes.
  • a key signal protein with a specific label is expressed in living cells, and specific antibodies are used to enrich the protein and its interacting proteins.
  • This method has been widely used to characterize the dynamic pTyr signaling protein complex when specific growth factors stimulate cells.
  • Another method is to use synthetic peptide sequences or full-length protein fragments containing tyrosine phosphorylated tyrosine sites as affinity probes to affinity purify pTyr protein complexes from cell or tissue lysates.
  • the AP-MS-based strategy can only detect a specific bait protein at a time.
  • This application provides a probe and its application.
  • the probe uses a bait protein to bind a target protein complex. After light radiation, the photoreactive group forms a covalent bond with the target protein complex, which significantly improves the interaction between proteins. Finally, the protein complex is obtained through the enrichment of the enrichment group, and the subsequent analysis of the interaction between proteins is carried out to realize the effective enrichment and identification of the interacting protein with weak binding force.
  • this application has developed a high-throughput and highly sensitive sample pre-processing process and separation and detection step to achieve efficient identification and analysis of enriched protein complexes.
  • the present application provides a probe that includes a spacer arm and a bait protein, a photoreactive group, and an enrichment group connected to the spacer arm.
  • the inventor fully investigated the challenges and problems faced by the research on protein-protein interactions, and designed probes containing functional groups, including bait proteins for targeting target proteins, and light for covalently binding target proteins.
  • Reactive groups and biotin groups for enrichment.
  • ultraviolet radiation promotes the covalent binding of the photoreactive group to the target protein, and significantly improves the bait protein and the target protein.
  • the binding power of the protein complex is effectively enriched by the enrichment group, which is conducive to the analysis of weak and transient interactions between proteins.
  • the probe not only realizes the enrichment and identification of interacting proteins with weak binding force, but also significantly improves the target protein in human or animal tissue samples and cell membranes by optimizing the length of the probe skeleton and different photoreactive groups.
  • the enrichment and identification capabilities of its complexes reduce the background interference of other non-specific adsorption proteins.
  • the inventor has developed a high-throughput, high-sensitivity sample pre-processing process and separation and detection step to achieve efficient identification and analysis of protein complexes.
  • the development and development of the above technologies have broad application prospects and huge market value.
  • the spacer arm includes lysine, preferably L-lysine.
  • the bait protein includes a protein and/or polypeptide containing an SH2 domain, preferably an SH2 domain.
  • the SH2 domain is used as the bait protein by using the specific binding ability of the SH2 domain to the tyrosine phosphorylated protein, and the weak dynamic change between the SH2 domain and the tyrosine phosphorylated protein after ultraviolet light irradiation
  • the enhanced interaction is a covalent binding interaction, which is conducive to the study of tyrosine phosphorylated protein complex in cell signal transduction.
  • the SH2 domain can be a wild-type SH2 domain or a mutant SH2 domain.
  • the mutant SH2 domain is a Src SH2 domain mutant, and the mutant replaces the 138th threonine in the SH2 domain with valine and 188th on the basis of the wild-type Src SH2 domain.
  • the cysteine of is replaced with alanine, and the lysine at position 206 is replaced with leucine.
  • the three-site mutant Src SH2 domain (Src superbinder) is used as the bait protein, which further improves the affinity for tyrosine phosphorylated proteins and significantly improves the sensitivity of the probe.
  • the photoreactive group includes any one of biphenyl dimethyl ketone, aryl azide or diazoxide.
  • biphenyl ketone, aryl azide and diazoxide are photoreactive groups, which are activated under ultraviolet light (365nm) to react with the peptide chain backbone of the protein to form a covalent bond.
  • Probes containing photoreactive groups can not only be used to detect the direct one-to-one weak interaction or transient interaction between the bait protein and the target protein, but also can play the long covalent bond of the photoreactive group Range, to detect indirect interactions between multiple proteins and protein complexes formed after protein interactions.
  • the enrichment group includes biotin.
  • biotin is used as the enrichment group
  • affinity of biotin and streptavidin can be utilized
  • streptavidin microspheres are used as the enrichment carrier, which is conducive to the effective realization of protein complexes. Extraction and enrichment.
  • the SH2 domain is connected to the NHS (N-hydroxysuccinimide) group and/or I group on the spacer arm through a free primary amino group and/or a sulfhydryl group.
  • the SH2 domain can be connected to the spacer arm through the combination of its free primary amino group and the NHS group, or it can be connected to the spacer arm through the combination of its free sulfhydryl group and the I group.
  • the NHS group and the I group are in the spacer.
  • the arms have the same function of binding bait proteins.
  • L-lysine is used as a spacer, and NHS group and/or I group, photoreactive group and biotin group are modified on it, and the probe skeleton obtained is used to connect SH2 domain, so
  • the probe framework that binds to the SH2 domain in the probe is shown in Formula I and/or Formula II:
  • n 1 is selected from 0, 1 or 2
  • n 2 is selected from 0, 1 or 2
  • R is selected from
  • n 1 is 0, n 2 is 0, and R is
  • n 1 is 1, n 2 is 2, and R is
  • n 1 is 1, n 2 is 2, and R is
  • n 1 is 1, n 2 is 2, and R is
  • the probe backbone does not include the bait protein.
  • the probe skeleton includes a spacer arm and an NHS group (or I group) connected to the spacer arm, a photoreactive group, and an enrichment group.
  • the present application provides a forward protein array, which is composed of a solid phase carrier and the probes as described in the first aspect immobilized on the solid phase carrier.
  • the probe is immobilized on a solid support through an enrichment group.
  • the solid phase carrier is modified with streptavidin.
  • the solid phase carrier includes any one of a 96-well cell culture plate, a 384-well cell culture plate or a glass slide, preferably a 96-well cell culture plate or a 384-well cell culture plate.
  • the present application connects the probe and the bait protein to a 96-well plate loaded with streptavidin to capture the protein containing phosphorylated tyrosine and its interacting protein complex.
  • This operation greatly reduces the amount of probe and increases its storage time.
  • the probe and the bait protein were fixed on a 96-well plate. It can be stored for one month in NP-40 (Nonidet P40) buffer containing 0.02% (w/v) NaN 3 at 4°C. It includes the following steps:
  • the bait protein refers to a protein containing an SH2 domain.
  • the BCA kit is used for concentration determination, and the final protein concentration is guaranteed to be 0.02 ⁇ g/ ⁇ L.
  • the incubation time in step (2) is 1.5 to 2.5 hours, for example, it can be 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2 hours, 2.1 hours, 2.2 hours, 2.3 hours.
  • Hours, 2.4 hours or 2.5 hours the incubation temperature is 0-4°C, for example, it can be 0°C, 1°C, 2°C, 3°C or 4°C.
  • the incubation process in step (2) is accompanied by uniform oscillation at a low speed to ensure the binding to streptavidin.
  • the stabilizer added in step (3) can optionally contain 0.02%-0.05% (w/v) NaN 3 , for example, it can be 0.02%, 0.03%, 0.04% or 0.05%.
  • the stabilizer in step (3), may be added in 50-100 ⁇ L of NP-40 buffer containing 0.02% (w/v) NaN 3 , for example, 50 ⁇ L, 60 ⁇ L, 70 ⁇ L, 80 ⁇ L, 90 ⁇ L or 100 ⁇ L.
  • the SH2 domain-containing probe is formed by a spacer arm and an SH2 domain connected to the spacer arm, a photoreactive group, and biotin.
  • the probe skeleton is described elsewhere in this article.
  • the probe backbone can have the structure of Formula I:
  • this application provides a sample pretreatment method for phosphorylated tyrosine protein complex, including:
  • the probe is formed by a spacer arm and an SH2 domain connected to the spacer arm, a photoreactive group and biotin;
  • the enzymolysis reagent is an ammonium bicarbonate solution containing trypsin.
  • this application provides a method suitable for detection of pTyr protein complex in a trace amount of cell lysate, aiming at the characteristics of a 96-well plate loaded with streptavidin, and optimizing conditions to achieve efficient sample capture and enzymatic analysis. It includes the following steps:
  • the incubation product is irradiated with ultraviolet light of 350-370nm, preferably 365nm, for 20-40min;
  • the elution buffer is a RIPA buffer salt containing 5-8M (for example, 5M, 6M, 7M or 8M) urea, and the preferred formula is 6M urea, 1M NaCl, 1% (w/v) deoxycholic acid Sodium, 1% (w/v) sodium dodecyl sulfonate, 1% (v/v) Triton X-100, and 50 mM Tris-HCl (pH 7.4).
  • the reagents (reducing agent, alkylating reagent, trypsin) and buffer salt solution used in other operations are all well-known in the art.
  • the reducing agent can be selected from tris(2-carboxyethyl) Yl)phosphine hydrochloride
  • the alkylating agent may be iodoacetamide.
  • the optimized trypsin concentration in the enzymolysis process is 0.1 ⁇ g/50 ⁇ L, which means that 0.1 ⁇ g trypsin and 50 ⁇ L of 30-60mM (for example, 30mM, 40mM, 50mM or 60mM) ammonium bicarbonate aqueous solution are added to each well. This greatly reduces the amount of trypsin and effectively avoids the impact of trypsin self-digestion on target protein detection.
  • this application provides a fast and efficient chromatographic separation method, combined with the latest Q Exactive HF-X mass spectrometer, the mass spectrometry analysis time for a single sample can be within 35 minutes.
  • the data-independent collection mode requires the use of the corresponding cell lysate after liquid chromatography fractionation to build a database in the data-dependent acquisition mode (DDA), and then digest 1 mg of HeLa cell lysate into peptides. , Using high performance liquid chromatography (C18 column, 5 ⁇ m packing, 2.1mm id ⁇ 15cm) divided into 12 fractions.
  • the peptide sample is re-dissolved in 0.1-1% (v/v), preferably 0.1% (v/v) formic acid solution (mobile phase A), mixed with iRT reagent in proportion to the operating requirements, and self-made C18 (1.9) at 15cm ⁇ 50 ⁇ m ⁇ m) chromatographic column, Easy-nLC 1200 chromatographic system (Thermo Fisher Scientific) for separation, the mobile phase gradient is shown in Table 1, the flow rate is 600 ⁇ 1000nL/min, preferably 800nL/min, and other chromatographic parameters are shown in Table 2. .
  • Proportion of mobile phase B (acetonitrile containing 0.1% (v/v) formic acid) time 0% To 0% 30s 3% 30s 9% 12min 30% 3min 45% 30s 98% 4min 98% 30s
  • the polypeptide sample processed by the method provided in this application is re-dissolved in 0.1% (v/v) formic acid solution, mixed with iRT reagent according to the operating requirements, and then subjected to the same liquid chromatography as in (1).
  • the separation conditions are chromatographic separation, and the Q Extractive HF-X mass spectrum is used for acquisition in DIA mode.
  • the scanning range of DIA is 350-1550m/z.
  • the scanning range is divided into 40 continuous windows with unequal divisions, and 3 full scans are inserted in them.
  • the acquisition window settings are shown in Table 3, and there is 1m between the windows. /z overlap.
  • the resolution of Full MS is 120,000, AGC target is 3e6, maximum IT is 50ms, DIA secondary scan resolution is 30000, AGC target is 3e6, and maximum IT is auto.
  • This application provides a probe containing functional groups, which includes a bait protein for targeting a target protein, a photoreactive group for covalently binding the target protein, and a biotin group for enrichment. group.
  • the probe does not affect the activity of the target protein.
  • ultraviolet radiation promotes the covalent binding of the photoreactive group to the target protein, and significantly improves the binding of the bait protein to the target protein.
  • the protein complex is obtained through the enrichment of the enrichment group, which not only realizes the enrichment and identification of the interacting protein with weak binding force, but also improves significantly by optimizing the length of the probe backbone and different photoreactive groups.
  • This application uses the SH2 domain as a bait protein probe to successfully enrich and identify the tyrosine phosphorylated protein complex of the EGFR signaling pathway.
  • the SH2 domain is optimized to Src superbinder as a probe for the formation of bait protein, which has efficient enrichment and identification capabilities for tyrosine phosphorylated protein complexes in different breast cancer cell lines and breast cancer tissues. It successfully enriched EGFR and ERBB2. Compared with traditional immunohistochemical technology, the sensitivity was increased by nearly 100 times, and the effect of monitoring the activity of 64 target protein kinases at one time was realized.
  • the forward protein array formed by immobilizing trifunctional probes on 96-well plates in this application can be used for high-throughput exploration of weak and dynamically changing tyrosine phosphorylated protein-mediated protein complexes in complex biological samples It has good quantitative properties, only a small amount of starting material is needed to detect the dynamic EGF-stimulated tyrosine phosphorylation protein complex, and it can detect the tyrosine phosphorylation in cancer cell lines stimulated by erlotinib with high throughput
  • the state of the protein provides new ideas for the more systematic application of photo-affinity forward protein arrays to clinical tumor tissue samples and the discovery of new diagnostic markers and drug targets. It has broad application prospects and huge market value.
  • This application provides a method for detecting and analyzing tyrosine phosphorylated protein complexes suitable for trace samples. For 5 ⁇ g of cell lysate, the target protein and related pathway proteins can be captured and accurately quantified. Compared with the previous method, a 300-fold increase in sensitivity was achieved with a 100-fold reduction in probe usage;
  • This application realizes the one-month storage of bait protein for the first time, by binding the bait protein with probe attached to a 96-well plate modified with streptavidin, in a buffer solution containing a protein stabilizer , Can extend the storage time of bait protein from one week to one month, which greatly improves the throughput, and provides a high-throughput solution for the enrichment and identification of large-scale and batch tyrosine phosphorylated protein complexes and drug screening Program;
  • This application uses a 150 ⁇ m inner diameter self-packed chromatographic column, a 20-minute mobile phase gradient, combined with variable window data non-dependent acquisition mode and the latest Q Extractive HF-X mass spectrometry to achieve no column temperature heating device, no sample loss
  • a single sample can be separated and detected within 35 minutes, which greatly reduces sample analysis time, improves throughput, and provides technical support for batch biomarker discovery and drug screening;
  • This application is used to dynamically detect the identification of transient changes in the phosphorylation conditions of related signaling pathway proteins in cells under the action of different EGF stimulation times and different concentrations of inhibitors, and accurately capture drug targets after treatment with tyrosine phosphorylation inhibitors , And accurate determination of changes in related pathway proteins can play an important role in the screening of biomarkers and drug targets.
  • Fig. 1A is a schematic diagram of the structure and application of the functional probe of the present application
  • Fig. 1B is the structural formula of the probe skeleton containing the NHS group
  • Fig. 1C is the structural formula of the probe skeleton containing the I group.
  • Figure 2 is a diagram showing the optimized mass spectrometry identification results of different probe spacer lengths for the enrichment and identification ability of the target protein in Example 5.
  • Figure 2A is the mass spectrometry identification result diagram of the probe 2 for the enrichment and identification ability of the target protein.
  • Fig. 2B is a mass spectrometry identification result diagram of the enrichment and identification ability of probe 1 for the target protein, and
  • Fig. 2C is a comparison result diagram of the enrichment ability of probe 2 and probe 1 for related proteins in the EGFR signaling pathway.
  • Figure 3 shows the optimized mass spectrometry identification results of the photoreactive groups of different probes for the enrichment and identification of the target protein in Example 6.
  • Figure 3A shows the tyrosine phosphorylation of the EGFR signaling pathway by the photoreactive groups of different probes The comparison result of the enrichment ability of protein complexes.
  • Fig. 3B is the comparison result of the non-specific protein adsorption ability of different probe photoreactive groups.
  • Figure 4 shows the results of enrichment and identification of the target protein and its complexes near the cell membrane by probe 2 in Example 7.
  • Figure 4A shows the tyrosine phosphorylation of EGFR signaling pathway by probe 2 containing different bait proteins The results of the enrichment effect of the complexes.
  • Figure 4B shows the interaction network of tyrosine phosphorylated proteins identified by probe 2 containing different bait proteins.
  • Figure 5 is a diagram of the enrichment and identification results of Src superbinder probe on tyrosine phosphorylated protein complexes in Example 8.
  • Figure 5A shows the effect of Src superbinder probe on tyrosine phosphorylated protein complexes of EGFR signaling pathway Enrichment effect result graph
  • Figure 5B is a comparison result graph of Src superbinder probe and N-PI3K probe for EGFR protein enrichment ability.
  • Figure 6 is a diagram showing the enrichment and identification results of Src superbinder probes for tyrosine phosphorylated protein complexes in different breast cancer cell lines in Example 9.
  • Figure 6A is the comparison result of the Src superbinder probe for the enrichment and identification of tyrosine phosphorylated protein complexes in BT-474 and MDA-MB-468 breast cancer cell lines
  • Figure 6B is the Src superbinder probe Comparison of the enrichment and identification capabilities of tyrosine phosphorylated protein complexes in MDA-MB-468 and MDA-MB-231 breast cancer cell lines.
  • Figure 6C shows the Src superbinder probe on breast cancer cell lines Enrichment and identification results of tyrosine phosphorylated proteins related to EGFR/ERBB2 signaling pathway.
  • FIG. 7A is a graph of the results of immunohistochemistry of Example 10
  • FIG. 7B is a graph of the results of mass spectrometry of Example 10
  • FIG. 7C is a graph of the results of 64 target protein kinases of Example 10.
  • Figure 8 is a schematic diagram of the detection of a light affinity forward protein array.
  • Figure 9 shows the optimization of washing conditions for streptavidin-coated 96-well plates by EGFR.
  • Figure 10A shows the optimization of the labeling photocrosslinking efficiency through different volumes of NP-40 lysate
  • Figure 10B shows the optimization of the photocrosslinking efficiency through different UV irradiation times.
  • Figure 11A shows the binding capacity of a 96-well plate coated with streptavidin by anti-4G10. Add 5 ⁇ g Src superbinder probe to each well and change the amount of protein added to explore the saturated binding capacity of the protein.
  • Figure 11B Add 300 ⁇ g protein to each of the wells and change the amount of Src superbinder probe to explore the saturation binding capacity of Src superbinder probe.
  • Figure 12 shows the quantitative performance of the Src superbinder probe based on the forward protein array platform.
  • Figure 13A is a known interaction diagram of EGFR, GRB2, SHC1 and CBL
  • Figure 13B is a forward protein array based on Src superbinder probes to capture tyrosine phosphorylated EGFR and tyrosine phosphorylated EGFR from 5 ⁇ g EGF-stimulated HeLa cell lysate. It regulates tyrosine phosphorylation of protein complexes.
  • Figure 14A shows the ability of the forward protein array based on the Src superbinder probe to capture tyrosine phosphorylated protein complexes in erlotinib-treated HeLa cell lysate.
  • Figure 14B shows the corresponding immunoprecipitation results, and
  • Figure 14C shows the corresponding protein Figure of blotting results.
  • Figure 15 is a schematic diagram of the application operation process.
  • Figure 16A shows the identification results of the number of matching spectra of proteins and peptides under different ammonium bicarbonate solution volumes.
  • Figure 16B shows the 0% missed cut ratio results of the number of matching spectra of proteins and peptides under different ammonium bicarbonate solution volumes.
  • Figure 16C It is the distribution of coefficient of variation under different ammonium bicarbonate solution volumes.
  • Figure 17A is a graph showing the quantitative results of phosphorylated tyrosine protein complexes under stimulating conditions when different cell dosages are used.
  • Figure 17B is the classification of enriched proteins.
  • Figure 17C is the method of this application with only 5 ⁇ g of cell lysate. Protein enrichment effect achieved by 1.4mg cells.
  • Figure 18A shows that the method can enrich and identify more protein complexes after EGF stimulation under the same amount of probe, bait protein and cell lysate.
  • Figure 18B shows the method of the application and the prior art Compared with the results, Fig. 18C shows the difference between the method of the application and the protein identification intensity multiples of the prior art, and Fig. 18D shows the steric hindrance caused by the detection process of the prior art.
  • Figure 19A is the quantitative protein quantity under different storage time
  • Figure 19B is the Pearson correlation coefficient graph among the samples under different storage time
  • Figure 19C is the result graph of the identification effect of the reported EGFR signaling pathway protein as the storage time increases.
  • Figure 20A shows the effect of different chromatographic column inner diameters on the identification
  • Figure 20B shows the optimization result of the data-independent acquisition mode window.
  • Figure 21A shows the dynamic changes of EGFR signaling pathway proteins under different EGF stimulation time conditions and different AG1478 inhibitor concentrations.
  • Figure 21B shows the results of changes in EGFR, CBL, and SHC1 proteins with the identification strength of the inhibitor.
  • This application has designed 7 probe backbones, which are connected to SH2 domains to prepare functional probes as shown in Figure 1A.
  • the functional probes can efficiently enrich and enrich tyrosine phosphorylated protein complexes. Identification role.
  • the structure of the probe skeleton is shown in Figure 1B and Figure 1C.
  • SH2 domain SEQ ID NO.1 (1mg/mL, dissolved in HEPES buffer)
  • probe backbone containing the photoreactive group and biotin group
  • the probe backbone is dissolved in dimethyl
  • the molar ratio of sulfoxide, SH2 domain and probe backbone is 1:10), react for 1 min at room temperature, and then add 5 ⁇ L of 1mol/L glycine solution to stop the activity of the NHS group to obtain the label as shown in Figure 1A Probe with bait protein SH2 domain.
  • Tris(2-carboxyethyl)phosphine hydrochloride TCEP, dissolved in 50mM ABC
  • IAA Iodoacetamide
  • TCEP TCEP
  • 5mM 5mM
  • step 5 Add the enzymatically hydrolyzed peptide samples collected in step 1) and centrifuge at 400 ⁇ g for 10-15 minutes to make all the samples pass through the C18 membrane;
  • the Orbitrap Fusion mass spectrometer connected with a nano-upgraded high performance liquid chromatograph (Easy-nLC 1000) was used for mass spectrometry identification.
  • the peptides were dissolved in 0.1% (v/v) formic acid solution and loaded In a C18 resin analytical column (100 ⁇ m inner diameter ⁇ 20cm), chromatographic separation and mass spectrometry were performed at a flow rate of 200nL/min and an effective gradient of 2h.
  • probe 1 and probe 2 were used to prepare probe 1 and probe 2 with different spacer lengths (the bait protein uses PI3K p85 ⁇ N-terminal SH2 domain (SEQ ID NO.1), named: N-PI3K);
  • probe 2 can effectively enrich and identify the tyrosine phosphorylation of 9 EGFR signaling pathways compared with probe 1.
  • Protein complexes such as EGFR, CBL, CBLB, SHC1 and GRB2 can be effectively enriched and identified; at the same time, as shown in Figure 2C, protein complexes with relatively small molecular weight EGFR signaling pathways in the cytoplasm, such as SHC1 For CBL, CBLB, GRB2 and other proteins, probe 2 can better enrich and identify the EGFR with larger molecular weight (about 130kDa) and close to the cell membrane than probe 1.
  • Example 6 The optimization of different probe photoreactive groups for the enrichment and identification of target protein
  • each probe can significantly enrich the protein that interacts with N-PI3K after being irradiated with ultraviolet light, but the number of enriched interacting proteins of each probe is significantly different, and probe 2 enriches the interaction The largest amount of protein.
  • This result shows that probe 2 with the photoreactive group of biphenyl dimethyl ketone has the best effect on the enrichment and identification of the dynamic tyrosine phosphorylated protein complex of the EGFR signaling pathway.
  • experimental group 1 added 1.4 mg of normal HeLa cell lysate to each probe; experimental group 2 was each probe Add 1.4 mg of normal HeLa cell lysate to the needle.
  • experimental group 2 was subjected to ultraviolet light irradiation according to Example 2
  • experimental group 1 was not subjected to ultraviolet light irradiation, and all experimental groups were subjected to subsequent treatment according to Example 3-4.
  • Example 7 The ability of probe 2 to enrich and identify target proteins and their complexes near the cell membrane
  • N-PI3K labeled with probe backbone 2 can effectively enrich and identify tyrosine phosphorylated protein complexes in the EGFR signaling pathway, for example, EGFR and SHC1 can be effectively enriched and identified. And GRB2 etc.
  • probe backbone 2 In order to better verify that these identified proteins are located in the EGFR tyrosine phosphorylated protein complex, we use probe backbone 2 to label the SHC1 SH2 domain and GRB2 SH2 domain to reversely verify whether these proteins can form an effective EGFR Tyrosine phosphorylates protein complexes.
  • Example 1 Prepare probe 2 of different bait proteins according to Example 1.
  • the bait proteins use N-PI3K, SH2 domain of GRB2 protein and SH2 domain of SHC1 protein respectively;
  • the results are shown in Figure 4A.
  • the SHC1 SH2 probe can effectively enrich and identify EGFR, GRB2 and PIK3C2B; the GRB2 SH2 probe can effectively enrich and identify EGFR and SHC1. It shows that SHC1 SH2 probe and GRB2 SH2 probe can effectively enrich and identify EGFR tyrosine phosphorylated protein complexes, and these complexes form an interactive network (Figure 4B).
  • Example 8 Enrichment and identification of tyrosine phosphorylated protein complexes by Src superbinder probe
  • the N-PI3K probe can effectively enrich the dynamic tyrosine phosphorylated protein complexes of the EGFR signaling pathway in HeLa cells stimulated by EGF.
  • the content of tyrosine phosphorylated protein in the EGFR signaling pathway is extremely low.
  • the content of tyrosine phosphorylated protein in the endogenous EGFR signaling pathway is also extremely low.
  • the detection and evaluation of these endogenous tyrosine phosphorylated proteins is of significant significance for the occurrence and development of diseases and for the treatment of diseases.
  • the Src superbinder probe can effectively enrich and identify 15 proteins in the dynamic tyrosine phosphorylated protein complex of the EGFR signaling pathway. Efficient enrichment and identification of the dynamic tyrosine phosphorylated protein complex of the EGFR signaling pathway.
  • the Src superbinder probe In order to detect the enrichment and identification effect of the Src superbinder probe on the endogenous EGFR protein in normal cultured cells, we first incubate the Src superbinder probe with the normal cultured HeLa cell lysate, and then perform UV light and streptavidin Microsphere enrichment. The results are shown in Figure 5B. Compared with the N-PI3K probe, the Src superbinder probe can effectively enrich and identify EGFR protein after ultraviolet light, which reflects its efficient enrichment and identification capabilities for EGFR protein.
  • Example 9 Enrichment and identification of tyrosine phosphorylated protein complexes in different breast cancer cell lines by Src superbinder probe
  • the Src superbinder probe can effectively enrich and identify tyrosine phosphorylated protein complexes related to the EGFR signaling pathway with very low content in normal cultured cells.
  • Src superbinder probes to enrich and identify specific endogenous tyrosine phosphorylated proteins and their corresponding protein complexes in the three normally cultured breast cancer cell lines.
  • the results are shown in Figure 6A and Figure 6B.
  • the Src superbinder probe can specifically enrich EGFR in the MDA-MB-468 cell line and ERBB2 in the BT-474 cell line, indicating that the Src superbinder probe is effective for different cell lines. (MDA-MB-468, BT-474 and MDA-MB-231) have good selectivity.
  • the Src superbinder probe also has a good ability to enrich and identify tyrosine phosphorylated protein complexes related to the EGFR and ERBB2 signaling pathways.
  • Example 10 Enrichment and identification of tyrosine phosphorylated protein complex in breast cancer tissue by Src superbinder probe
  • Fig. 7A is an immunohistochemical sample image
  • the + sign indicates that the expression of ERBB2 is detected
  • the more the + sign the stronger the expression
  • the-sign indicates that the expression of ERBB2 is not detected.
  • FIGS. 7A and 7B that, compared with traditional immunohistochemical techniques, the probe of this application can significantly improve the identification sensitivity of functional protein complexes. Taking the ERBB2 receptor membrane protein as an example, the sensitivity is increased by nearly 100 times;
  • FIG. 7C the probe of the present application can also perform high-throughput evaluation of the activity of multiple target proteins, and can monitor the activity of 64 target protein kinases at one time.
  • Example 10 Using Src superbinder as the SH2 domain, biphenyl benzophenone as the photoreactive group, and biotin as the enrichment gene, the Src superbinder probe was prepared according to the strong affinity between biotin and streptavidin , Src superbinder probes are fixed in a 96-well cell culture plate modified with streptavidin to construct a photoaffinity forward protein array, and then compound the tyrosine phosphorylated proteins of the EGFR signaling pathway in EGF-stimulated HeLa cells For enrichment and identification.
  • the Src superbinder probe is assembled by the chemical reaction between the free primary amino group of the Src superbinder and the NHS group of the trifunctional chemical probe, and then the Src superbinder probe is coated with streptavidin. Incubate the well plate and bind to the 96-well plate through the biotin label to form a photo-affinity forward protein array; then, add the cell lysate to the 96-well plate and incubate with the photo-affinity forward protein array to bind to The Src superbinder on the 96-well plate will recognize and interact with tyrosine phosphorylated proteins and their mediated protein complexes; under UV irradiation, the Src superbinder probes can be covalently cross-linked directly or indirectly The tyrosine phosphorylated protein complex that interacts with it, especially the weak, dynamically changing tyrosine phosphorylated protein complex.
  • the cell lysate (fixed 50 ⁇ g) was set to 30 ⁇ L, 50 ⁇ L, 100 ⁇ L, 150 ⁇ L and 200 ⁇ L respectively and added to the 96-well plate.
  • the other operations were the same as the "Optimization of photoaffinity forward protein array washing solution conditions" in Example 12.
  • the photoaffinity forward protein array method has the highest photocrosslinking efficiency when the volume of the cell lysate is 30 ⁇ L; as shown in Figure 10B: the photoaffinity forward protein array method is exposed to ultraviolet light for 30 min. The crosslinking efficiency is the highest, so this condition is used in subsequent experiments.
  • the amount of Src superbinder probe added to each well is 0.01 ⁇ g, 0.05 ⁇ g, 0.1 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 5 ⁇ g, and 10 ⁇ g, respectively.
  • add 300 ⁇ g protein to each well set the cell lysate to 30 ⁇ L optimized before photocrosslinking, and change the primary antibody from anti-EGFR to anti-phosphotyrosine clone 4G10.
  • the other operations are the same as those in Example 12 for “positive photoaffinity”. To optimize the conditions of the protein array washing solution”.
  • the photoaffinity forward protein array method shows good quantitative ability (R 2 >0.999) and high sensitivity. Only 5 ⁇ g of cell lysate can be used to achieve tyrosine phosphorylated protein in complex samples. Detection.
  • Example 13 The photoaffinity forward protein array is used for the identification of protein complexes
  • the Src superbinder probe bound to a 96-well plate can efficiently capture EGFR and its interacting proteins GRB2, SHC1 and CBL, and the results are related to EGF stimulation. It is important that the Src superbinder probe can bind more membrane receptor protein EGFR after UV cross-linking.
  • Known EGFR-related protein complexes such as GRB2, SHC1 and CBL are also easily recognized and captured by the photoaffinity forward protein array. These proteins interact to form EGFR-related protein complexes, which proves that the photoaffinity forward protein array has high-throughput performance to capture tyrosine phosphorylated protein complexes from complex biological samples.
  • a stable FLAG-GRB2 cell line was constructed.
  • the stable cell line was induced by doxycycline (1 ⁇ g/mL) for 24h to make the cells express FLAG-GRB2, and erlotinib was added at the same time as doxycycline.
  • the FLAG-GRB2 cell line was stimulated with EGF (100ng/mL) for 5 min.
  • EGF 100ng/mL
  • the cell lysate and 30 ⁇ L anti-FLAG M2 microspheres were incubated overnight at 4°C; the anti-FLAG M2 particles were washed three times with NP-40, and then SDS-PAGE electrophoresis and Western blotting were performed.
  • erlotinib can inhibit the activation of EGFR and its downstream signaling pathway proteins; in addition, as shown in Figure 14A, cells treated with erlotinib use photo-affinity forward protein array technology to capture EGFR, GRB2 and SHC1 Significant reduction; as shown in Figure 14C: erlotinib reduces the interaction of EGFR and GRB2.
  • This embodiment aims to realize the capture and analysis of related tyrosine phosphorylated proteins in a trace amount of cell lysate, and to reduce the concentration of the cell lysate to detect the feasibility and sensitivity of the method.
  • the overall operation process is shown in Figure 15.
  • probe to the bait protein SH2 domain (1 mg/mL, dissolved in HEPES buffer) at a molar ratio of 1:10 (protein: probe), and incubate for 2 minutes at room temperature. Subsequently, a 1mol/L glycine solution was added at a molar ratio of 1:10 (probe:glycine) to terminate the reaction between the probe and the bait protein.
  • the obtained bait protein labeled with the probe was ultrafiltered 7 times to remove unreacted probe and glycine molecules. Determine the protein concentration and dilute to 0.02 ⁇ g/ ⁇ L.
  • the bait protein labeled with the probe was added to a 96-well plate modified with streptavidin at 50 ⁇ L/well, and incubated at 4° C.
  • trypsin solution for in-well enzymolysis (In-well enzymolysis) to ensure that each microwell contains 0.1 ⁇ g trypsin, corresponding to the volume of 50mM ammonium bicarbonate solution of 30 ⁇ L, 50 ⁇ L, 100 ⁇ L and 200 ⁇ L.
  • 16C shows that the coefficient of variation (CV) of the three repetitions under different ammonium bicarbonate solution volumes is less than 0.2, indicating that the pretreatment process has good repeatability.
  • Example 15 According to the method of Example 15, 5 ⁇ g, 50 ⁇ g, 300 ⁇ g of HeLa cell lysate before and after EGF stimulation (including 3 technical repetitions) were added to the streptavidin-coated 96-well plate connected with the probe and bait protein, respectively. ), and then perform subsequent treatments according to the general conditions described in Example 15, and compare the enrichment and identification of protein complexes under EGF stimulation conditions.
  • Amino acid protein; the 68 proteins enriched in 50 ⁇ g cell lysate include 15 reported enriched proteins, 42 phosphorylated tyrosine proteins and 10 reported proteins with phosphorylated tyrosine Interacting proteins; when the amount of cell lysate is increased to 300 ⁇ g, the enriched protein contains 51 phosphorylated tyrosine proteins and 8 have been reported to interact with phosphorylated tyrosine proteins.
  • the role of protein shows that using the integrated sample pretreatment method described above, only 5 ⁇ g of cell lysate can be used to achieve the protein enrichment effect achieved by 1.4 mg of cells in the previous method. It shows that this method is highly sensitive and suitable for the enrichment and identification of phosphorylated tyrosine protein complexes in large quantities of samples. In the subsequent experiments, in order to ensure the identification effect, 50 ⁇ g cell lysate was used for experiments.
  • Example 15 50 ⁇ g of HeLa cell lysate before and after EGF stimulation was added to the streptavidin-coated 96-well plate connected with the probe and bait protein (including 3 times technique Repeat), and then follow the general conditions described in Example 15 for subsequent processing.
  • the bait protein of the same quality with the probe attached to the 1.5mL centrifuge tube 50 ⁇ g of HeLa cell lysate before and after EGF stimulation (including 3 technical repetitions), and use 10 ⁇ L of Streptomyces Avidin beads are captured, and then subsequent processing is performed according to the general conditions described in the patent 201910599017.6.
  • the samples processed by the two methods were compared with the enrichment and identification of protein complexes under EGF stimulation conditions.
  • the identification strength of most proteins in the method of this application is much higher than the previous method, and the maximum difference can reach 11 times.
  • the probe connected to the bait protein is first connected to streptavidin, and then the protein complex in the cell lysate is captured
  • the protein complex in the cell lysate was first captured by the probe connected to the bait protein, and then enriched with streptavidin. This sequence change may cause the connection
  • the biotin group in the probe of the protein complex is encapsulated, resulting in a steric hindrance effect, which makes it difficult to be enriched by streptavidin (as shown in Figure 18D).
  • the previous method involves two solution transfers, which will cause sample loss.
  • the reduction in protein enrichment identification caused by this loss will be particularly obvious.
  • all operations in the method of this application All are integrated in a 96-well plate loaded with streptavidin to avoid sample loss. It shows that compared with the previous method, this application has higher sensitivity and can identify more tyrosine phosphorylated protein complexes, which is very advantageous for the identification and capture of protein complexes with lower abundance in trace samples.
  • Example 18 The influence of bait protein and probe storage time on protein identification ability
  • Example 15 the 96-well plates integrated with bait proteins were stored in NP-40 buffer containing 0.02% (w/v) NaN 3 and placed at 4°C. Store under the conditions for 1-30 days. Then, the protein complexes in the HeLa cell lysate after EGF stimulation were enriched and identified in 96-well plates containing bait proteins stored for different days, and compared with the increase of storage time, whether the enrichment ability of protein complexes Will change.
  • the quantitative detection effect of these proteins is less affected within the storage time range of 1-10 days; the time increases to 15 days.
  • the strength of part of the protein will decrease by about 50%; the time will continue to increase, and the strength will not decrease.
  • the method of the present application greatly extends the storage time of the bait protein and the probe, and provides strong support for high-throughput, rapid, and batch sample detection.
  • the samples were prepared according to the methods determined in Example 15 and Example 16, and at the same time, 1 ⁇ g of HeLa cells were lysed and the peptides were enzymatically hydrolyzed, and the samples were detected using nanoLC combined with Q Extractive HF-X mass spectrometry.
  • the optimized results of chromatographic separation conditions are shown in Figure 20A.
  • the increase in the inner diameter of the chromatographic column can increase the flow rate from 250nL/min to 800nL/min, which can greatly reduce the time for chromatographic equilibrium and sample loading.
  • the increase in the inner diameter of the chromatographic column will inevitably cause the loss of a part of the quantitative protein.
  • 1 ⁇ g of HeLa cell lysate was used to analyze the peptides enzymatically, and the results also showed that the 100 ⁇ m i.d. column can quantitatively detect the most proteins.
  • the processed pulldown sample due to the reduced sample complexity, it will not be affected by this.
  • the total quantitative protein and the number of quantitatively detected pTyr protein are unchanged, and the total separation and detection time is shortened from 60 minutes to 35 minutes. Minutes, while maintaining the original qualitative effect and quantitative depth, greatly improving the efficiency of analysis and detection.
  • a mass spectrum independent data acquisition mode is adopted, and a variable window condition containing 40 windows is selected for mass spectrometry identification, and the number of MS1 scans added therein is optimized to increase the average number of points collected by a single chromatographic peak.
  • the experimental results are shown in Figure 20B. The more points collected on an average single chromatographic peak, the higher the credibility of the data.
  • variable window acquisition mode can quantitatively detect more target proteins with better repeatability. Based on the above results, a 150 ⁇ m i.d. chromatographic column was used for chromatographic separation, and 40 variable window data including 3 MS1 scans were collected independently for subsequent experiments.
  • Example 21 High-throughput phosphorylated protein complex identification. Proteomics methods. Identification and screening of targeted drug-action protein complexes. When the HeLa cell density reaches 70%, tyrosine kinase inhibitor AG1478 is added to stimulate for 20 hours. Then, EGF with a final concentration of 100 ⁇ g/mL was added to stimulate for 5 minutes, and the other steps were the same as in Example 19. This example aims to detect the changes of EGFR protein complexes in cells after inhibition by AG1478.
  • AG1478 can inhibit the activation of EGFR and its downstream signaling pathway proteins.
  • the inhibitory effect on EGFR and related proteins is better; for most proteins in the upstream of the EGFR pathway, when the administration concentration is 0.1 ⁇ M, the identified protein strength decreases slightly .
  • the intensity dropped significantly, and then the dose concentration continued to increase, the intensity did not change significantly.
  • the intensity changes of EGFR, CBL and SHC1 follow the aforementioned rules, and the error bars show that the method has good repeatability.
  • This application provides a functional group-containing probe, which includes a bait protein for targeting a target protein, a photoreactive group for covalently binding the target protein, and a biotin group for enrichment.
  • the probe does not affect the activity of the target protein.
  • ultraviolet radiation promotes the covalent binding of the photoreactive group to the target protein, and significantly improves the binding of the bait protein to the target protein.
  • the protein complex is enriched by the enrichment group, which not only realizes the enrichment and identification of the interacting protein with weak binding force, but also improves the human body by optimizing the length of the probe backbone and different photoreactive groups.
  • the SH2 domain was used as a probe for the bait protein to successfully enrich and identify the tyrosine phosphorylated protein complex of the EGFR signaling pathway.
  • Src superbinder as a probe for the formation of bait proteins, it has high efficiency in enrichment and identification of tyrosine phosphorylated protein complexes in different breast cancer cell lines and breast cancer tissues, and achieves the enrichment of EGFR and ERBB2 Compared with the traditional immunohistochemical technology, the sensitivity is improved by nearly 100 times, and the effect of monitoring the activity of 64 target protein kinases at one time is realized.
  • the forward protein array formed by immobilizing the probe on a 96-well plate can be used for high-throughput exploration of weak tyrosine phosphorylated protein-mediated protein complexes in complex biological samples, especially those located near the insoluble cell membrane. Things.
  • the forward protein array technology has good quantitative properties. It only needs a small amount of starting material to detect dynamic tyrosine phosphorylation protein complexes, and can accurately reflect the tyrosine phosphorylation in tumor cells after targeted drugs.
  • the state of chemical protein complexes provides new ideas for the more systematic application of photo-affinity forward protein arrays to clinical tumor tissue samples and the discovery of new diagnostic markers and drug targets. It has broad application prospects and great promise. Market value.
  • the application also provides a high-throughput and high-sensitive phosphorylated tyrosine protein complex enrichment sample pretreatment method and a fast and accurate chromatographic separation and mass spectrometry identification method.
  • This sample pretreatment method can increase the sensitivity by 300 times under the condition that the amount of probe and bait protein is reduced by 100 times compared with the previous method; the storage time of bait protein in buffer solution containing stabilizer can be extended to one month; in EGF Under stimulating conditions, only 5 ⁇ g of cell lysate can be used to achieve the amount of protein enriched in 1.4mg of cell lysate in the previous method, which provides stable, sensitive and high-throughput identification of pTyr protein and interacting protein complexes
  • a 150 ⁇ m id self-packing chromatographic column, a 20-minute chromatographic gradient, 40 variable window DIA acquisition mode and the latest Q Extractive HF-X mass spectrometer without using an external column temperature stabilization device, without losing qualitative Under the premise of quantitative detection performance
  • This method is used to capture the dynamic changes of protein complexes under different EGF stimulation times, and the dynamic changes of signal pathway proteins and transient and weakly interacting protein complexes can be accurately captured.
  • Dosage experiments for phosphorylated tyrosine kinase inhibitors show that this method is suitable for screening biomarkers and drug targets, and can capture the dynamic changes of downstream signaling pathway proteins affected by drugs with high sensitivity.
  • This method provides a high-throughput and highly sensitive detection method for the identification of phosphorylated tyrosine protein complexes, and provides new ideas for the screening of new diagnostic markers and drug targets. It has broad application prospects and huge market value.

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Abstract

L'invention concerne une sonde comprenant un bras espaceur, et une protéine appât, un groupe photoréactif et un groupe d'enrichissement fixés au bras espaceur. La protéine appât comprend un domaine protéique qui reconnaît la modification post-traductionnelle de protéines spécifiques, par exemple, un domaine SH2 qui reconnaît la phosphorylation par la tyrosine. La sonde n'affecte pas l'activité de la protéine cible, ce qui est avantageux pour une analyse d'interaction faible et transitoire entre des protéines, ce qui améliore significativement les capacités d'enrichissement et d'identification d'échantillons de tissu humain ou animal et de protéines cibles et de leurs complexes à proximité de la membrane cellulaire, et ce qui assure la réussite de l'enrichissement et de l'identification de complexes protéiques phosphorylés par la tyrosine dans différents tissus et lignées cellulaires du cancer du sein. L'invention concerne en outre un procédé de protéomique à haut rendement et intégré pour l'enrichissement et l'identification de complexes protéiques phosphorylés.
PCT/CN2020/100173 2019-07-04 2020-07-03 Sonde et procédé d'analyse d'un complexe protéique l'utilisant WO2021000946A1 (fr)

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