CN115947862B - SH2 super parent protein and conjugate formed by conjugation of SH2 super parent protein and solid phase - Google Patents
SH2 super parent protein and conjugate formed by conjugation of SH2 super parent protein and solid phase Download PDFInfo
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- CN115947862B CN115947862B CN202211040518.9A CN202211040518A CN115947862B CN 115947862 B CN115947862 B CN 115947862B CN 202211040518 A CN202211040518 A CN 202211040518A CN 115947862 B CN115947862 B CN 115947862B
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Abstract
The invention relates to the technical field of proteomics, in particular to SH2 super parent protein and a conjugate formed by the SH2 super parent protein and solid phase conjugation. The conjugate of SH2 super parent and solid phase is prepared, and the thiol crosslinking method is adopted to increase the space flexibility of SH2 protein coupling to the solid phase, so that the conjugate is more beneficial to combining tyrosine phosphorylated peptide fragments in complex protein enzyme digestion products.
Description
Technical Field
The invention relates to the technical field of proteomics, in particular to SH2 super parent protein and a conjugate formed by the SH2 super parent protein and solid phase conjugation.
Background
Protein phosphorylation plays an important regulatory role in vital activities, and is a hotspot for post-translational modification research in proteomics at present. Studies have shown that about 1/3 of the proteins in eukaryotic cells are in a phosphorylated state in the resting state. Protein kinases regulate a variety of biological processes in cells, such as cell cycle, cell growth, and cell differentiation, by catalyzing the phosphorylation of specific substrates. Protein phosphorylation can occur on a wide variety of amino acids, with serine, threonine and tyrosine being the most common phosphorylation sites. Protein phosphorylation is a reversible dynamic process, regulated by competing activities of protein kinases and phosphatases. Phosphorylation and dephosphorylation are switches that activate key regulatory proteins and control signaling pathways. Once an abnormality occurs in the phosphorylation process, the associated signaling pathway is dysfunctional. Thus, abnormalities in protein phosphorylation are associated with the occurrence of a variety of diseases, such as inflammatory diseases, diabetes, infectious diseases, cardiovascular diseases, and cancers. At present, a large number of protein kinases and phosphatases have become hot targets for drug development.
Protein kinase/phosphatase regulated signaling pathways are involved in the development and progression of almost all types of cancers, and as protein kinase/phosphatases become more well known, drug development targeting protein kinase/phosphatases has become more and more important. Taking tyrosine kinase as an example, EGFR can be combined with ligands to form activated dimers, and the dimers regulate survival, proliferation and metastasis of tumor cells by activating intracellular kinase pathways, phosphorylating activation sites such as Y992, Y1045, Y1068, Y1148 and Y1173, and further activating Ras/Raf/MAPK or PI3K/Akt signaling pathways. Many inhibitors against the EGFR receptor are one of the most successful examples of targeted cancer treatments to date, such as Erlotinib, gefitinib, lapatinib, and the like. Sorafenib is also a typical representative of tyrosine kinase inhibitors for tumor therapy, and is a multi-target tyrosine kinase inhibitor, comprising vascular endothelial growth factor receptor (involved in angiogenesis and growth, wound repair, inflammation and other processes), c-Kit kinase, raf kinase and the like, which has significant effects on anti-tumor angiogenesis and induction of tumor cell apoptosis. Although the status of tyrosine kinases is so important, there is still a lack of adequate targeted drugs on the market, the limiting factor being the far insufficient number of tyrosine kinase assays, which are difficult to assay because tyrosine phosphorylation is usually in a very low metering range and distributed over multiple sites. In order to increase the identification depth of tyrosine phosphorylation sites, tyrosine phosphorylated peptide fragments are usually enriched and then detected, and the conventional flow is as follows: 1. extracting and quantifying protein; 2. enzymatically fragmenting the protein into polypeptides; 3. enrichment from complex polypeptides to tyrosine phosphorylated polypeptides of interest; 4. tyrosine phosphorylated polypeptides were analyzed by mass spectrometry.
Removing the disadvantage of low stoichiometric amounts of protein phosphorylation, the non-phosphorylated modified peptide fragments contained in the complex polypeptide mixture will also inhibit the response of the phosphopeptide signal. On the other hand, multiple trials are required to obtain a satisfactory modification profile, so that sufficient protein samples are required to meet the demand. Therefore, the separation and enrichment method of the phosphopeptide is important before analysis.
Taking the enrichment of phosphorylated tyrosine peptide fragments as an example, a variety of commercially available tyrosine phosphorylated antibodies have been developed on the market, such as: PT66, anti-pTyr-4G10, pY99, P-Tyr-100. However, these antibodies are expensive, have poor lot-to-lot reproducibility and small amounts, and expensive antibodies are difficult to meet given the large number of tyrosine phosphorylation sites to identify.
SH2 super parent (SH 2-super parent) based on phage display technology is utilized, and through evolutionary mutation of wild SH2 domains, SH2 structural variants with 1000-fold improvement of phosphotyrosine capture capacity are finally obtained. Thus, SH2 superparents can be used well as an antibody replacement for complex sample tyrosine phosphorylation proteome analysis. Three amino acid mutations are introduced into a binding pocket of an SH2 structural domain and a tyrosine phosphorylation site, so that the dissociation constant value reaches a nanomolar level equivalent to that of an antibody antigen, and the purification of an SH2 super-parent for tyrosine phosphorylation peptide segment becomes a new choice. However, a method for facilitating the phosphorylation and enrichment of butyric acid by not only maintaining the protein activity but also immobilizing SH2 super-parents to a support is lacking.
Disclosure of Invention
The invention aims to provide an improved SH2 super parent protein, the amino acid sequence of which is shown in SEQ ID NO: 1.
The protein is obtained by modifying the original SH2 super parent protein, the modification process does not influence the activity of the SH2 super parent protein, and the modified SH2 super parent protein can be crosslinked with a solid phase through sulfhydryl groups.
The invention also provides nucleic acids, vectors and host cells containing the SH2 super-parent protein.
The invention also provides a conjugate formed by conjugation of disulfide bond formed by cysteine at the C-terminal of SH2 super parent protein and a solid phase, a kit containing the conjugate and application thereof.
According to the invention, when the cysteine residue at the C-terminal of the SH2 super-parent protein is incubated with a coupling solid phase, the solid phase and the exposed sulfhydryl (-SH) react specifically and efficiently, so that a covalent and irreversible thioether bond for permanently connecting the SH2 super-parent protein to the resin is formed. The space flexibility of SH2 protein coupling to a solid phase is maintained by a sulfhydryl crosslinking mode, and tyrosine phosphorylation peptide segments can be effectively captured from complex protein digestion products.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of the activity detection of SH2 super-parent proteins before and after transformation according to an embodiment of the present invention;
FIG. 2 shows the number of SH2-Beads enriched peptide fragments immobilized by different crosslinking methods according to one embodiment of the present invention.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).
The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.
Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%.
In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
The term "peptide" or "polypeptide" as used herein is defined as a chain of amino acid residues that are bound by peptide bonds and generally have a defined sequence. The term "peptide" or "polypeptide" as used herein may, but need not, refer to a chain of amino acid residues that do not have any N-terminal and/or C-terminal amino acid residues. That is, as used herein, a "peptide" or "polypeptide" may refer to an amino acid chain that is embedded in a longer amino acid chain. The term "peptide" as used herein includes the terms "polypeptide", "peptide" and "protein".
"peptide fragment mix sample" refers to a mixture comprising a plurality of peptides as defined above. The source may be any sample for which a profile of protein tyrosine phosphorylation is desired, including a profile of protein kinase activity or immunoreceptor phosphotyrosine signaling. Thus, the sample can be any sample comprising biological material, and which comprises or is suspected of comprising an active protein kinase or a peptide modified by an active protein kinase (e.g., a pTyr-containing peptide), including within a kinase (e.g., a kinase activation loop), within a phosphatase regulatory region, within an ITRM, and within a target downstream of the kinase and phosphatase. Samples may include, but are not limited to: an established cell line; cell cultures, including primary cell cultures; biological fluids such as serum, plasma, urine or blood; a tissue sample; or tissue extracts. The sample may be of human or non-human origin or may comprise human or non-human protein kinase activity or human or non-human pTyr-containing peptides. The peptide fragment mixture sample can be obtained by a known method, for example, a product obtained by subjecting a protein to enzymatic hydrolysis by an enzyme (e.g., pancreatin, etc.).
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Unless otherwise contradicted by purpose and/or technical solution of the present application, the cited documents related to the present invention are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present invention, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are incorporated into the present application by reference, but are not limited to the embodiments that can be implemented. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.
The invention relates to SH2 super parent protein, the amino acid sequence of which is shown in SEQ ID NO: 1.
In some embodiments, the SH2 super-parent protein has an N-terminal linked tag protein.
The tag is attached to the protein for various purposes, e.g., to facilitate purification, to aid in proper folding of the protein, to prevent precipitation of the protein, to alter chromatographic properties, to modify the protein or to label or tag the protein. Examples of tags include one or more of Arg tag, his tag, strep tag, flag tag, T7 tag, V5-peptide tag, GST tag and c-Myc tag. A preferred tag in the present invention is a His tag consisting of six histidine residues.
According to a further aspect of the invention, it also relates to a nucleic acid encoding an SH 2-super-parent protein as described above.
The nucleic acid is typically RNA or DNA, and the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are preferably used when they are incorporated into vectors. Furthermore, the nucleic acid molecules may be codon optimized according to the different host cells.
In one aspect, the SH2 super-parent protein/nucleic acid further comprises variants thereof that are mutated within a suitable range, e.g., as set forth in SEQ ID NO:1, and the amino acid sequence has an amino acid sequence/nucleotide sequence that is greater than 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and no cysteine is produced at any of the sites after mutation. The term "% identity" in the context of two or more nucleotide sequences or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example,% identity is the entire length of the coding region relative to the sequences to be compared.
The SH2 super-parent protein has substantially the same affinity as before the engineering.
According to a further aspect of the invention, it also relates to a vector comprising a nucleic acid as described above.
The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).
According to a further aspect of the invention, it also relates to a host cell comprising a nucleic acid as described above, or a vector as described above.
In the research field of cancer phosphorylation proteomics, the improvement of the number of phosphorylated tyrosine identification is the basis of the whole histology, for example, by improving the identification number, the research and development of new drugs for comprehensive assistance and accurate medical treatment in the aspects of target spot discovery, target spot identification, drug repositioning, biomarker discovery and the like can be realized. The improvement of the enrichment method for phosphorylated tyrosine is of great significance for the whole phosphorylated proteomics. In the experimental process, the inventor researches different crosslinking modes to fix SH2 super parent proteins, and surprisingly discovers that SH2 super parent proteins are crosslinked on a solid phase through sulfhydryl groups, so that the number of phosphorylated tyrosine identifications can be effectively improved, and the accuracy and efficiency of SH2 super parent proteins in phosphorylated tyrosine identifications can be greatly improved.
The term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, a prokaryotic cell such as E.coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 drosophila cell or Sf9, or an animal or human cell such as a fibroblast, CHO cell, NSO cell, heLa cell, HEK 293 cell, etc. The host cell is preferably a prokaryotic cell, more preferably E.coli.
According to a further aspect of the invention, it also relates to a conjugate formed by conjugation of the cysteine at the C-terminus of SH2 super-parent protein as described above with a solid phase via disulfide bonds.
The term "solid phase" as used herein refers to and includes any support capable of binding the affinity reagents disclosed herein. Well known supports or carriers include any of glass (e.g., borosilicate glass), agar, agarose derivatives, magnetic beads, silica, titania, alginate, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters and gelatin, silicon, ceramic, glass, polyurethane, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone, or copolymers or mixtures of any of the several. The support material may have virtually any structural configuration so long as the coupled affinity agent is capable of binding peptides and/or proteins. Thus, the support structure may be an SPE solid phase extraction column, centrifuge tube with separation membrane, EP tube, separation membrane, microplate well, microsphere, column, pellet, or multiwell plate. Those skilled in the art will be aware of many other suitable carriers for binding affinity agents, or will be able to determine such vectors using routine experimentation.
In some embodiments, the solid phase is agarose microspheres. The particle diameter of the microspheres is preferably in the range of 0.1 μm to 1mm, for example 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 500 μm, 700 μm.
The invention also relates to a kit containing the conjugate.
The term "kit" refers to any article of manufacture (e.g., package or container) comprising at least one device, which may further comprise instructions, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
In some embodiments, the kit may further comprise a wash buffer, an eluent, and the like.
The invention also relates to a method for enriching tyrosine phosphorylated peptide, comprising the following steps:
a) Incubating the peptide fragment mixture sample with a conjugate as described above to bind tyrosine phosphorylated peptide fragments therein to a solid phase;
b) Washing the solid phase and separating the tyrosine phosphorylated peptide fragment from the solid phase.
According to yet another aspect of the present invention, there is also provided a method of testing tyrosine phosphorylated peptide fragments comprising:
the tyrosine phosphorylated peptide fragments are enriched and isolated tyrosine phosphorylated peptide fragments are identified using the methods described above.
In some embodiments, the method further comprises quantifying the isolated tyrosine phosphorylated peptide fragment.
In some embodiments, the method of identifying or the quantifying comprises mass spectrometry techniques.
Mass spectrometry techniques may include, for example, multiplex Reaction Monitoring (MRM), selective Reaction Monitoring (SRM), or Parallel Reaction Monitoring (PRM) techniques.
Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
In the examples below, the experimental materials used mainly include:
semi-lysate of A-bacteria (50 mM NaH) 2 PO 4 150mM NaCl,20mM imidazole)
Formula of B-cell lysate (91.5% of semi-cell lysate, 1% of PMSF,0.5% of TECP,1% of lysozyme, 1% of ribozyme, 5% of Triton X-100)
C IAP buffer (self-assembling reagent, formulation: pH 7.6, 50mM Tris-HCl,50mM NaCl,10mM NaHPO) 4 )
D Washing buffer (configurable stock solution, formulation: pH 8.0, 50Mm NaH) 2 PO 4 500mM NaCl,20mM imidazole)
E Elutation buffer (for use at present, formula: pH 8.0, 50Mm NaH) 2 PO 4 150mM NaCl,250mM imidazole)
F (beams-SH 2) coupling buffer (self-matching reagent, formula: pH 8.0, fungus semi-lysate)
G mercapto Cross-linking (Beads-SH 2) washing buffer (self-assembling reagent, formulation: pH 8.0, 50mM Tris-HCl,1M NaCl)
H amino crosslinking (Beads-SH 2) washing buffer (self-assembling reagent, formula: pH 8.0, 50mM NaH) 2 PO 4 ，1M NaCl)
I mercapto crosslinking (Beads-SH 2) Blocking buffer (self-assembling reagent, formulation: pH 8.0, 50mM L-Cysteine)
J amino crosslinking (Beads-SH 2) Blocking buffer (self-assembling reagent, formulation: pH 8.0, 50mM NH) 4 Cl)
K (loads-SH 2) storage buffer (self-assembling reagent, formula: pH 8.0,1 ×IAP,0.2% sodium azide)
L-nickel column purification material
(SulfoLink TM Coupling resin product number: 20401 Branding): thermo Scientific TM
(AminoLink TM Plus coupling resin cat No.: 20505 Branding): thermo Scientific TM
M fluorescent peptide fragment
Determination of pY peptide fragment binding Activity of FITC-GGpYGG for detecting SH2 super-parent
Comparative examples
The functional group target point at the junction of the SH2 super parent and the solid support is a non-selective target point or any one selected from amino, carboxyl, sulfhydryl, aldehyde, hydroxyl and azide. Examples of common crosslinking reactive groups for protein coupling are as follows:
the solid phase support containing chemical reaction groups is used for crosslinking, and the free ends of the functional groups are coupled with the immobilized modification carrying the chemical reaction groups;
analysis of SH2 superparent protein cross-linked to immobilization carrier: the cross-linking process with primary amino and mercapto as cross-linking functional groups is suitable for cross-linking SH2 super parent protein to coupled resin particle via coupling reaction, and EDC one-step process with carboxyl functional groups cross-linking can connect amino and carboxyl in protein simultaneously and may produce side reactions. Hydroxyl functional crosslinking is commonly used in other crosslinking processes such as sugars. If the SH2 super-parent protein is crosslinked by a sulfhydryl functional group, the SH2 super-parent protein has two cysteines and is positioned in the protein, so that the crosslinking site is uncertain, and the effect is not expected.
In combination with the above analysis, coupling to a stationary support by amino reaction is currently the most preferred solution.
The SH2 super-parent protein is coupled through amino reaction, and the SH2 super-parent protein has free primary amino, so that the protein can be directly crosslinked to a support without further modification, and the detailed flow is as follows:
1. expression of SH2 proteins
Transforming the recombinant plasmid inserted with the original SH2 super parent protein into DH5 alpha escherichia coli, screening positive single strains by using an antibody, sequencing the strains, selecting positive strains with the DNA sequence meeting the conditions, shaking, preserving the bacteria, extracting the plasmid, transforming the plasmid into BL21 (DE 3) expression strains, and expressing the protein.
Original SH2 super-parent sequence (His tag added to enable purification of protein):
MKHHHHHHPMS (His tag) -DYDIPTTENLYFQGAMDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETVKGAYALSVSDFDNAKGLNVRHYLIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSK (SEQ ID NO: 2)
Protein expression, firstly, adding corresponding antibiotics into BL21 positive strains to perform small shaking, turning into large shaking after 16 hours, preparing 1L of LB culture solution, and adding the corresponding antibiotics to obtain 1:1000 E.coli is transferred in proportion, shaking is carried out for 3-4 hours at 37 ℃, OD value is 0.6-0.8, IPTG is added to induce protein expression, the protein expression is carried out overnight at 16 ℃, the next day, 5000g of cell sediment is collected at 4 ℃ by a centrifuge, and the cell sediment is stored in a refrigerator at-80 ℃.
2. Cleaning Beads
Protein purification, preparing bacterial lysate and bacterial half lysate, re-suspending escherichia coli sediment expressing SH2 super parent protein by using the bacterial lysate, crushing escherichia coli by using an ultrasonic crusher, centrifuging at a high speed, collecting supernatant, balancing nickel columns, washing nickel column purification Beads, co-incubating protein extract and nickel column purification Beads at 4 ℃ for 30min, passing through the columns, washing the Beads by using a Washing buffer, eluting non-specifically bound hybrid protein until the Broaded is detected, collecting an effluent liquid sample when the hybrid protein is not contained in the effluent liquid, detecting the effluent liquid sample by using an gel, and eluting SH2 super parent protein from the Beads by using an gel buffer. Protein samples produced during the extraction process were collected, run out and stained to see if the purified protein was consistent with the expected size.
3. Amino reactive crosslinking
1) BCA measured protein concentration;
2) By aminoLink TM Plus coupling treePreparing the fat to prepare the Beads;
3) Washing the affinity chromatographic column with PBS buffer solution, balancing pH value of the column, adding Beads suspension in corresponding proportion into the column, and washing Beads for 3 times;
4) Binding the Beads with SH2 protein, rotating at 4deg.C for 15 min, and standing on ice for 30min;
5) Collecting Beads by affinity chromatography column, and collecting Beads with NH-containing column 4 Blocking buffer of Cl seals unbound primary amino binding sites on the Beads, and sealing is completed;
6) Washing SH2 super parent Beads 3 times by using 20ml washing buffer;
7) Transfer the Beads to a 15ml centrifuge tube with 1×IAP, add sodium azide to a final concentration of 0.2%, and store at 4deg.C.
4. Enrichment of pY peptide fragments with linked SH2-Beads
Experimental samples: the detected object is human A549 tumor cell polypeptide treated by sodium alum for 10 minutes, and the human A549 tumor cell polypeptide is obtained according to the pretreatment operation in the SH2-super binder-based tyrosine phosphorylation enrichment method.
200ug of humanized A549 tumor cell polypeptide is treated by sodium alum for 10 minutes, and then phosphorylated tyrosine enrichment is carried out according to the following technical scheme. The kit used for the concentration measurement was BCA kit from thermo company, and the product number was 23225.
(1) IAP-dissolved polypeptide dry powder was added to 200ug polypeptide dry powder.
(2) The Agarose microsphere Agarose Beads with SH2-super binder protein was washed to prepare a 40% suspension.
(3) Mu.l of Agarose Beads were pipetted into the samples and incubated.
(4) The supernatant was discarded by centrifugation.
(5) And adding a high-salt cleaning solution for cleaning.
(6) Washing was performed by adding 50mM ammonium bicarbonate aqueous solution.
(7) Elution was performed by adding 0.5% trifluoroacetic acid.
(8) The eluted samples were desalted for C18.
And (3) pumping the obtained peptide solution, re-dissolving, and performing liquid phase tandem mass spectrometry (LC-MS/MS) on the solution according to the method to obtain identification and quantification results.
The enriched peptide fragment dry powder is detected by the following method:
the resulting peptide fragments were detected using liquid phase coupled tandem mass spectrometry, wherein a liquid chromatograph was used, thermo Ultimate model 3000, and a mass spectrometer was model orbitrap exploris, model 480, from Thermo Scientific.
3 technical replicates were made, each replicate injected with 1 needle, each run for 2 hours, and the total test duration was 6 hours.
The main parameters of mass spectrum are:
the ion source voltage is set to 2.1kV; the scanning range of the primary mass spectrum is 400-1200 m/z; resolution was set to 60,000; the cycle time of the DDA acquisition mode of the secondary mass spectrum is 3s. Ion fragmentation mode is high energy collision dissociation mode (HCD), and fragment ions are detected in Orbitrap. The dynamic exclusion time was set to 30s. The AGC is set to: primary 3E6, secondary 1E5.
The software used for mass spectrometry data analysis was Proteome Discoverer 2.5, searching for identification major parameters: the database is a homo protein database of swissprot; the mass error range of the primary mass spectrum is 10ppm; the mass error range of the secondary mass spectrum is 0.02Da; the fixation modification is 'Carbamidomethyl (C)'; variable modification is "Oxidation (M), acetyl (Protein N-terminal), phosphorylation (STY)", enzyme cutting method Trypsin, and maximum allowable leakage cut number is 2.
The number of phosphorylated tyrosine peptide fragments obtained by detecting SH2 super parent enrichment is about 600, and the number of phosphorylated tyrosine sites is not more than 500. This effect was not expected by the inventors.
EXAMPLE 1 modification of SH2 super parent
To further improve the enrichment of SH2 superparents for the number of phosphorylated tyrosine peptide fragments, the inventors tried to crosslink with thiol functional groups.
Firstly, through plasmid transformation, the original SH2 super-parent body is subjected to mutation to eliminate cysteine in the original SH2 super-parent body, and a peptide segment containing the cysteine is introduced into the C end of the SH2 super-parent body.
The sequence of the modified SH2 super parent is as follows:
MKHHHHHHPMS (His tag) -DYDIPTTENLYFQGAMDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETVKGAYALSVSDFDNAKGLNVRHYLIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLSHRLTTVSPTSKGGSGGCLE (SEQ ID NO: 1)
The modified SH2 super-parent protein was expressed using the method in the comparative example.
To verify whether the affinity with the PY peptide after transformation of the SH2 super-parent is affected, we designed a fluorescence polarization binding experiment aiming at the problem so as to evaluate the affinity of the original SH2 super-parent protein and the modified SH2 super-parent protein, the experiment uses the FITC-GGpYGG fluorescent peptide as a substrate, respectively binds with the original SH2 super-parent (shown as SEQ ID NO: 1) and the modified SH2 super-parent, incubation is performed in Phosphate Buffered Saline (PBS) buffer with pH of 7.4 at room temperature, the fluorescent polarization binding signal is measured on an EnVision HTS multi-label plate reader (Perkin Elmer), the dissociation constant (Kd) of the original SH2 super-parent and the phosphorylated tyrosine fluorescent peptide is 1.8 mu M, and the affinity is strong as the dissociation constant is smaller, so that the affinity of the modified SH2 super-parent protein is not affected obviously by the transformation of the SH2 super-parent protein.
EXAMPLE 2 thiol reactive Cross-linking
The modified SH2 super parent protein is crosslinked with a solid phase through sulfhydryl groups, and the crosslinking process is as follows:
1) BCA measured protein concentration;
2) By SulfoLink TM Preparing SH2 super parent Beads by coupling resin;
3) Firstly, cleaning an affinity chromatography column, balancing the pH value of the column, adding Beads suspension in a corresponding proportion into the column, and cleaning Beads for 3 times;
4) The Beads are combined with SH2 protein, rotated and combined for 15 minutes at 4 ℃, and kept static on ice for 30 minutes;
5) Through an affinity chromatographic column, collecting the Beads, and Blocking unbound cysteine binding sites on the Beads by using a Blocking buffer containing cysteine;
6) Washing SH2 super parent Beads 3 times by using 20ml washing buffer;
7) The Beads were transferred to a 15ml centrifuge tube using 1×IAP, sodium azide was added at a final concentration of 0.2%, and stored at 4 ℃.
The thiol-linked SH 2-beams were enriched for pY peptide fragments using the method of the comparative example, and the mass spectrum results were compared and are shown in Table 1 and FIG. 2.
TABLE 1 results of mass spectra of SH2-Beads enriched pY peptide fragments using different crosslinking modes
From the results in the table, it is shown that using the same materials, using sulfhydryl cross-linked resins for the Beads, and amino cross-linked Beads, for the enrichment of pY peptide fragments, there is a significant difference in the number of pY peptide fragments and the number of sites identified by mass spectrometry, and the SH2-Beads enrichment efficiency of sulfhydryl cross-linking is nearly doubled.
After analytical studies, we hypothesize that amino groups, although capable of immobilizing SH2 proteins on the coupled resin Beads, greatly reduce the conformational flexibility of SH2 proteins once the N-terminal or primary amino group-containing amino acid side chains of proteins are attached to Beads, which is detrimental to binding tyrosine phosphorylated peptides.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.
Claims (10)
- A conjugate formed by conjugation of cysteine at the C-terminal end of SH2 super-parent protein with a solid phase through disulfide bonds, wherein the amino acid sequence of the SH2 super-parent protein is as shown in SEQ ID NO: 1.
- 2. The conjugate of claim 1, wherein the SH2 super parent protein has an N-terminal linked tag protein.
- 3. The conjugate of claim 2, wherein the tag protein comprises one or more of an Arg tag, his tag, strep tag, flag tag, T7 tag, V5-peptide tag, GST tag, and c-Myc tag.
- 4. A conjugate according to any one of claims 1 to 3, wherein the solid phase comprises any one of glass, agar, agarose derivatives, magnetic beads, silica, titania, alginate, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya, locust bean gum, xanthan gum, pectin, mucin, liver thioester and gelatin, silicon, ceramic, polyurethane, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone, or a copolymer or mixture of any of the above.
- 5. The conjugate of claim 4, wherein the solid phase is an SPE solid phase extraction column, a centrifuge tube with a separation membrane, an EP tube, a separation membrane, a microplate well, a microsphere, a column, a pellet, or a multiwell plate.
- 6. A kit comprising the conjugate of any one of claims 1 to 5.
- 7. A method of enriching tyrosine phosphorylated peptide fragments comprising:a) Incubating the peptide fragment mixture sample with the conjugate of any one of claims 1 to 5 to bind tyrosine phosphorylated peptide fragments therein to a solid phase;b) Washing the solid phase and separating the tyrosine phosphorylated peptide fragment from the solid phase.
- 8. The use of a conjugate according to any one of claims 1 to 5 for the preparation of a kit for testing tyrosine phosphorylated peptide fragments,wherein the conjugate is used for enriching tyrosine phosphorylated peptide fragments, and the kit further comprises a reagent for identifying the separated tyrosine phosphorylated peptide fragments.
- 9. The use of claim 8, wherein said kit further comprises reagents for quantifying said isolated tyrosine phosphorylated peptide.
- 10. The use of claim 8 or 9, the method of identifying or the quantifying comprising mass spectrometry techniques.
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| CN108693348A (en) * | 2017-04-11 | 2018-10-23 | 中国科学院大连化学物理研究所 | A kind of tyrosine-phosphorylated protein quantitative analysis method |
| CN109564215A (en) * | 2016-06-10 | 2019-04-02 | 李顺成 | Method for being summarized with variant SH2 structural domain protein tyrosine phosphorylation |
| WO2019241308A1 (en) * | 2018-06-11 | 2019-12-19 | The Regents Of The University Of Colorado, A Body Corporate | Effector protein identification by sh2 domain affinity chromatography coupled mass spectrometry |
| CN114524871A (en) * | 2022-01-28 | 2022-05-24 | 青岛赛诺基因科技有限公司 | Variant SH2 domains with high affinity for tyrosine-containing sulfated-modified peptides |
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| CN108414606A (en) * | 2017-02-10 | 2018-08-17 | 中国科学院大连化学物理研究所 | The tyrosine-phosphorylated protein group analysis method of tyrosine phosphatase peptide |
| CN108693348A (en) * | 2017-04-11 | 2018-10-23 | 中国科学院大连化学物理研究所 | A kind of tyrosine-phosphorylated protein quantitative analysis method |
| WO2019241308A1 (en) * | 2018-06-11 | 2019-12-19 | The Regents Of The University Of Colorado, A Body Corporate | Effector protein identification by sh2 domain affinity chromatography coupled mass spectrometry |
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