CN110873772B - Probe and synthesis method and application thereof - Google Patents
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Abstract
The invention provides a probe, a synthesis method and application thereof, wherein the method comprises the following steps: (1) Removing Boc from the compound A, and reacting with the compound B to obtain a compound C; (2) Condensing the compound C in the step (1) with N-hydroxysuccinimide to obtain a probe skeleton; (3) Mixing the decoy protein with the obtained probe skeleton, connecting, adding glycine solution to terminate the reaction, and filtering to obtain the probe. The synthesis method of the invention has simple operation, low cost and environmental protection, the prepared probe adopts SH2 structural domain as bait protein, which is favorable for carrying out weak interaction and instantaneous interaction analysis between proteins, is especially favorable for carrying out enrichment and identification of SH2 structural domain and tyrosine phosphorylated protein, obviously improves enrichment and identification capability of target proteins and complexes thereof near human or animal tissue samples and cell membranes, and reduces background interference of other nonspecific adsorption proteins.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a probe and a synthesis method and application thereof, and in particular relates to a probe containing three functional groups and a synthesis method and application thereof.
Background
Proteins are the basic stones that constitute living organisms, and are generally composed of modular domains that can be folded independently, exerting different biological functions. Protein interaction domains play an important role in protein interactions and in the formation of protein complexes. Interactions between proteins are often found during studies of post-translational modifications of proteins, such as phosphorylation, acetylation, methylation, and ubiquitination. The Src homolog 2 (SH 2) domain is a representative example of a protein interaction domain, and SH2 domains can specifically recognize tyrosine phosphorylated (pTyr) proteins in a variety of signal transduction pathways. Currently, more than 120 different SH2 domains have been found in the human proteome. Kinases or phosphatases can dynamically regulate the activity of pTyr proteins, and SH2 domains can be used to enrich for identification of these dynamic protein complexes and to participate in signal transduction in different signaling pathways. Thus, the SH2 domain can be used as a universal detector for characterizing dynamic signaling processes under different physiological or pathological conditions.
Protein complexes that bind SH2 domains often have a dynamic nature, are weak in binding forces (binding constants of the order of micromolar to nanomolar) and are mostly present near poorly soluble cell membranes and are difficult to separate using standard adsorption purification methods. Protein chip technology can identify the binding specificity of SH2 domain and pTyr polypeptide with high flux, but can not accurately identify protein, and is difficult to apply to complex biological samples. At present, the affinity enrichment combined mass spectrometry identification technology (affinity purification and mass spectrometry, AP-MS) is widely applied to high-throughput identification of protein complexes in complex systems by virtue of the characteristic that the protein can be qualitatively and quantitatively researched. However, AP-MS has great limitations in identifying weak interactions and protein complexes in the vicinity of the cell membrane, because these interacting proteins are easily lost during the adsorption purification of AP-MS. Thus, there is an urgent need to develop a technique capable of overcoming the short plates of the AP-MS technique and identifying dynamic protein interactions in a complex system.
In recent years, chemical probe technology has played an increasingly important role in the study of protein complex interactions. These probes typically bind covalently to proteins that interact with the protein of interest using a controlled chemical or photocatalytic reaction. However, the currently reported probe technology generally requires exposure of cells or proteins to oxidative or short wavelength ultraviolet (254 nm) environments, causing irreversible damage to the interacting protein complexes.
US005532379a discloses a Sulfo-SBED probe comprising a biotin group, a NHS group and an aryl azide photoreactive group, wherein the aryl azide group has a significant non-specific labelling effect and cannot be directly applied to the study of protein-protein interactions; US20140011212A1 discloses a trifunctional probe for the study of glycoprotein receptors and their ligand interactions but not for the study of other protein interactions.
Therefore, the molecular probes containing multifunctional groups are developed and utilized to study weak interaction or transient interaction between proteins, so that the range and the field of research on protein interaction can be remarkably promoted, a novel method for proteomics research is facilitated to be developed and developed, powerful support is provided for researching a signal transduction network of interaction between proteins, finally, a scientific research method is provided for systematically researching an intercellular signal transduction process in a tumor microenvironment and for disease, particularly pathogenesis of tumor diseases.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a probe, and a synthesis method and application thereof, wherein the synthesis method is simple to operate, low in cost and environment-friendly, and the prepared probe adopts SH2 structural domain as bait protein, so that enrichment and identification of interaction proteins with weak binding force are realized, and enrichment and identification capability of target proteins near cell membranes are remarkably improved.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method of synthesizing a probe, the method comprising the steps of synthesizing a probe scaffold:
(1) Removing Boc from the compound A, and reacting with the compound B to obtain a compound C;
(2) Condensing the compound C in the step (1) with N-hydroxysuccinimide to obtain a probe skeleton;
wherein n is 1 Select 0,1 or 2, n 2 Selecting 0,1 or 2, R is selected from
The synthesis method of the invention has simple operation, low cost and environmental protection, and the prepared probe adopts SH2 structural domain as bait protein, thus not only realizing enrichment and identification of interaction protein with weak binding force, but also obviously improving the enrichment and identification capability of target protein near cell membrane.
Preferably, the molar ratio of compound A to compound B in step (1) is 1 (1-2), which may be, for example, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0, preferably 1 (1.5-1.6).
Preferably, the temperature of the reaction in step (1) is 20-30℃and may be, for example, 20℃21℃22℃23℃24℃25℃26℃27℃28℃29℃or 30 ℃.
Preferably, the reaction time of step (1) is 18-24h, and may be, for example, 18h, 19h, 20 h, 21h, 22h, 23h or 24h.
Preferably, the pH of the reaction in step (1) is not less than 9, and may be, for example, 9, 9.5, 10, 10.5, 11, 11.5 or 12, preferably 9 to 10.
Preferably, the molar ratio of compound C to N-hydroxysuccinimide of step (2) is 1 (1.5-2.5), which may be, for example, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:1.98, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4 or 1:2.5, preferably 1 (1.98-2).
Preferably, the time of the condensation in step (2) is 18-24h, and may be, for example, 18h, 19h, 20 h, 21h, 22h, 23h or 24h.
Preferably, after step (2), the step of attaching the bait protein is further comprised of: mixing the decoy protein with the obtained probe skeleton, connecting, adding glycine solution to terminate the reaction, and filtering to obtain the probe.
In the present invention glycine is used to terminate the activity of NHS groups.
Preferably, the molar ratio of the decoy protein to the probe scaffold is 1 (2-10), which may be, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.
Preferably, the temperature of the connection is 20-30℃and may be, for example, 20℃21℃22℃23℃24℃25℃26℃27℃28℃29℃or 30 ℃.
Preferably, the time of the connection is 1-10min, for example, 1min, 2min, 3min, 4 min, 5min, 6min, 7min, 8min, 9min or 10min.
Preferably, the concentration of the glycine solution is 0.5 to 2mol/L, and may be, for example, 0.5mol/L, 0.6 mol/L, 0.7mol/L, 0.8mol/L, 0.9mol/L, 1.0mol/L, 1.1mol/L, 1.2mol/L, 1.3 mol/L, 1.4mol/L, 1.5mol/L, 1.6mol/L, 1.7mol/L, 1.8mol/L, 1.9mol/L or 2.0 mol/L, preferably 1mol/L.
Preferably, the molar ratio of the probe scaffold to the glycine solution is 1 (5-20), which may be, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.
Preferably, the time for stopping the reaction is 5-80min, for example, 5min, 10min, 15 min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min or 80min.
Preferably, the temperature of the termination reaction is 20-30℃and may be, for example, 20℃21℃22℃23℃24℃25℃26℃27℃28℃29℃or 30 ℃.
Preferably, the termination reaction is carried out under light-protected conditions.
Preferably, the filtration is performed using ultrafiltration tubing.
As a preferred technical scheme, the invention provides a synthesis method of a probe, which comprises the following steps:
(1) Removing Boc from the compound A, and reacting with the compound B at 20-30 ℃ and pH of not less than 9 for 18-24h, wherein the molar ratio of the compound A to the compound B is 1 (1-2), so as to obtain a compound C;
(2) Condensing the compound C and the N-hydroxysuccinimide in the step (1) for 18-24 hours according to the mol ratio of the compound C to the N-hydroxysuccinimide of 1 (1.5-2.5) to obtain a probe framework;
(3) Mixing the decoy protein with the probe skeleton obtained in the step (2) according to the molar ratio of the decoy protein to the probe skeleton of 1 (2-10), and connecting for 1-10min at 20-30 ℃;
(4) Adding glycine solution with concentration of 0.5-2mol/L according to the mol ratio of the probe skeleton to the glycine solution of 1 (5-20), terminating the reaction for 5-80min at 20-30 ℃ under the condition of light shielding, and filtering by an ultrafiltration tube to obtain the probe.
In a second aspect, the present invention provides a probe prepared by the method of the first aspect.
Preferably, the probe skeleton in the probe is shown as a general formula I:
wherein n is 1 Select 0,1 or 2, n 2 Selecting 0,1 or 2, R is selected from
Preferably n 1 Is 0, n 2 Is 0, R is
Preferably n 1 Is 1, n 2 Is 2, R is
Preferably n 1 Is 1, n 2 Is 2, R is
Preferably n 1 Is 1, n 2 Is 2, R is
Preferably, the decoy proteins in the probes comprise proteins and/or polypeptides comprising SH2 domains, preferably SH2 domains, further preferably mutated SH2 domains.
In the invention, the specific binding capacity of SH2 domain to tyrosine phosphorylating protein is utilized, SH2 domain is adopted as bait protein, weak dynamic change interaction between SH2 domain and tyrosine phosphorylating protein is enhanced into covalent binding interaction after ultraviolet light radiation, and the research of tyrosine phosphorylating protein complex in cell signal transduction is facilitated.
Preferably, the mutant SH2 domain is a Src SH2 domain mutant, which is based on the wild-type Src SH2 domain, having a substitution of valine for threonine at position 138, alanine for cysteine at position 188, and leucine for lysine at position 206 within the SH2 domain.
In the invention, a three-site mutant Src SH2 structural domain (Src superbinder) is adopted as a bait protein, so that the affinity for tyrosine phosphorylating protein is further improved, and the sensitivity of the probe is remarkably improved.
Preferably, the SH2 domain is linked to the NHS group on the probe backbone by a free primary amino group.
In the present invention, the SH2 domain may be linked to the probe backbone by its free primary amino group(s) bound to NHS groups.
In a third aspect, the present invention provides the use of a probe according to the second aspect for detecting protein-protein interactions, preferably tyrosine phosphorylating residue-containing protein-protein interactions.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, a simple and convenient-to-operate, low-cost and environment-friendly organic method is adopted to synthesize a probe skeleton comprising photoreactive groups and enrichment groups, after an NHS group is connected with an SH2 structural domain, a functional probe Photo-pTyr-scaffold capable of enriching and identifying tyrosine phosphorylated protein complexes is prepared, when the SH2 structural domain interacts with tyrosine phosphorylated proteins, ultraviolet radiation promotes covalent binding of photoreactive groups and target tyrosine phosphorylated proteins, and protein complexes are obtained through effective enrichment of enrichment groups, so that weak interaction and instantaneous interaction analysis between proteins are facilitated, enrichment and identification capability of target proteins and complexes near human or animal tissue samples and cell membranes are remarkably improved, and background interference of other nonspecific adsorbed proteins is reduced.
Drawings
FIG. 1 is a structural formula of a probe backbone containing NHS groups;
FIG. 2 (A) is a Western blot experimental result diagram of example 6, FIG. 2 (B) is a Western blot experimental result diagram of example 8, FIG. 2 (C) is a Western blot experimental result diagram of example 9, and FIG. 2 (D) is a Western blot experimental result diagram of example 10;
FIG. 3 is a graph showing the Western blot analysis results of example 11.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Materials and instrumentation:
thin Layer Chromatography (TLC) using 60F254 silica gel plates, detection using UV detector or carbonization of 10% (w/v) ethanol solution of 4- (dimethylamino) cinnamaldehyde, separation of intermediates and chemical probes using semi-preparative high performance liquid chromatography;
MS analysis used a high resolution Q-Exactive Orbitrap mass spectrometer;
the solvent of NMR (400MHz 1H NMR,100MHz 13C NMR) was chloroform, with Tetramethylsilane (TMS) as an internal standard;
the unit of chemical shift is ppm, the unit of coupling constant is Hz, delta represents chemical shift, s represents singlet, d represents doublet, t represents triplet, and m represents multiplet.
Example 1 Synthesis of Probe skeleton 1
According to literature [ Ouchi T, yamame E, hara K, hirai M, ohya Y (2004) Design of attachment type of drug delivery system by complex formation of avidin with biotinyl drug model and biotinyl saccharide. J Control Release 94 (2-3): 281-291; frei AP, wollscheid B, jeon OY, carreira EM U.S. Pat. No. 5,54/0011212A 1 ] synthesizes compound c1;
a mixture of c1 (896 mg,1 mmol) and diethylamine (2.19 g,30 mmol) in acetonitrile was stirred at room temperature and after the disappearance of starting material c1 (monitored by TLC), the solvent was removed; the residue was dissolved in dimethylformamide (DMF, 10 mL), N-diisopropylethylamine (258 mg,2 mmol) and succinic anhydride (200 mg,2 mmol) were added, stirred for 12 hours or more, DMF was removed, and the residue was isolated by silica gel to obtain c2 (387 mg) in 50% yield;
HRMS(m/z)：[M-H] - C 35 H 61 O 11 N 6 s calculated 773.4125, measured 773.4125;
trifluoroacetic acid (TFA, 1.026g,8.9 mmol) was added to a solution of c2 (70 mg,0.09 mmol) in dichloromethane (5 mL) at 0deg.C, stirred for 2h, and TLC monitoring found the disappearance of starting material c 2; after evaporating the mixture in vacuo, dissolving in DMF (5 mL), adding triethylamine to adjust pH above 9; 4-Benzoylbenzoic acid-2, 5-dioxopyrrolidin-1-yl ester (43.6 mg,0.135 mmol) was added, stirred overnight, the solvent was removed in vacuo and the residue was isolated with silica gel to give c3 (47 mg) in 59% yield;
HRMS(m/z)：[M-H] - C 44 H 61 O 11 N 6 s calculated 881.4125, measured 881.4130;
to a mixture of compound c3 (47 mg,0.053 mmol) and hydroxysuccinimide (12 mg,0.105 mmol) in DMF (5 mL) was added N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDCI, 30mg,0.158 mmol), stirred overnight, when compound c3 disappeared (monitored by TLC), the solvent was removed under vacuum, and the residue was isolated and purified by semi-preparative high performance liquid chromatography to give probe scaffold 1 (13 mg) in 25% yield;
1 H NMR(500MHz,DMF-d 7 )δ(ppm)8.71(t,J＝5.5Hz,1H),8.17-8.12(m, 3H),7.90-7.83(m,5H),7.76-7.73(m,2H),7.64-7.61(m,2H),6.42(s,1H),6.32(s,1H),4.48-4.45(m,1H),4.36-4.28(m,2H),3.59-3.57(m,5H),3.48-3.38(m,7H), 3.24-3.18(m,5H),2.99-2.92(m,5H),2.72-2.69(m,3H),2.16(t,J＝7.5Hz,2H),1.86-1.36(m,16H)。
13 C NMR(126MHz,DMF-d 7 )δ(ppm)196.3,172.9,172.4,170.8,169.7,166.3, 163.7,140.2,139.1,137.9,133.6,130.5,130.3,129.3,128.0,70.9,70.6,69.2,69.1,62.2,60.6,56.5,54.0,40.8,40.2,36.9,36.8,36.2,32.8,29.2,29.1,26.8,26.3,26.2, 23.9。
HRMS(m/z)：[M+H] + C 48 H 66 O 13 N 7 s calculated 980.4434, found 980.4421.
Example 2 Synthesis of Probe skeleton 2
According to literature [ Ouchi T, yamame E, hara K, hirai M, ohya Y (2004) Design of attachment type of drug delivery system by complex formation of avidin with biotinyl drug model and biotinyl saccharide. J Control Release 94 (2-3): 281-291; frei AP, wollscheid B, jeon OY, carreira EM US 2014/0011212A1; srinivasan B, huang X (2008) Functionalization of magnetic nanoparticles with organic molecules: loading level determination and evaluation of linker length effect on immobization. Chirality 20 (3-4): 265-277 ] to synthesize compound c4;
TFA (1.026 g,9 mmol) was added to a solution of c4 (100 mg,0.09 mmol) in dichloromethane (5 mL) at 0deg.C, stirred for 2 hours, and TLC monitoring found the disappearance of starting material c4; after evaporating the mixture in vacuo, dissolving in DMF (5 mL), adding triethylamine to adjust pH above 9; 4-Benzoylbenzoic acid-2, 5-dioxopyrrolidin-1-yl ester (43.6 mg,0.135 mmol) was added, stirred overnight, the solvent was removed in vacuo and the residue was isolated by silica gel to give c5 (65 mg) in 59% yield;
HRMS(m/z)：[M+Na] + C 62 H 95 O 14 N 9 SNa calculated 1244.6611, found 1244.6584;
EDCI (30 mg,0.158 mmol) was added to a mixture of compound c5 (65 mg,0.053 mmol) and hydroxysuccinimide (12 mg,0.105 mmol) in DMF (5 mL), stirred overnight, and when compound c5 disappeared (monitored by TLC), the solvent was removed under vacuum, and the residue was isolated and purified by semi-preparative high performance liquid chromatography to give probe scaffold 2 (17 mg) in 24% yield;
1 H NMR(500MHz,DMF-d 7 )δ8.71(br s,1H),8.13(d,J＝8.0Hz,2H), 8.01-7.83(m,7H),7.75-7.73(m,4H),7.62(t,J＝7.5Hz,2H),6.42(s,1H),6.33(s,1H),4.48-4.46(m,1H),4.30-4.22(m,2H),3.66-3.57(m,7H),3.49-3.39(m,8H), 3.23-3.20(m,5H),3.17-3.12(m,6H),2.95-2.93(m,3H),2.72-2.68(m,4H),2.56-2.42(m,4H),2.18-2.12(m,6H),1.78-1.55(m,18H),1.51-1.28(m,16H)。
13 C NMR(126MHz,DMF-d 7 )δ196.2,172.9,172.8,172.6,172.5,170.9,169.8, 166.3,162.9,162.7,162.5,140.2,139.1,137.8,133.6,130.5,130.3,129.3,128.0,70.9,70.6,69.2,62.2,60.6,56.5,54.0,40.8,40.3,39.4,39.3,39.2,37.0,36.8,36.4, 36.2,32.3,31.8,31.6,31.0,30.0,29.9,29.2,29.1,27.2,26.5,26.4,26.2,26.1,26.0,25.0,23.9。
HRMS(m/z)：[M+H] + C 66 H 99 O 16 N 10 s calculated 1319.6956, found 1319.6957.
EXAMPLE 3 Synthesis of Probe skeleton 3
TFA (1.026 g,9 mmol) was added to a solution of c4 (100 mg,0.09 mmol) in dichloromethane (5 mL) at 0deg.C, stirred for 2 hours, and TLC monitoring found the disappearance of starting material c4; after evaporation of the mixture in vacuo, it was dissolved in DMF (5 mL) and the pH was adjusted to greater than 9 by addition of triethylamine; 2, 5-Dioxopyrrolidin-1-yl 4-azidobenzoate (35.2 mg,0.14 mmol) was added, stirred overnight, the solvent removed in vacuo and the residue isolated by silica gel to give c6 (63 mg) in 61% yield;
HRMS(m/z)：[M-H] - C 55 H 89 O 13 N 12 s calculated 1157.6398, measured 1157.6417;
EDCI (30 mg,0.158 mmol) was added to a mixture of compound c6 (61 mg,0.053 mmol) and hydroxysuccinimide (12 mg,0.106 mmol) in DMF (5 mL), stirred overnight at room temperature, and when compound c6 disappeared (monitored by TLC), the solvent was removed under vacuum, and the residue was isolated and purified by semi-preparative high performance liquid chromatography to give probe scaffold 3 (16 mg) in 24% yield.
1 H NMR(500MHz,DMF-d 7 )δ8.50(s,1H),8.03-8.01(m,3H),7.97-7.90(m, 2H),7.77-7.73(m,3H),7.24-7.21(m,2H),6.43(s,1H),6.34(s,1H),4.48-4.46(m, 1H),4.31-4.22(m,2H),3.66-3.57(m,9H),3.49-3.45(m,6H),3.24-3.19(m,6H),3.16-3.11(m,5H),2.93-2.91(m,5H),2.71-2.68(m,2H),2.54-2.43(m,4H), 2.18-2.12(m,6H),1.75-1.67(m,7H),1.61-1.26(m,27H)。
13 C NMR(126MHz,DMF-d 7 )δ172.9,172.8,172.6,172.1,172.0,171.5,171.0, 170.8,169.8,166.1,163.7,143.2,132.4,129.8,119.5,70.9,70.6,69.2,62.2,60.6,56.5,54.0,51.8,40.8,40.2,39.3,39.2,38.2,37.0,36.8,36.4,36.2,34.2,32.3,31.8, 31.6,31.0,29.2,29.1,27.2,26.5,26.4,26.2,26.1,25.0,23.9。
HRMS(m/z):[M+H] + C 59 H 94 O 15 N 13 S calculated 1256.6708, found 1256.6705.
EXAMPLE 4 Synthesis of Probe skeleton 4
TFA (1.026 g,9 mmol) was added to a solution of c4 (100 mg,0.09 mmol) in dichloromethane (5 mL) at 0deg.C, stirred for 2 hours, and TLC monitoring found the disappearance of starting material c4; the mixture was evaporated in vacuo, dissolved in DMF (5 mL) and triethylamine was added to adjust pH above 9; 2, 5-dioxopyrrolidin-1-yl 3- (3-methyl-3H-diazepan-3-yl) propionate (30 mg,0.135 mmol) was added, stirred overnight, the solvent removed in vacuo and the residue isolated by silica gel to give c7 (61 mg) in 61% yield;
HRMS(m/z)：[M+H] + C 53 H 94 O 13 N 11 s calculated 1124.6748, measured 1124.6478;
EDCI (30 mg,0.158 mmol) was added to a mixture of compound c7 (60 mg,0.053 mmol) and hydroxysuccinimide (12 mg,0.105 mmol) in DMF (5 mL), stirred overnight at room temperature, and when compound c7 disappeared (monitored by TLC), the solvent was removed under vacuum, and the residue was isolated and purified by semi-preparative high performance liquid chromatography to give probe scaffold 4 (16 mg) in 25% yield.
1 H NMR(500MHz,DMF-d 7 )δ8.12-8.04(s,1H),7.97-7.95(m,1H),7.93-7.91 (m,1H),7.84-7.83(m,1H),7.78-7.73(m,3H),6.43-6.34(m,2H),4.48-4.46(m,1H),4.31-4.29(m,1H),4.27-4.22(m,1H),3.59-3.57(m,3H),3.54-3.53(m,5H), 3.49-3.45(m,5H),3.24-3.11(m,13H),2.93-2.91(m,4H),2.73-2.69(m,3H),2.56-2.43(m,4H),2.18-2.12(m,6H),2.08-2.06(m,2H),1.78-1.51(m,19H), 1.50-1.27(m,17H),1.01(s,3H)。
13 C NMR(126MHz,DMF-d 7 )δ172.9,172.8,172.7,172.6,171.5,171.0,170.9, 169.8,163.7,70.9,70.7,69.2,62.3,60.6,56.6,54.0,51.9,51.7,40.8,39.5,39.4,39.3,37.0,36.9,36.4,36.2,32.3,31.8,31.7,31.0,30.9,30.7,29.3,29.1,27.2,26.5, 26.4,26.3,26.1,25.0,24.0,19.6。
HRMS(m/z):[M+H] + C 57 H 97 O 15 N 12 S calculated 1221.6912, found 1221.6886.
EXAMPLE 5 expression, purification and buffer replacement of SH2 Domain
Expression of SH2 domain:
constructing a plasmid for expressing SH2 domain containing GST tag, transferring into escherichia coli BL21 cells, taking 2mL of cell culture solution after overnight culture, adding into 1L of fresh LB culture medium, and culturing the LBThe culture medium contains ampicillin with final concentration of 50mg/L, and is cultured at 37deg.C and 200rpm until OD 600 =0.6, IPTG was added at a final concentration of 0.25mM and induction was continued for 12h at 16 ℃;
purification of SH2 domain:
BL21 cell culture broth (6000 Xg, 30 min) was collected by centrifugation, and 50mL of 1 XPBS (137mM NaCl,2.7mM KCl,10mM Na) was added to the cell pellet 2 HPO 4 ，1.7mM KH 2 PO 4 Ph=7.4) were washed once and the cells were collected by centrifugation; 50mL of lysate (1% (v/v) Triton X-100, 1.5mM DTT,2mM EDTA, dissolved in 1 XPBS, pH=8.0) was added to the cell pellet and sonicated (ice bath, 400 Watts, 5/15 seconds, 30 times); centrifuging at 14000 Xg for 30min, transferring the supernatant to a protein purification column containing 5mL Glutathione Sepharose 4B, and slowly shaking at 4deg.C for incubation for 1 hr; adding 2 column volumes of lysate, washing for 2 times; adding 20mL of eluent (50 mM Tris-HCl,150mM NaCl,10mM reduced glutathione, pH=8.0), and collecting the eluent;
buffer replacement of SH2 domain:
the buffer for the protein of interest was replaced with HEPES buffer (50mM HEPES,150mM NaCl,1mM DTT,1mM EDTA,pH =8.0) using a ultrafiltration tube (3 kDa cut-off Amicon Ultra centrifugal filter device, merck Millipore) and the protein concentration of the SH2 domain was adjusted to 1mg/mL.
EXAMPLE 6 SH2 bait protein-labeled probe backbone 2
The invention designs 4 probe frameworks, which are used for preparing functional probes after being connected with SH2 domains, and the structure of the probe frameworks is shown in figure 1.
Adding 0.5 mu L of probe skeleton 2 containing photoreactive groups and biotin groups (the molar ratio of the probe skeleton 2 to the probe skeleton 2 is 1:2, 1:5, 1:10 and 1:30 respectively) to 50 mu L of SH2 domain (1 mg/mL in HEPES buffer), reacting for 1min, 2min, 5min and 10min at room temperature, and then adding 5 mu L of glycine solution of 1mol/L to terminate the activity of NHS groups to obtain probe 2 marked with SH2 bait protein;
to the obtained probe 2, 14. Mu.L of a 5 XSDS-PAGE loading buffer was added, and the mixture was heated at 96℃for 10 minutes to conduct SDS-PAGE electrophoresis and Western blotting experiments.
As a result, as shown in FIG. 2 (A), the electrophoresis behavior of SH2 domain on SDS-PAGE was significantly shifted with the increase in the concentration of probe scaffold 2 and the labeling time, and in addition, the result of streptavidine-HRP showed that the change in the electrophoresis behavior was caused by the labeling of probe scaffold 2.
The invention adopts the SH2 structural domain and the probe skeleton 2 with the reaction time of 1min as the subsequent experimental conditions.
Example 7 SH2 decoy protein labelling of Probe frameworks 1, 3 and 4
In comparison with example 6, the conditions were the same as in example 6 except that the probe skeleton 2 was replaced with the probe skeletons 1, 3 and 4.
Since the results of labeling SH2 domains with probe frameworks 1, 3 and 4 are substantially identical to those of probe framework 2, the experimental data representative of probe framework 2 are not described in detail herein.
Example 8 Activity assessment of SH2 Domain
To probe 2 prepared in example 6 was added 100mM sodium Pervanadate (PV) -stimulated HeLa cell lysate, incubated with slow shaking at 4℃for 2h, 30. Mu.L streptavidin magnetic beads, and incubated with slow shaking at 4℃for 2h;
1mL of RIPA buffer (50 mM Tris-HCl,150mM NaCl,1% (v/v) Triton X-100,1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, pH=7.4) was added for 3 washes, followed by 30. Mu.L of 2 XSDS-PAGE loading buffer, heating at 96℃for 10min, and then SDS-PAGE electrophoresis and Western blotting experiments were performed.
As a result, as shown in FIG. 2 (B), the activity of SH2 domain was not affected when the molar ratio of SH2 domain to probe backbone 2 was less than 1:30, as compared to the normal GST pull-down.
The invention adopts the molar ratio of SH2 structural domain to probe skeleton 2 of 1:10 as the subsequent experimental condition.
Example 9 optimization of photoreactive group reaction conditions
To 5. Mu.L of a 1mol/L glycine solution was added 0.5. Mu.L of probe skeleton 2 (probe skeleton 2 was dissolved in dimethyl sulfoxide), and the reaction was conducted at room temperature in the dark for 1 hour to neutralize the reactivity of the NHS group of probe skeleton 2. mu.L of SH2 domain (1 mg/mL in HEPES buffer, 1:10 molar ratio of protein to probe) was added, then the above solution was transferred to a quartz dish, irradiated with 365nm UV light for 10min, 30min and 60min, added with 14. Mu.L of 5 XSDS-PAGE loading buffer, heated at 96℃for 10min, and then subjected to SDS-PAGE electrophoresis and Western blotting experiments.
As a result, as shown in FIG. 2 (C), the probe was covalently bonded to the SH2 domain after ultraviolet irradiation.
The invention adopts the ultraviolet irradiation time of 30min as the subsequent experimental condition.
Example 10 optimization of streptavidin pull-Down reaction conditions
To probe 2 prepared in example 6, 30. Mu.L of streptavidin magnetic beads were added, incubated at 4℃for 2 hours with slow shaking, 1mL of optimized RIPA buffer (50 mM Tris-HCl,1M NaCl,1% (v/v) Triton X-100,1% (w/v) desalted sodium cholate, 1% (w/v) SDS, pH=7.4) was added for 3 times, then 30. Mu.L of 2 XSDS-PAGE loading buffer was added, heating was carried out at 96℃for 10 minutes, and SDS-PAGE electrophoresis and Western blotting experiments were carried out on the magnetic beads, running-through liquid and washing liquid obtained during the experiment together, and as a result, as shown in FIG. 2 (D), 30. Mu.L of streptavidin magnetic beads were completely able to enrich the probe labeled with SH2 domain.
Example 11 enrichment and identification of target proteins by probes
Because of the extremely low levels of tyrosine phosphorylated proteins in the endogenous EGFR signaling pathway, to increase the levels of these proteins, we use EGF to stimulate cells to activate the relevant tyrosine phosphorylated proteins in the EGFR signaling pathway. At the same time we set a control group, which was not stimulated with EGF.
To probe 2 prepared in example 6, 100. Mu.g of EGF-stimulated or unstimulated cell lysate was added, and incubated with slow shaking at 4℃for 2 hours, followed by transfer to a quartz dish, irradiation with 365nm ultraviolet light was performed for 30 minutes, while a control group was set, and the control group was not subjected to ultraviolet light irradiation. Then 30 mu L of streptavidin magnetic beads are added into each group, the mixture is slowly shaken and incubated for 2 hours at 4 ℃, and 1mL of optimized RIPA buffer solution is adopted for cleaning for 3 times;
30. Mu.L of 2 XSDS-PAGE loading buffer was added, and the mixture was heated at 96℃for 10min, and SDS-PAGE electrophoresis and Western blotting were performed.
As a result, as shown in fig. 3, probe 2 can significantly enrich the tyrosine-phosphorylated protein EGFR that interacts with it (fig. 3, lane 2), and the enrichment effect of probe 2 on the tyrosine-phosphorylated protein EGFR is greatly enhanced after uv irradiation (fig. 3, lane 4). Indicating that the proteins interacting with probe 2 are more enriched. Meanwhile, we found that probe 2 also had a significant enrichment effect on cell-background EGFR even in the absence of EGF stimulation after uv irradiation (fig. 3, lane 3), indicating that probe 2 has very high sensitivity to poorly soluble membrane proteins located near the cell membrane.
In summary, the invention synthesizes the probe skeleton comprising the photoreactive group and the enrichment group by adopting the organic method with simple operation, low cost and environmental friendliness, and after the SH2 structural domain is connected by the NHS group, the functional probe Photo-pTyr-scaffold capable of enriching and identifying the tyrosine phosphorylated protein complex is prepared, when the SH2 structural domain interacts with the tyrosine phosphorylated protein, the ultraviolet radiation promotes the covalent bonding of the photoreactive group and the target tyrosine phosphorylated protein, and the enrichment group effectively enriches the protein complex to facilitate the analysis of weak interaction and transient interaction between proteins, thereby remarkably improving the enrichment and identification capability of the target protein and the complex near human or animal tissue samples and cell membranes and reducing the background interference of other nonspecific adsorbed proteins.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (27)
1. The probe is characterized in that a probe skeleton in the probe is shown as a general formula I:
wherein n is 1 Is 0, n 2 Is 0, R is
Alternatively, n 1 Is 1, n 2 Is 2, R is
Alternatively, n 1 Is 1, n 2 Is 2, R is
Alternatively, n 1 Is 1, n 2 Is 2, R is
The decoy protein in the probe is SH2 structural domain;
the probe skeleton is prepared by a synthesis method comprising the following steps:
(1) Removing Boc from the compound A, and reacting with the compound B to obtain a compound C;
(2) Condensing the compound C in the step (1) with N-hydroxysuccinimide to obtain a probe skeleton;
2. the probe of claim 1, wherein the molar ratio of compound A to compound B in step (1) is 1 (1-2).
3. The probe of claim 2, wherein the molar ratio of compound A to compound B in step (1) is 1 (1.5-1.6).
4. The probe of claim 1, wherein the temperature of the reaction of step (1) is 20-30 ℃.
5. The probe of claim 1, wherein the reaction time of step (1) is 18-24 hours.
6. The probe of claim 1, wherein the pH of the reaction of step (1) is not less than 9.
7. The probe of claim 6, wherein the reaction in step (1) has a pH of 9 to 10.
8. The probe of claim 1, wherein the molar ratio of compound C to the N-hydroxysuccinimide of step (2) is 1 (1.5-2.5).
9. The probe of claim 8, wherein the molar ratio of compound C to the N-hydroxysuccinimide of step (2) is 1 (1.98-2).
10. The probe of claim 1, wherein the time of the condensation of step (2) is 18-24 hours.
11. The probe of claim 1, wherein the probe is prepared by ligating a decoy protein after step (2), in particular: mixing the decoy protein with the obtained probe skeleton, connecting, adding glycine solution to terminate the reaction, and filtering to obtain the probe.
12. The probe of claim 11, wherein the molar ratio of the decoy protein to the probe scaffold is 1 (2-10).
13. The probe of claim 11, wherein the temperature of the ligation is 20-30 ℃.
14. The probe of claim 11, wherein the time of ligation is 1-10min.
15. The probe of claim 11, wherein the glycine solution has a concentration of 0.5 to 2mol/L.
16. The probe of claim 15, wherein the glycine solution has a concentration of 1mol/L.
17. The probe of claim 11, wherein the molar ratio of the probe backbone to the glycine solution is 1 (5-20).
18. The probe of claim 11, wherein the time to terminate the reaction is from 5 to 80 minutes.
19. The probe of claim 11, wherein the termination reaction is at a temperature of 20-30 ℃.
20. The probe of claim 11, wherein the termination reaction is performed in the absence of light.
21. The probe of claim 11, wherein the filtration is performed using an ultrafiltration tube.
22. The probe of claim 1, wherein the probe is prepared by a synthetic method comprising the steps of:
(1) Removing Boc from the compound A, and reacting with the compound B at 20-30 ℃ and pH of not less than 9 for 18-24h, wherein the molar ratio of the compound A to the compound B is 1 (1-2), so as to obtain a compound C;
(2) Condensing the compound C and the N-hydroxysuccinimide in the step (1) for 18-24 hours according to the mol ratio of the compound C to the N-hydroxysuccinimide of 1 (1.5-2.5) to obtain a probe framework;
(3) Mixing the decoy protein with the probe skeleton obtained in the step (2) according to the molar ratio of the decoy protein to the probe skeleton of 1 (2-10), and connecting for 1-10min at 20-30 ℃;
(4) Adding glycine solution with concentration of 0.5-2mol/L according to the mol ratio of the probe skeleton to the glycine solution of 1 (5-20), terminating the reaction for 5-80min at 20-30 ℃ under the condition of light shielding, and filtering by an ultrafiltration tube to obtain the probe.
23. The probe of claim 1, wherein the decoy protein in the probe is a mutant SH2 domain.
24. The probe of claim 23, wherein the mutant SH2 domain is a Src SH2 domain mutant that replaces threonine at position 138 with valine, cysteine at position 188 with alanine, and lysine at position 206 with leucine within the SH2 domain on the basis of the wild-type Src SH2 domain.
25. The probe of claim 1, wherein the SH2 domain is attached to the NHS group on the probe backbone by a free primary amino group.
26. Use of a probe according to any one of claims 1 to 25 for detecting protein-protein interactions.
27. The use of claim 26, wherein the probe is used to detect interactions between proteins containing tyrosine phosphorylated residues.
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