Metal ion chelating structure applicable to mass spectrometric flow cytometry and preparation method and application thereof

Abstract

The application discloses a metal ion chelating structure applicable to mass spectrometry flow detection, and discloses a preparation method and application of the metal ion chelating structure. The metal ion chelating structure can efficiently chelate metal ions such as lanthanide metal ions and transition metal ions, and can directly connect the metal ions such as the lanthanide metal ions and the transition metal ions to antibodies, has high application performance, and meets the application requirement of mass spectrometry flow detection. The metal ion chelating structure has wide applicability, can improve the number of metal markers of mass spectrometry flow detection, and expands more detection channels. The preparation method of the metal ion chelating structure is simple in process, easy to synthesize, and low in cost.

Description

Technical Field

[0001] This invention relates to the field of metal ion chelation structure technology, specifically to metal ion chelation structures applicable to mass spectrometry flow cytometry detection, their preparation methods, and applications. Background Technology

[0002] Standard Biotools' commercial kits, the Maxpar X8 Antibody Labeling Kit and Maxpar MCP9 Antibody Labeling Kit, use 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA) and divinyltriaminepentaacetic acid (DTPA) as metal ion chelating units. These DOTA / DTPA structures require the addition of polyacrylic acid macromolecules to be used in mass spectrometry flow cytometry to label metal elements onto flow cytometry antibodies. The synthesis method for obtaining the final product involves multiple steps of grafting metal ion chelating units onto polyacrylic acid macromolecules, which is complex, costly, and generally has limited chelating effect on lanthanide metal ions, and is difficult to effectively chelate transition metal ions.

[0003] Invention CN116041257A discloses a new structure that can chelate metal ions, but this structure cannot be directly linked to flow cytometry antibodies. When linking lanthanide metal ions or transition metal ions to flow cytometry antibodies, it is still necessary to indirectly load the metal ions onto the flow cytometry antibodies by linking them to polyacrylic acid macromolecules provided by Standard BioTools as a carrier backbone.

[0004] This invention aims to develop a metal ion chelating structure that can chelate metal ions, including lanthanide metal ions and transition metal ions, and can be directly linked to flow cytometry antibodies for mass spectrometry flow cytometry detection. Summary of the Invention

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A metal ion chelating structure applicable to mass spectrometry flow cytometry detection has the following chemical structure:

[0007]

[0008] The above-mentioned method for preparing metal ion chelate structures applicable to mass spectrometry flow cytometry detection includes the following steps:

[0009] In the presence of a condensing reagent and a basic reagent, ethylenediaminetetraacetic acid and N-(4-aminophenyl)maleimide were subjected to a condensation reaction in a solvent, followed by post-treatment to obtain the metal ion chelate structure.

[0010] Furthermore, the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0011] Furthermore, the alkaline reagent is N,N-diisopropylethylamine.

[0012] Furthermore, the solvent is N,N-dimethylformamide.

[0013] Furthermore, the condensation reaction is carried out at a temperature of room temperature to 75°C for a reaction time of 12-18 hours.

[0014] Furthermore, the post-processing includes the following steps:

[0015] After the condensation reaction was completed, water was added for extraction, the organic phase was retained, the solvent was dried by rotary evaporation, and then vacuum dried to obtain the metal ion chelate structure.

[0016] The above-mentioned metal ion chelating structures, which can be applied to mass spectrometry flow cytometry detection, are used in the detection of metal antibody labels based on mass spectrometry flow cytometry technology.

[0017] Furthermore, the detection of target protein expression levels was performed using mass spectrometry flow cytometry.

[0018] Furthermore, in application, the mass number of the metal ions chelated by the metal ion chelating structure is distributed in the range of 75-209, including lanthanide metals, noble metals and other rare earth metals or transition metals.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] This invention relates to a metal ion chelating structure that uses ethylenediaminetetraacetic acid (EDTA), a metal ion chelating agent with stronger chelating ability and a wider range of chelating metal ions, as a gripper group. Through chemical coupling, it connects to N-(4-aminophenyl)maleimide, which can link free thiol groups of biomolecules. On one hand, EDTA possesses a polycarboxylic acid group, enabling efficient chelation of lanthanide and transition metal ions. On the other hand, this invention's metal ion chelating structure can directly link lanthanide and transition metal ions to antibodies, exhibiting high efficiency and meeting the requirements of mass spectrometry flow cytometry detection applications. This invention's metal ion chelating structure has broad applicability, increasing the number of metal markers detectable by mass spectrometry flow cytometry and expanding detection channels.

[0021] The method for preparing the metal ion chelate structure of the present invention is simple, easy to synthesize, and inexpensive. Attached Figure Description

[0022] Figure 1The hydrogen NMR spectrum of the metal ion chelate structure prepared for the example.

[0023] Figure 2 To label the noble metal platinum using the Standard BioTools Maxpar X8 Antibody Labeling Kit commercial kit and the metal ion chelation structure described in this invention, respectively. 198 Comparative data on the CD8 expression levels of the same peripheral blood immune cell sample after Pt ions were introduced into CD8 monoclonal flow cytometry antibody.

[0024] Figure 3 To label lanthanide metals using the Standard BioTools Maxpar X8 Antibody Labeling Kit (a commercial kit) and the metal ion chelation structure described in this invention, respectively. 141 Pr) ions to CD56 monoclonal flow cytometry antibodies, and labeled lanthanide metals ( 175 Comparative data on the expression levels of CD16 and CD56 in the same peripheral blood immune cell sample after Lu ions were introduced into the CD16 monoclonal flow cytometry antibody. Detailed Implementation

[0025] The technical solution of the present invention will now be clearly and completely described with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention and do not represent all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the protection scope of the present invention.

[0026] Unless otherwise specified, all chemical reagents mentioned in the embodiments of this invention are purchased from the market.

[0027] Example 1

[0028] A method for preparing metal ion chelate structures applicable to mass spectrometry flow cytometry detection includes the following steps:

[0029] Using 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) as the condensing agent and N,N-diisopropylethylamine (DIPEA) as the basic reagent, ethylenediaminetetraacetic acid (EDTA) and N-(4-aminophenyl)maleimide were condensed in N,N-dimethylformamide (DMF). The reaction route is as follows:

[0030]

[0031] Specifically, the following steps are included:

[0032] Weigh 290 mg EDTA and 570 mg HATU into a 50 mL single-necked flask, add 8 mL DMF, and then add 260 mg DIPEA. Stir the reaction system at room temperature (350 r / min) for 30 min to activate it.

[0033] 188 mg of N-(4-aminophenyl)maleimide was weighed and added directly to the reaction system, and the mixture was stirred at room temperature for 12 h.

[0034] After the reaction was complete, 8 mL of deionized water was added for extraction. The organic phase was retained and the solvent was dried in a rotary evaporator at 60 °C and 50 Pa. Then, it was transferred to a vacuum oven and dried at room temperature for 24 h to obtain 388 mg of the metal ion chelate structure, with a yield of 84% (yield = actual yield / theoretical yield × 100%). The chemical structure of the metal ion chelate structure is shown below:

[0035]

[0036] Its 1H NMR spectrum is as follows Figure 1 As shown, the characteristic peaks of the hydrogen on the methylene group in the EDTA group are between 2.0 and 4.0 ppm, the characteristic peaks of the four H on the benzene ring are between 7.2 and 7.8 ppm, and the single peak with a chemical shift of around 7.9 is the characteristic peak of the two H on maleimide.

[0037] Example 2

[0038] Preparation of chelated noble metal platinum using metal ion chelation structure ( 198 Metal antibodies against Pt ions include the following steps:

[0039] 1. Antibody-linked metal ion chelate structure:

[0040] ① Take 100 μg of CD8 monoclonal flow cytometry antibody to be labeled and add it to a pre-labeled 50 kDa ultrafiltration tube. Centrifuge at 12000g for 20 min, rinse, add 100 μL of PBS buffer (10 mM pH 7.4) containing 4 mM TCEP reducing agent, and incubate at 37°C for 30 min. After incubation, add 400 μL of PBS buffer (10 mM pH 7.4, the same below) and centrifuge at 12000g for 20 min, rinse, and repeat rinsing 3 times.

[0041] ② Add 10 μL of the 1 mM metal ion chelate structure prepared in Example 1 (dissolved in DMF), and add PBS buffer to make up to a 50 μL system. Incubate at 37°C for 90 min. After incubation, add 400 μL of PBS buffer and centrifuge at 12000g for 20 min to rinse. Rinse 3 times.

[0042] 2. Antibody chelation of noble metal platinum (through a metal ion chelation structure) 198 Pt ions:

[0043] ① Add 50 μL of PBS buffer to the antibody linking the metal ion chelate structure in the 50 kDa ultrafiltration tube, then add 13 μL of 50 mM platinum (… 198 Pt ion solution (platinum ions provided by platinum dichloride) was repeatedly blown and aspirated to mix thoroughly.

[0044] ②Incubate in a 37℃ water bath for 60 min. After incubation, add 400 uL of PBS buffer and centrifuge at 12000g for 20 min, then rinse at least 5 times.

[0045] 3. Recovery of antibodies labeled with precious metal platinum ions:

[0046] Take a new sample tube and label it with the platinum-labeled antibody. Slowly add 100 μL of PBS buffer along the filter membrane to a 50 kDa ultrafiltration tube. Invert the 50 kDa ultrafiltration tube onto the labeled sample tube and centrifuge at 1000 g for 10 min to recover the platinum-labeled antibody (i.e., the antibody). 198 Pt is marked CD8).

[0047] Example 3

[0048] Preparation of chelated lanthanides using metal ion chelation structures ( 141 Pr、 175 Metal antibodies against Lu ions include the following steps:

[0049] 1. Antibody-linked metal ion chelate structure:

[0050] Using the same steps as in Example 2, the antibody to be labeled with metal is linked to the metal ion chelate structure. Specifically, it is proposed to... 141 Pr lanthanide metal ion-labeled CD56 antibody 175 Lu lanthanide metal ion-labeled CD16 antibody.

[0051] 2. Antibody chelation of lanthanide metal ions via metal ion chelation structures:

[0052] The same steps as in Example 2 were used, with the only adjustment: 5 μL of 50 mM lanthanide metal ( 141 Pr and 175 Lu) ion solution ( 141 Pr and 175 Lu (provided by the corresponding trichloride) is added to the antibody linking the metal ion chelating structure. Lanthanides ( 141 Pr) ions and lanthanide metals ( 175 Lu ions were tested separately.

[0053] 3. Recovery of lanthanide metal ion-labeled antibodies:

[0054] Using the same steps as in Example 2, the lanthanide metal ion-labeled antibody (i.e., 141 Pr marks CD56 and 175 Lu (marked CD16).

[0055] Example 4

[0056] Platinum metal antibodies labeled with the Standard BioTools Maxpar X8 Antibody Labeling Kit were used as a performance control group for the metal ion chelate structure-labeled platinum metal antibodies disclosed in this invention. The method is as follows:

[0057] 1. Polymer conjugates capable of chelating metal ions, chelating the noble metal platinum (… 198 Pt ions:

[0058] ① Add 87 μL of L buffer (provided in the commercial kit) to the PCR tube to resuspend the polymer (i.e., DOTA / DTPA structure combined with polyacrylic acid macromolecule, provided in the commercial kit), and pipette repeatedly to mix thoroughly.

[0059] ② Add 13 μL of 50 mM platinum to the PCR tube. 198 Pt ion solution (platinum ions provided by platinum dichloride) was repeatedly blown and aspirated to mix thoroughly.

[0060] ③Incubate in a 37℃ water bath for 30 minutes.

[0061] ④ Take a 3kDa ultrafiltration tube and its matching collection tube. Add 200uL of C buffer (provided in the commercial kit, the same below) to the 3kDa ultrafiltration tube. Then transfer the polymer that chelates platinum ions in the PCR tube to the 3kDa ultrafiltration tube. Centrifuge the 3kDa ultrafiltration tube at 12000g for 20min and discard the filtered waste liquid after centrifugation in the collection tube.

[0062] ⑤ Add 300 μL of C buffer to the 3 kDa ultrafiltration tube, centrifuge at 12000 g for 20 min, rinse at least 5 times to remove excess platinum ions that did not participate in chelation.

[0063] 2. Antibody replacement buffer and reduction:

[0064] Take 100 μg of CD8 monoclonal flow cytometry antibody to be labeled and add it to a pre-labeled 50 kDa ultrafiltration tube. Centrifuge at 12000g for 20 min, rinse, and add 100 μL of R buffer containing 4 mM TCEP reducing agent (R buffer is provided in the commercial kit). Incubate at 37°C for 30 min. After incubation, add 400 μL of C buffer, centrifuge at 12000g for 20 min, and rinse three times.

[0065] 3. Antibody-linked precious metal platinum ( 198 Pt ions:

[0066] The polymer chelating platinum ions from the 3kDa ultrafiltration tube in step 1 of this embodiment was added to the 50kDa ultrafiltration tube containing the reduced antibody in step 2 of this embodiment, and incubated in a water bath at 37°C for 90 min. After incubation, the tube was washed three times by centrifugation at 12000g for 20 min with 400 μL W buffer (provided in the commercial kit) to remove unreacted polymer chelating platinum ions.

[0067] 4. Recovery of antibodies labeled with precious metal platinum ions:

[0068] Take a new sample tube and label it with the platinum-labeled antibody. Slowly add 100 μL of PBS buffer along the filter membrane to the 50 kDa ultrafiltration tube. Invert the 50 kDa ultrafiltration tube into the sample tube and centrifuge at 1000 g for 10 min to recover the platinum-labeled antibody (i.e., the antibody). 198 Pt is marked CD8).

[0069] Example 5

[0070] Lanthanide metal antibodies labeled with the Standard BioTools Maxpar X8 Antibody Labeling Kit were used as a performance control group for labeling lanthanide metal antibodies with the metal ion chelation structure disclosed in this invention. The method is as follows:

[0071] 1. Polymer conjugates capable of chelating metal ions, chelating lanthanides ( 141 Pr、 175 Lu) metal ions:

[0072] The same steps as in Example 4 were used, with the only adjustment: 5 μL of 50 mM lanthanide metal was taken ( 141 Pr and 175 Lu) ion solution ( 141 Pr and 175Lu (provided by the corresponding trichloride) was added to the polymer resuspended in 95 μL of L buffer, and the incubation time in step ③ was 60 min.

[0073] 2. Antibody replacement buffer and reduction:

[0074] The same steps as in Example 4 are used. Specifically, it is proposed to... 141 Pr lanthanide metal ion-labeled CD56 antibody 175 Lu lanthanide metal ion-labeled CD16 antibody.

[0075] 3. Antibody-linked lanthanide metals ( 141 Pr and 175 Lu) ions:

[0076] The same steps as in Example 4 are used.

[0077] 4. Recovery of lanthanide metal ion-labeled antibodies:

[0078] Using the same steps as in Example 4, the lanthanide metal ion-labeled antibody (i.e., 141 Pr marks CD56, and 175 Lu (marked CD16).

[0079] Using mass cytometry, antibodies labeled with platinum ions (Example 4) and lanthanide ions (Example 5) from the Standard BioTools Maxpar X8 Antibody Labeling Kit were compared on the same peripheral blood immune cell sample. Additionally, antibodies labeled with platinum ions using the metal ion chelation structure described in this invention (Example 2) and lanthanide ions (Example 3) were compared. Figure 2 As can be seen, the metal ion chelate structure prepared in Example 1 can effectively chelate platinum ions and label them onto CD8 antibodies, enabling the recognition and labeling detection of immune cell surface antigens via mass cytometry. In contrast, the StandardBioTools Maxpar X8 Antibody Labeling Kit cannot label platinum ions onto CD8 antibodies. Meanwhile, as... Figure 3 As shown, the metal ion chelate structure prepared in Example 1, like the StandardBioTools Maxpar X8 Antibody Labeling Kit, can effectively label lanthanide metal ions onto corresponding antibodies. It can also identify and label immune cell surface antigens by mass spectrometry and flow cytometry, achieving comparable target protein detection results.

Claims

1. A compound that can be detected by mass spectrometry flow cytometry, characterized in that, Its chemical structure is as follows: 。 2. The method for preparing the compound applicable to mass spectrometry flow cytometry detection according to claim 1, characterized in that, Includes the following steps: In the presence of a condensing agent and a basic reagent, ethylenediaminetetraacetic acid and N-(4-aminophenyl)maleimide were subjected to a condensation reaction in a solvent, followed by post-treatment to obtain the compound.

3. The preparation method according to claim 2, characterized in that, The condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

4. The preparation method according to claim 2, characterized in that, The alkaline reagent is N,N-diisopropylethylamine.

5. The preparation method according to claim 2, characterized in that, The solvent is N,N-dimethylformamide.

6. The preparation method according to any one of claims 2-5, characterized in that, The condensation reaction was carried out at a temperature of room temperature to 75°C for 12-18 hours.

7. The preparation method according to any one of claims 2-5, characterized in that, The post-processing includes the following steps: After the condensation reaction was completed, water was added for extraction, the organic phase was retained, the solvent was dried by rotary evaporation, and then dried under vacuum to obtain the compound.

8. The application of the compound of claim 1, which can be applied to mass spectrometry flow cytometry detection, in the detection of metal antibody labeling based on mass spectrometry flow cytometry technology.

9. The application according to claim 8, characterized in that, Detection of target protein expression levels at the single-cell level using mass spectrometry flow cytometry.

10. The application according to claim 8 or 9, characterized in that, When applied, the mass number of the metal ions chelated by the compound is distributed in the range of 75-209, including lanthanides and Pt.

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