Mass spectrum flow element tag based on polypeptide skeleton and preparation method and application thereof

Abstract

The invention discloses a mass spectrum flow element tag based on a polypeptide skeleton as well as a preparation method and application of the mass spectrum flow element tag. The polypeptide biological macromolecular chain is prepared from amino acid in a biological expression mode. Then a polypeptide chain serves as a skeleton structure, active groups (sulfydryl, amino, carboxyl and the like) on amino acid are efficiently coupled with a metal chelate, and the polypeptide skeleton element labeling probe with high sensitivity, high water solubility and biocompatibility is synthesized. The element tag of the polypeptide skeleton prepared by the invention is low in cost, and can meet the requirements of different complex biological analysis application scenes when being used as a matched reagent and combined with a mass spectrometry flow cytometry.

Description

Technical Field

[0001] The present invention belongs to the technical field of mass cytometry analysis, and particularly relates to a mass cytometry element tag based on a polypeptide backbone, a preparation method thereof, and an application thereof. Background Art

[0002] Single-cell mass cytometry (Mass Cytometry, CyTOF) is a high-throughput single-cell analysis technology that combines flow cytometry and mass spectrometry analysis, and is prospectively regarded by Nature as one of the 8 most anticipated biotechnologies. This technology overcomes the defect of fluorescence overlap in flow cytometry. The detection channels can theoretically reach up to hundreds of channels, and there is no interference between them, greatly improving the upper limit of multi-channel detection. It is widely used in immunology, oncology, and clinical research.

[0003] The core of mass cytometry is the labeling technology of metal elements. In 2001, Professor Xinrong Zhang first proposed an immunoassay strategy using rare earth metal (europium, Eu) stable isotopes as tags for the detection of thyroid-stimulating hormone (TSH) in blood. Subsequently, in 2002, an immunoassay strategy based on gold nanoparticle tags was proposed. The idea of metal stable isotope labeling was first proposed by Professor Xinrong Zhang. Foreign scholars took the lead in developing a mass cytometry instrument based on the metal stable isotope labeling strategy. Now, mass cytometry has become a revolutionary technology and has achieved great success in biomolecule detection. In theory, more than a hundred isotopes with mass numbers ranging from 75 to 209 can be detected. Currently, the commercial element tags used in mass cytometry are high-polymer element tags based on bifunctional chelating agents, which are exclusively produced by Fluidigm Corporation in the United States and are expensive. In addition, since the water solubility of the polymer decreases sharply with the increase of the chain length, only about 100 metal ions can be conjugated to each polymer tag, resulting in limited sensitivity and inability to detect low-abundance cell markers.

[0004] In order to improve the sensitivity of element tags, metal nanoparticles (silver nanoparticles, tantalum oxide nanoparticles, metal-organic frameworks, etc.) have been developed as element tags for mass cytometry. Nanoparticles can contain tens of thousands of metal ions, greatly increasing the metal ion loading capacity. Therefore, nanoparticles have a high signal response when used as metal element tags. However, with the development of time, the problems of nanoparticles themselves have gradually emerged. For example: (1) Non-specific adsorption and endocytosis phenomena result in a high background signal; (2) The stability of nanomaterials in phosphorus-containing biological buffers is poor; (3) The types of available metal nanoparticles are limited. Therefore, the popularization and application of such tags are restricted. In summary, developing mass cytometry element tags with higher sensitivity to meet the needs of different scenarios such as cell biology, clinical medicine, and immunology has great scientific and commercial value.

[0005] Polypeptide molecules are biological macromolecular chains constructed by amino acids through biological expression or solid-phase synthesis reactions, with excellent water solubility and biocompatibility. There are more than 20 kinds of natural amino acid molecules, which have different functional groups, different polarities, and hydrophilic and hydrophobic properties. For example, there is a naked amino group at the end of lysine, a carboxyl group in aspartic acid, and a stable mercapto group in cysteine. These functional groups enable polypeptide molecules to efficiently conjugate metal chelates and antibodies simultaneously. In addition, amino acids can be divided into hydrophilic amino acids and hydrophobic amino acids, polar amino acids and non-polar amino acids. Therefore, by regulating the type, quantity, and position of amino acids, the metal atom loading rate can be increased. However, the solid-phase synthesis method generally has difficulty in synthesizing long-chain polypeptides with repeating units, especially polypeptides with repeating structures containing some special functional functional groups, such as polylysine and polycysteine. Therefore, this limits its ability to load metal ions. In addition, even if a large amount of raw materials are used for synthesis, the cost is extremely high, which cannot meet the analysis and application requirements of mass cytometry. Therefore, using the Escherichia coli expression system to synthesize functional long-chain polypeptide skeletons has important value for constructing high-performance mass cytometry tags. Summary of the Invention

[0006] Aiming at the problems existing in the existing mass cytometry element tags, the purpose of the present invention is to provide a polypeptide molecular backbone element probe synthesized by a bacterial expression system and its preparation method.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a preparation method of a polypeptide molecular backbone element probe.

[0008] The preparation method of the polypeptide molecular backbone element probe provided by the present invention includes the following steps: combining a macrocyclic metal ion complex with a polypeptide rich in lysine and / or aspartic acid and / or cysteine through a chemical coupling reaction to obtain the polypeptide molecular backbone element tag probe; The macrocyclic metal ion complex is a complex of a macrocyclic chelating agent and a metal ion; Alternatively, after combining a macrocyclic chelating agent with a polypeptide rich in lysine and / or aspartic acid and / or cysteine through a chemical coupling reaction, it is then complexed with a metal ion to obtain the polypeptide molecular backbone element tag probe.

[0009] In the above method, the polypeptide contains at least two active groups R and R', and the R and R' groups are different active groups.

[0010] The lysine-rich polypeptide has a large number of R groups: -NH2. The -NH2 is used to bind to a macrocyclic metal ion complex or a macrocyclic chelating agent, and the R' group -COOH at the C-terminus is used to bind to an antibody through orthogonal coupling. The aspartic acid-rich polypeptide has a large number of R groups: -COOH. The -COOH is used to bind to a macrocyclic metal ion complex or a macrocyclic chelating agent, and the R' group -NH2 at the N-terminus is used to bind to an antibody through orthogonal coupling. The cysteine-rich polypeptide has a large number of R groups: -SH. The -SH is used to bind to a macrocyclic metal ion complex or a macrocyclic chelating agent, and the R' group, namely -NH2 at the N-terminus or -COOH at the C-terminus, is used to bind to an antibody through orthogonal coupling.

[0011] In the above method, the number of amino acids forming the lysine- and / or aspartic acid- and / or cysteine-rich polypeptide is not less than 30, and further, the number of amino acids is 30 - 70.

[0012] In the above method, the number of lysine and / or aspartic acid and / or cysteine in the lysine- and / or aspartic acid- and / or cysteine-rich polypeptide accounts for not less than 50% of the number of amino acids forming the polypeptide chain.

[0013] In the above method, the lysine- and / or aspartic acid- and / or cysteine-rich polypeptide can be obtained by a biosynthetic method.

[0014] Further, the biosynthetic method can be a bacterial prokaryotic expression method.

[0015] In an embodiment of the present invention, the polypeptide is obtained by expression through an Escherichia coli system.

[0016] In the above method, the lysine- and / or aspartic acid- and / or cysteine-rich polypeptide is a linear polypeptide.

[0017] According to an embodiment of the present invention, for the lysine-rich polypeptide, its amino acid sequence (from the N-terminus to the C-terminus) is: CKKGKKKGKKKAKAKSKKKKKGKQKKKGKSKRKLKKGKKAKKVKKGKSKKKSGGG (SEQ ID No.1).

[0018] In the above method, the lysine-rich polypeptide needs to be prepared through a biological expression reaction.

[0019] In an embodiment of the present invention, the lysine-rich polypeptide (SEQ ID No.1) is obtained by introducing a prokaryotic expression plasmid carrying the DNA sequence shown in SEQ ID No.2 into Escherichia coli and performing expression.

[0020] In the above method, the macrocyclic metal ion complex can be prepared by various conventional methods well-known in the art.

[0021] Further, the macrocyclic chelating agent can be DOTA or its derivatives or DTPA or its derivatives.

[0022] Still further, the DOTA derivatives can be DOTA-NCS, DOTA-NHS, DOTA-MAL or DOTA-NH2 or DOTA-COOH; Still further, the DTPA derivatives can be DTPA-NCS, DTPA-NHS, DTPA-MAL or DTPA-NH2 or DOTA-COOH.

[0023] Further, the metal ion can be a rare earth metal ion, and specifically can be selected from the ions of at least one of the following rare earth metals: La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu and Yb.

[0024] Further, the chemical coupling reaction can be the reaction of -NH2 and -NHS, the reaction of -NH2 and -NCS, the EDC-NHS reaction of -NH2 and -COOH or the reaction of -SH2 and -MAL (maleimide group).

[0025] In the above method, the molar ratio of the macrocyclic metal ion complex to lysine and / or aspartic acid and / or cysteine in the polypeptide is 10:1; In the above method, the molar ratios of the macrocyclic chelating agent, lysine and / or aspartic acid and / or cysteine in the polypeptide, and the metal ion are 10:1:100 in sequence.

[0026] After synthesizing the polypeptide rich in lysine or aspartic acid or cysteine, the present invention uses a macrocyclic chelating agent containing functional groups such as -NH2, -COOH, -NCS or maleimide to act with rare earth metal ions to obtain a macrocyclic metal ion complex. Through common covalent coupling reactions, such as: the reaction of -NH2 and -NHS, the reaction of -NH2 and -NCS, the EDC-NHS reaction of -NH2 and -COOH or the reaction of -SH2 and -MAL (maleimide group), the metal ion complex reacts with the active group R group in the polypeptide to obtain a polypeptide-metal ion complex.

[0027] After the synthesis of the polypeptide molecular backbone element probe, the present invention covalently couples another active group R' contained in the polypeptide with an antibody (e.g., through the bifunctional coupling agent NHS-PEG-MAL: the amino group of the polypeptide binds to the -NHS active group of the coupling agent, and the -SH group of the antibody binds to the -MAL active group of the coupling agent), and then applies the antibody-polypeptide complex to antigen-antibody immunoassay. The signal intensities of the antigen and metal elements are obtained by mass cytometry, and then applied to actual serum or biological samples.

[0028] In a second aspect, the present invention provides a polypeptide molecular backbone element probe prepared by the method described in the first aspect.

[0029] In a third aspect, the present invention provides an antibody-polypeptide complex obtained by covalently coupling the polypeptide molecular backbone element probe with an antibody.

[0030] The antibody-polypeptide complex is obtained by covalently coupling the active group R' in the polypeptide molecular backbone element probe with an antibody.

[0031] Further, the antibody can be an antibody against cell surface CD proteins (such as CD3, CD4, CD8, CD45, CD19, etc.).

[0032] In a fourth aspect, the present invention provides the application of the above-mentioned polypeptide molecular backbone element probe in the preparation of element tags for mass cytometry.

[0033] In a fifth aspect, the present invention provides the application of the above-mentioned polypeptide molecular backbone element probe and antibody-polypeptide complex in at least one of the following aspects: cell biology, clinical medicine, immunology.

[0034] Further, the immunology can be the immunoassay of biological macromolecules; Furthermore, the detection method is mass cytometry.

[0035] Furthermore, the biological reaction between the polypeptide molecular backbone element probe and the biological macromolecule to be detected is an antigen-antibody immune reaction.

[0036] Compared with the prior art, the present invention has the following beneficial effects: (1) The polypeptide molecule is a biological macromolecule chain constructed by amino acids through the Escherichia coli protein expression system, with excellent water solubility and biocompatibility.

[0037] (2) Compared with solid-phase synthesis, the Escherichia coli expression system has low synthesis cost and high yield, and is very suitable for the synthesis of polypeptide backbones with long chains and a large number of repeating functional groups.

[0038] (3) The positions and quantities of lysine, aspartic acid, or cysteine containing reactive functional groups in the polypeptide molecule can be controlled, thereby improving the labeling efficiency and detection accuracy of the probe.

[0039] (4) The types of amino acids in the polypeptide backbone, especially some non-polar amino acids, can be adjusted by designing plasmids to reduce the background adsorption of metal tags. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the principle for synthesizing polylysine peptide chains by biological expression methods. Among them, (A) is a schematic diagram of the key elements of the recombinant expression vector in Example 1. (B) is a flow chart for prokaryotic expression of polylysine peptide chains using Escherichia coli.

[0041] Figure 2 It is a gel electrophoresis diagram of polylysine peptide chains synthesized by biological expression methods. Among them, lane A (red frame) is polylysine peptide chains with various tags such as MBP fusion tag, Twin-Strep II tag, and 6×His tag, with a molecular weight of 46.3 kDa. Lane B (blue frame) is the tag strip (6×His tag, Twin-Strep II tag, and MBP fusion tag located at the N-terminal of the TEV cleavage site), with a molecular weight of 40.2 kDa.

[0042] Figure 3 It is a schematic diagram of the reaction principle for labeling polylysine peptide chains with metal complexes and MAL end groups.

[0043] Figure 4 It is a mass cytometry result diagram of polypeptide backbone element tags. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0045] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0046] Example 1: Synthesis of polylysine peptide chains by biological expression methods In order to more accurately control the degree of polymerization of polypeptides, synthetic biology methods are used, using E. coli as a medium and polypeptide production factory, to effectively synthesize polypeptides of any sequence and length. By regulating the number and position of lysine, polylysine peptide chains can be synthesized, which has low production costs and is easy to industrialize. Take the synthesis of a polypeptide containing 34 lysines and a total length of 55 amino acids as an example: The deoxynucleotide coding sequence of the polylysine peptide chain to be expressed was inserted between the restriction sites BamHI and XhoI of the pET28-MBP-TEV plasmid (Addgene, #69929) (the amino acid sequence of the polylysine peptide chain and the corresponding deoxynucleotide coding sequence are shown in Table 1) to construct a recombinant expression vector ( Figure 1 (A)). The polylysine peptide chain expressed using the recombinant expression vector has a cysteine residue at its N-terminus and is linked to the vector's native MBP protein, a Twin-Strep II tag, and a 6×His tag. The MBP protein is used to increase the water solubility of the E. coli protein product, while the Twin-Strep II tag and 6×His tag are used to obtain a high-purity polylysine peptide tag. A TEV cleavage site is located between the MBP tag and the target polypeptide for removal of the MBP protein.

[0047] The constructed plasmid was transformed into Escherichia coli for culture and induction of protein expression ( Figure 1 (B) Then the bacteria were broken, the protein was extracted, and the target protein was purified for the first time using the Twin-Strep II tag. The Strep-Tag II tag purification products are: polylysine peptide chains with various tags such as MBP fusion tag, Twin-Strep II tag, 6×His tag, etc. ( Figure 2 Middle lane A). TEV enzyme was used to cut the MBP protein and the polypeptide-6×His peptide at the TEV cleavage site ( Figure 2 Lane B (center) shows the cleaved N-terminal 6×His tag, Twin-Strep II tag, and MBP fusion tag. The peptide-6×His tag is then extracted from the solution using a Ni column. Dialysis is performed against a 3 kDa column (18 mm diameter) to remove the imidazole present during Ni elution. The target peptide is then freeze-dried to yield the desired peptide (the 6×His tag does not affect the use of the peptide backbone). Approximately 10 mg of peptide backbone is extracted from 1 L of bacterial culture.

[0048] Table 1. Amino acid sequence of polylysine polypeptide

[0049] Example 2: Polylysine peptide chain labeling metal complexes and modification of MAL end groups (1)Polylysine polypeptide-labeled metal complex: Dissolve 100 mg of polylysine (prepared in Example 1) in N,N-dimethylformamide solution, then add 200 mg of DOTA-NCS macrocycle and react overnight at 45 °C. The reaction product is evaporated to remove N,N-dimethylformamide by vacuum rotary evaporation. Redissolve in water and use dialysis (5K Mw c.o., diameter 18 mm) to remove the excess DOTA-NCS macrocycle. The number of macrocycle labels is characterized by 1H NMR to be 34, and 150 mg is obtained by weighing.

[0050] (2)Dissolve the above polypeptide (50 mg) in a suitable solvent, usually deionized water or PBS (pH 7.4) buffer. Add EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to the reaction system. The dosage of EDC is 1-3 times the molar ratio of the polypeptide, and the dosage of NHS is 1 times the molar ratio of EDC. Dissolve EDC and NHS in deionized water or PBS solution to ensure complete dissolution. Add these reagents at room temperature and stir well. Add the EDC and NHS solutions to the polypeptide solution, and the reaction temperature is room temperature. Stir and let the reaction proceed for 1 hour to activate the carboxyl group in the polypeptide. Add a bifunctional coupling agent: NH2-PEG-MAL (MadChemExpress, Cat. No.: HY-35261) to the reaction mixture. The addition amount of NH2-PEG-MAL is 2-3 times the molar ratio of the polypeptide. Continue to stir the reaction mixture at room temperature for 2-4 hours to ensure the completion of the coupling reaction. The coupling reaction occurs through the amidation reaction of the amino group with the activated carboxyl group. Remove the unreacted chemical reagents and by-products by dialysis to obtain the purified coupling product: 40 mg.

[0051] Example 3: Polylysine metal polypeptide-labeled antibody Take 100 μg of polylysine polypeptide (prepared in Example 2), dissolve it in 100 μL of 500 mM acetate buffer, add 10 μL of 50 mM rare earth metal ion (175 Lu), react at 37 °C for 30 min, then ultrafilter 3 times using a 3 k ultrafiltration tube and wash with PBS buffer to remove excess metal ions. The obtained polylysine metal polypeptide is stored at 4 °C for later use. Take 100 μg of antibody (CD-45 antibody) in 100 μL of PBS buffer, add 4 mM tris(2-carboxyethyl)phosphine (TCEP), and react at 37 °C for 30 min. After the reaction, ultrafilter 3 times using a 3 k ultrafiltration tube to remove excess TCEP, then add 100 μg of the above-prepared polylysine metal polypeptide and react at room temperature for 2 h. Wash 3 times using a 50 k ultrafiltration tube, and store the obtained polylysine polypeptide backbone antibody element label at 4 °C for later use.

[0052] Example 4: Mass cytometry detection of polylysine polypeptide backbone antibody element label Take 3×10 6 MCF-7 cells or Jurkat T cells, wash them twice with DPBS and resuspend them in 1 mL of DPBS. Add cisplatin to a final concentration of 0.5 uM, incubate at room temperature for 2 min, then add cell staining buffer and wash twice to remove the supernatant. Then resuspend the cells in 50 μL of cell staining buffer, add 5 μL of TrueStain FcX, incubate at room temperature for 15 min, add 1 μg of the polypeptide backbone element label labeled with CD-45 antibody (i.e., the polylysine metal polypeptide-labeled antibody prepared in Example 3), and continue to incubate for 30 min. Subsequently, wash the cells twice with cell staining buffer. Then, stain the cell nuclei with an Ir-DNA intercalator for 1 h to obtain nucleus-labeled cells. Finally, wash the cells twice with cell staining buffer and cell loading buffer respectively, and perform detection and analysis using a mass cytometry (MSFLO mass cytometry). Before cell analysis, use 169Tm and 169Tb to debug the sensitivity and stability of the instrument to make its sensitivity reach more than 150000 CPS and RSD < 3%; reduce the oxide ratio 155LaO / 139La to less than 5%. After debugging, detect the prepared cell sample in single particle mode. The cell sample is introduced into a digital pump controlled by the instrument software, and the injection flow rate is 30 μL / min, and then the metal element signals on the cells are collected.

[0053] The detection results are as Figure 4 shown. By Figure 4It can be seen that this label can perfectly distinguish Jurkat T cells containing the CD45 protein marker (red cell population) from MCF-7 cells without CD45 (blue cell population).

[0054] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses or improvements to the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A method for preparing a polypeptide molecular backbone element probe, comprising the following steps: combining a macrocyclic metal ion complex with a polypeptide rich in lysine and / or aspartic acid and / or cysteine through a chemical coupling reaction to obtain the polypeptide molecular backbone element tag probe; the macrocyclic metal ion complex is a complex of a macrocyclic chelating agent and a metal ion; Alternatively, after combining the macrocyclic chelating agent with the polypeptide rich in lysine and / or aspartic acid and / or cysteine through a chemical coupling reaction, it is then complexed with a metal ion to obtain the polypeptide molecular backbone element tag probe.

2. The preparation method according to claim 1, wherein: The polypeptide contains at least two reactive groups R and R', and the R and R' groups are different reactive groups; The polypeptide rich in lysine has a large number of R groups: -NH2. The -NH2 is used to bind the macrocyclic metal ion complex or the macrocyclic chelating agent, and the R' group -COOH at the C-terminus is used to bind the antibody through orthogonal coupling; The polypeptide rich in aspartic acid has a large number of R groups: -COOH. The -COOH is used to bind the macrocyclic metal ion complex or the macrocyclic chelating agent, and the R' group -NH2 at the N-terminus is used to bind the antibody through orthogonal coupling; The polypeptide rich in cysteine has a large number of R groups: -SH. The -SH is used to bind the macrocyclic metal ion complex or the macrocyclic chelating agent, and the R' group, i.e., -NH2 at the N-terminus or -COOH at the C-terminus, is used to bind the antibody through orthogonal coupling.

3. The preparation method according to claim 1 or 2, characterized in that: The number of amino acids forming the polypeptide rich in lysine and / or aspartic acid and / or cysteine is at least 30, and further is 30 - 70.

4. The preparation method according to any one of claims 1-3, characterized in that: In the polypeptide rich in lysine and / or aspartic acid and / or cysteine, the number of lysine and / or aspartic acid and / or cysteine accounts for at least 50% of the number of amino acids forming the polypeptide chain.

5. The preparation method according to any one of claims 1-4, characterized in that: The macrocyclic chelating agent is DOTA or its derivative or DTPA or its derivative; And / or, the metal ion is a rare earth metal ion, specifically selected from the ions of at least one of the following rare earth metals: La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu, and Yb; And / or, the chemical coupling reaction is selected from at least one of the following: -NH2 and -NHS reaction, -NH2 and -NCS reaction, -NH2 and -COOH EDC-NHS reaction, -SH2 and maleimide group reaction.

6. The preparation method according to any one of claims 1-5, characterized in that: The polypeptide rich in lysine and / or aspartic acid and / or cysteine is obtained through a biological expression method.

7. The polypeptide molecular backbone element probe prepared by the method according to any one of claims 1 - 6.

8. An antibody-polypeptide complex, which is obtained by covalently coupling the polypeptide molecular backbone element probe according to claim 7 with an antibody.

9. Use of the polypeptide molecular backbone element probe according to claim 7 in the preparation of an element tag for mass cytometry.

10. Use of the polypeptide molecular backbone element probe according to claim 7 or the antibody-polypeptide complex according to claim 8 in at least one of the following aspects: cell biology, clinical medicine, immunology.

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