WO2021120451A1 - Flow cytometric analysis technique for organic mass spectrometry - Google Patents

Abstract

Provided is an organic mass spectrometry flow apparatus used for single-cell analysis and an analysis method thereof, the apparatus comprising: a peristaltic pump, a cell dispersion and alignment apparatus, an ionization needle, and a mass spectrum detector; the peristaltic pump, the cell dispersion and alignment apparatus, and the ionization needle are connected in sequence by means of a pipeline, and the outlet of the ionization needle is aligned with the inlet of the mass spectrum detector; during detection, a cell suspension sample is first configured and a cell target protein is labeled, and organic mass spectrum detection is performed on the cell sample using an organic mass spectrometry flow apparatus. A flow mass spectrometry probe applies a large number of organic labels to the target protein such that detection sensitivity reaches the single-cell level, and thus qualitative and quantitative analysis of multiple target proteins and a large number of metabolites at the single-cell level, obtaining a large amount of single-cell level information.

Description

A flow cytometric analysis technique for organic mass spectrometry Technical field

The invention relates to a flow analysis technique based on organic mass spectrometry with single cells as the analysis object, and in particular to a method for preparing a universal protein-labeled flow mass spectrometry probe and a cell labeling method, cell monodispersion and arrangement technology, and single cell Mass spectrometry detection ionization interface design, and single-cell level protein and metabolite detection and quantification methods based on mass spectrometry signals and their applications in single-cell identification and typing.

Background technique

Cells are the basic unit of life activities, and one of the main research contents of life analysis is to analyze the mechanism of biological regulation at the cell level. Single cell analysis refers to the monitoring of life activities at a single cell or even a smaller level. The main analysis object is the content and dynamic changes of important biological molecules in a single cell, such as DNA, RNA, protein, and small molecule metabolites. Due to the existence of cell heterogeneity, many traditional analysis methods take a large number of cells as the analysis object, and obtain only average results, and lose important information about cell heterogeneity. However, the study of cell heterogeneity has important biology The significance of life science, especially in the research fields of systems biology, tumor cells, stem cells, cell drug resistance, etc. Therefore, single-cell analysis methods provide important analysis methods for cell heterogeneity and many important biological issues. However, due to the characteristics of single cells: small size, low substance content, many kinds of substances, large differences in substance content, and wide dynamic range, it is truly a single-cell analysis and an analysis method to obtain as much information as possible from the genome to the metabolome at the single-cell level. It needs to be based on high sensitivity, while meeting the requirements of wide dynamic range, fast analysis speed, and high analysis throughput, which undoubtedly poses a huge challenge to the analysis method.

Cell flow analysis based on fluorescence detection is a representative high-throughput single cell analysis method. The target protein or nucleic acid in the cell is fluorescently labeled and the protein, nucleic acid and other targets in the single cell are obtained by fluorescence detection. Content information. Due to the overlap of the bandwidth of the fluorescent signal, the number of targets simultaneously detected by this type of flow cytometry technology is greatly limited, and it is impossible to detect unlabeled small molecule metabolite information. Flow cytometry analysis based on inorganic mass spectrometry takes full advantage of the high resolution of mass spectrometry. The traditional fluorescent label is replaced with heavy metal element label to mark the cell surface or the target in the cell. It has 40 parameters in a single cell at the same time. The detection capabilities have successfully achieved cell typing, cell diversity analysis, and cell behavior analysis. However, heavy metal labels are expensive and difficult to separate and prepare. The single ionization method is also the main bottleneck for the development of inorganic mass spectrometry flow cytometry. At the same time, there are inherent limitations based on inorganic mass spectrometry. Molecular metabolites are detected.

Organic mass spectrometry relies on its high sensitivity, qualitative and quantitative, simultaneous detection of multiple substances, and the ability to provide a large amount of molecular structure information. It plays an increasingly important role in single cell detection, especially the use of organic mass spectrometry to detect a large number of single cells. Unlabeled small molecule metabolite information. Combining the commonly used labeling techniques in traditional flow cytometry with organic mass spectrometry, the development of organic mass spectrometry cell flow analysis can greatly utilize the advantages of flow cytometry in analysis throughput and macromolecule detection, and the advantages of organic mass spectrometry The advantages of multi-target analysis and small molecule detection at the single cell level, so as to obtain a large number of comprehensive material information from large molecules to small molecules at the single cell level.

The key point of the organic mass spectrometry cell flow analysis technology is the design of highly sensitive mass spectrometry tags and the cell analysis ionization interface that can realize online tag dissociation and online cell metabolite release. The ideal cell flow label needs to have functions such as specific recognition, efficient online dissociation and signal amplification, and needs to be economical, expandable and universal, and be suitable for high-throughput cell analysis. Literature (Y. Wang, R. Du, Qiao and B. Liu. Chem. Commun., 2018, 54, 9659-9662 and W. Ma, S. Xu, H. Nie, B. Hu, Y. Bai and H .Liu.Chem.Sci., 2019,10,2320-2325) reported a typical organic mass spectrometry label that can label macromolecules in cells, but the sensitivity of this type of probe analysis is not up to the single-cell level, and The dissociation method requires special chemical reactions or lasers, so it is difficult to meet the demand for online rapid dissociation of single-cell flow cytometry. Literature (G.Li, S. Yuan, S. Zheng, Y. Liu and G. Huang. Anal. Chem., 2018, 90, 3409-3415 and EK Neumann, TJComi, SSRubakhin and JVSweedler.Angew.Chem .Int.Ed.2019,58,5910–5914) reported a typical ionization method that can release the contents of a single cell and the design of an ionization device. However, the method is complicated to operate and the analysis time of a single cell is difficult. Meet the speed requirements of streaming analysis. Literature (Q. Huang, S. Mao, M. Khan, L. Zhou and J. Lin. Chem. Commun., 2018, 54, 2595-2598 and H. Yao, H. Zhao, X. Zhao, X. Pan ,J.Feng,F.Xu,S.Zhang and X.Zhang.Anal.Chem.,2019,91,9777-9783) reported that the ionization interface that can initially realize flow analysis can detect metabolite information However, simultaneous detection of proteins cannot be achieved.

Summary of the invention

In order to realize the function of organic mass spectrometry flow cytometry for high-throughput cell analysis and simultaneous detection of information from large molecules to small molecules at the single cell level, to make up for the lack of sensitivity of markers in the prior art and the inability to achieve online dissociation. The analysis throughput of the ionization method is low, and the problem of simultaneous detection of macromolecules and metabolites cannot be achieved at the same time. The present invention aims to provide high-throughput, high-sensitivity, strong applicability, and organic mass spectrometry flow analysis that can obtain a large amount of information in single cells. The method can qualitatively and quantitatively analyze the content of a large number of single cells in the cell dispersion from small molecular metabolites to large molecular proteins.

In the first aspect of the present invention, an organic mass spectrometry flow device for single cell analysis is provided, including a peristaltic pump, a cell dispersion and arrangement device, an ionization spray needle, and a mass detector, wherein the peristaltic pump, cell dispersion and The arranging device and the ionization spray needle are connected in sequence through a pipeline, and the outlet of the ionization spray needle is aligned with the entrance of the mass spectrometer; the cell sample with the mass spectrometer label is mixed and enters the cell dispersion and arranging device under the action of the peristaltic pump to obtain a single The dispersed and orderly arranged cell effluent flows into the ionization spray needle to realize the online dissociation of the cell mass spectrum label and the online release of the cell content. After ionization, it is detected by a mass detector.

In the above-mentioned organic mass spectrometry flow device for single cell analysis, the peristaltic pump is preferably a low-flow peristaltic pump with an adjustable flow rate ranging from 0.1 μL/min to 50 μL/min. The cell sample is mixed well under low-speed vortex and enters the peristaltic pump. The low-speed vortex can be achieved by a vortex mixer.

The cell dispersing and arranging device may be a microchannel chip or other single cell dispersing device. The microchannel chip may be a multi-turn spiral channel with a rectangular cross section, and the cross section is a rectangle with a height of 20-40 μm and a width of 30-80 μm, The number of spiral turns is 3-10 turns. Preferably, the material of the channel is polydimethylsiloxane.

The ionized spray needle is a hollow tube made of glass or metal, the overall inner diameter is less than 100μm, one end is a tip, the tip inner diameter is 20-30μm, the outer diameter is less than 100μm, the other end is a non-tip, and the non-tip is dispersed and arranged with cells The outlet end of the device is connected. The ionized spray needle may also be an integrated spray needle of a cell dispersion and arrangement device, which extends outward along the outlet end of the cell dispersion and arrangement device, and has a cone shape at the foremost end, with an inner diameter of 20-30 μm and an outer diameter of less than 100 μm.

The mass spectrometer detector is suitable for ion trap mass spectrometry, quadrupole mass spectrometry, triple quadrupole mass spectrometry, time-of-flight mass spectrometry, electrostatic field orbitrap mass spectrometry, and/or Fourier transform ion cyclotron resonance mass spectrometry.

In the second aspect of the present invention, an organic mass spectrometry flow analysis method for single cell analysis is provided. The implementation includes using flow mass spectrometry probes to label target proteins such as surface antigens in cells, and sample injection of cells. And arrangement control, cell ionization and mass spectrometry detection, data processing and other steps. details as follows:

1) Incubate the cell suspension samples C ₁ , C ₂ ,..., C _{x with a} mixture of various flow mass spectrometry probes G ₁ , G ₂ ,..., Ga to label the cell target protein, where x is Natural number, representing the serial number of the cell sample, a is a natural number, representing the number of the flow mass spectrometry probe, the flow mass spectrometry probe includes a precious metal nanoparticle core and the formation of MS bonds (M=Au, Ag and other precious metals) through the sulfhydryl group and the precious metal self-assembly Antibody/nucleic acid aptamer and mass spectrum label on the surface of precious metal nanoparticles;

2) Using the above-mentioned organic mass spectrometry flow cytometer to perform mass spectrometry detection on the labeled cell sample in step 1);

3) Data analysis: Extract multiple single cell mass spectra from the mass spectrometry detection results of step 2), and normalize the mass spectrometry signal according to the internal standard signal intensity in each mass spectrum to obtain the markers and metabolites in each cell The normalized intensity signal of the signal. The intensity signals of these markers and metabolites are used as the basis for cell typing, cell identification, and cell differentiation analysis.

The above step 1) To achieve the labeling of the cell surface and/or the target protein in the cell, first add a certain number of certain types of flow mass spectrometry probes to a certain concentration of cell suspension, and incubate at a certain temperature (such as 37°C) for a period of time Time (usually 10min-30min); wash the cells after incubation to remove unidentified components. The washing cells usually use phosphate buffer solution. The supernatant is discarded by centrifugation and the cells are resuspended to remove the remaining flow cytometry Mass spectrometer probe. After washing, the cells are dispersed in a dispersion suitable for mass spectrometry flow analysis by centrifugation and resuspension to obtain a cell sample to be tested.

Preferably, the concentration of the aforementioned cell sample to be tested is 10-1,000,000 cells/mL, and the cell sample to be tested may be cells of a single cell line, cells of mixed cell lines, unknown cell suspension samples, and the like.

Among them, the types of flow mass spectrometry probes incubated with the cells are the same as the types of cell target proteins of interest, and the number of each probe is added according to the ratio of the number of probes to cells 1:100000-1:1000000. The flow mass spectrometry probe used is a highly sensitive, universal multifunctional nanoprobe, which includes a precious metal nanoparticle core and self-assembled on precious metals through sulfhydryl groups and precious metals to form MS bonds (M=Au, Ag and other precious metals) Antibody/nucleic acid aptamer and mass spectrum label on the surface of the nanoparticle (see Figure 1).

The invention also provides a method for preparing the flow-type mass spectrometry probe. The flow cytometry mass spectrometer probe is prepared by the "one-pot reaction method". The sulfhydryl antibody/nucleic acid aptamer and the mass spectrometer label are bonded to the precious metal nanoparticles by self-assembly. The specific steps are: adding the precious metal nanoparticles to the aqueous solution First add the sulfhydryl antibody/nucleic acid aptamer solution, and react for a period of time (such as 10-16h) in the dark at room temperature; then add the mass spectrum labeling solution to the system (the mass spectrum label molecules are pre-dissolved in an aprotic solvent, for example Acetonitrile), react for a period of time in the dark at room temperature (such as 10-16h); centrifuge and wash 3-5 times to remove unbound antibody/nucleic acid aptamer and mass spec label; finally, disperse the nanoparticles in a buffer solution (commonly used phosphate Buffer solution). Among them, the molar ratio of the precious metal nanoparticles to the antibody/nucleic acid aptamer can be selected from 1:10-1:1000; the molar ratio of the precious metal nanoparticles to the mass spectrum tag can be 1:1000-1:10000.

In the above-mentioned preparation method of the flow mass spectrometry probe, the precious metal nanoparticles used are preferably gold nanoparticles or silver nanoparticles. The gold nanoparticles can be prepared by the method of reducing chloroauric acid by trisodium citrate, and the silver nanoparticles can be prepared by the method of reducing silver nitrate by trisodium citrate and sodium borohydride. The particle size of the noble metal nanoparticles is preferably 15nm-25nm.

The antibody/nucleic acid aptamer used is preferably a sulfhydryl modified antibody/nucleic acid aptamer, and an antibody/nucleic acid aptamer that can specifically bind to the target protein needs to be selected. The sulfhydrylation of the antibody can be done by pre-connecting the antibody with one end of the sulfhydryl group and the end of the N-hydroxysuccinimide linking arm (such as 4,7,10,13,16,19,22,25,32,35,38,41, 44,47,50,53-hexadecyloxa-28,29-dithiapentahexadecanedioic acid di-N-succinimide ester) according to the molar ratio of 2:1 in N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) sodium salt buffer solution was reacted for 1-12h in the dark at room temperature, and the amino terminal of the protein was thiolated. The nucleic acid aptamer used is preferably a 5'-end or 3'-end HS-(CH ₂ ) ₆ -sulfhydryl modified nucleic acid aptamer, which can be a deoxyribonucleic acid aptamer or a ribonucleic acid aptamer.

The mass spectrum tag used is a kind of small organic molecules that can achieve online dissociation and have a good mass spectrum response. The molecular weight is within 1500Da, and its molecular structure is shown in Formula I:

In formula I, n is an integer of 6-15, m is an integer of 0-8, and R is a group with mass spectrometry sensitizing ability and a molecular weight of 50-1000 Da, which contains, but is not limited to, quaternary ammonium, pyridyl, and quinolinyl. , Amino, alkyl-substituted amino (such as dimethylamino, diethylamino) and other electropositive groups or one or more of strong proton affinity groups, and preferably a molecular group with an aromatic ring conjugated structure group.

The mass spectrum tag shown in formula I has three components: one end of the structure is a sulfhydryl group, which can be self-assembled on the surface of precious metal nanoparticles through MS (M=Au, Ag and other precious metals) bonds; the sulfhydryl group is connected by a rigid alkyl chain and a flexible poly The chain-like structure composed of ethylene glycol chains can improve the efficiency of self-assembly of the mass spectrometer label on the surface of the nanoparticle, and increase the binding number of the mass spectrometer label on the surface of the nanoparticle; the other end of the structure is an R-based structure.

Preferably, the R group in formula I may be a structure with formula II as the main body:

In formula II, x is an integer from ₁ to 5, and R 1, R ₂ , and R _{3 are the} same or different, and are C1-C4 short-chain alkyl groups (such as methyl, ethyl, propyl, etc.).

Preferably, the R group in formula I can also be a structure with formula III as the main body:

In formula III, R ₁ , R ₂ , and R _{3 are the} same or different, and are C1-C4 short-chain alkyl groups (such as methyl, ethyl, propyl, etc.), and R ₄ represents one or more substitutions on the phenyl group. The group is hydrogen or a C1-C4 short-chain alkyl group (such as methyl, ethyl, propyl, etc.). The connection position of the polyethylene glycol chain in formula I and formula III can be at the ortho, meta or para _{position of the phenyl group; the connection position of R 4} can be at other unsubstituted positions on the phenyl group.

Preferably, the R group in formula I can also be a structure with formula IV as the main body:

In formula IV, R ₁ represents one or more substituents on the pyridyl group, and is hydrogen or a C1-C4 short-chain alkyl group (such as methyl, ethyl, propyl, etc.). The connection position of the polyethylene glycol chain in formula I and formula IV can be on the 1N atom of the pyridyl group, or on the 2C, 3C, 4C, 5C or 6C position, and _{the connection position of R 1} can be on the pyridyl group. Substitution on N atoms or other unsubstituted C atoms.

Preferably, the R group in formula I can also be a structure with formula V as the main body:

In formula V, R ₁ represents one or more substituents on the quinolinyl group, and is hydrogen or a C1-C4 short-chain alkyl group (such as methyl, ethyl, propyl, etc.). The linking position of the polyethylene glycol chain in formula I and formula V can be on the 1N atom of the quinolinyl group, or be connected to the 2C, 3C, 4C, 5C, 6C, 7C or 8C position. The attachment position of R ₁ may be on the unsubstituted N atom on the quinolinyl group or on other unsubstituted C atoms.

Preferably, the R group in formula I can also be a structure with formula VI as the main body:

In formula VI, R ₁ and R _{2 are the} same or different, and are hydrogen or C1-C4 short-chain alkyl (such as methyl, ethyl, propyl, etc.); R ₃ represents one or more substituents on the phenyl group , Is hydrogen or a C1-C4 short chain alkyl group. The connection position of the polyethylene glycol chain in formula I and formula VI can be at the ortho, meta or para position of the phenyl group, and the connection position of _{R 3 can be at other unsubstituted positions on the phenyl group.}

Preferably, the R group in formula I can also be a structure with formula VII as the main body:

In formula VII, R ₁ , R ₂ , R ₃ , and R _{4 are the} same or different, and are hydrogen or C1-C4 short-chain alkyl groups (such as methyl, ethyl, propyl, etc.); R ₅ represents 1C, 3C, and /Or one or more substituents at 4C position is hydrogen or C1-C4 short-chain alkyl; R ₆ represents one or more substituents at 5C, 7C and/or 8C position, which is hydrogen or C1-C4 C4 short-chain alkyl; R ₇ represents one or more substituents at 9C, 10C, 11C, 12C or 13C, and is hydrogen or a C1-C4 short-chain alkyl; wherein, R ₁ and R ₂ , R ₃ and R ₄ , R ₁ and 1C position, R ₂ and 3C position, R ₃ and 5C position, R ₄ and 7C position can form a ring. The connection position of the polyethylene glycol chain in formula I and formula VII may be at the 9C, 10C, 11C, 12C or 13C position of the phenyl group.

The R-based partial structure of the above-mentioned mass spectrometry tag has the characteristics of high ionization efficiency and high response of mass spectrometry in electrospray mass spectrometry detection. A large number of mass spectrometry tags are self-assembled on precious metal nanoparticles and labeled on each target protein, which has a signal amplification function.

The mass spectrum tag used can be efficiently dissociated online during the electrospray process to realize the rupture of the M-S bond, and generate two mass spectrum tags connected by disulfide bonds. During the preparation process, the molar ratio of the nanoparticles to the mass spectrum label can be selected from 1:1000 to 1:10000.

The mass spectrum tag used in the present invention has strong scalability. Using the mass spectrum tag shown in formula I, a series of mass spectrum tags with similar response capabilities but different m/z can be generated by changing the length of the short-chain alkyl chain in the R group. Used for simultaneous labeling and detection of multiple protein targets. Generally, the mass-to-nucleus ratios of mass spectrometry tags bound to the surface of different flow mass spectrometry probes for different target proteins differ by more than one.

In some embodiments of the present invention, gold nanoparticles with a diameter of 20 nm are used as the core, and 4, 7, 10, 13, 16, 19, 22, 25, 32, 35, 38, 41, 44, 47, 50 are selected. 53-hexadecoxaoxa-28, 29-dithiapentahexadecanedioic acid di-N-succinimidyl ester linked CA125 antibody, the structure of the mass spectrum label is shown in formula VIII; gold nanoparticles, antibodies The molar ratio of preparation to mass spectrum label is 1:24:9000.

The mass spectrometer labeled probe used in step 1) can be prepared first and stored at 4-8°C for later use.

In step 1), the final dispersion liquid used to disperse the cells can be an aqueous solution or pure water containing a volatile salt with a cell isotonic concentration (such as ammonium acetate) and an organic solution with a cell fixation effect (such as methanol) in a certain proportion (such as 1:1, 2:3) The mixed solution. A certain amount of internal standard substance is also added to the dispersion liquid for quantification by internal standard method. The optional internal standard substance is a compound whose structure is similar to that of a mass spectrometry tag, but the mass-to-nucleus ratio produced in mass spectrometry detection can be clearly distinguished from the mass spectrometry tag. For example, a molecule of formula I whose structure is represented by formula VII with an R group is a mass spectrometry tag. When the internal standard substance is selected as crystal violet, methyl violet and other compounds, the concentration of the internal standard substance in the spray solvent can be 0.05-0.5μmol/L. In the obtained mass spectrum, the intensity of the internal standard signal is normalized to the signal intensity of other mass spectra.

Step 2) is used to realize the mass spectrometry detection of the labeled cell sample, and the organic mass spectrometry flow device used is as described above. The specific analysis process of cell samples using this device is as follows: Place the sample tube containing the cell dispersion on the vortex mixer, make the cells vortex at a low speed (such as 400rpm), insert the peristaltic pump tube into the cell sample tube , Set the flow rate of the peristaltic pump, and use the peristaltic pump to inject samples; turn on the mass spectrometry scan, apply a certain voltage to the ionization needle, and collect mass spectrometry data. The cell sample is first sent to the cell dispersion and arrangement device by the peristaltic pump to realize the monodispersion of the cells and make the cells flow into the ionization needle in a more orderly arrangement state, and realize the dissociation and metabolism of the mass spectrum label under the electrospray condition The release of substances, as well as the mass spectrometry detection of tags and metabolites, record the time-varying mass spectrum ion chromatogram and the mass spectrum collected at each moment.

Preferably, the cell sample concentration for each analysis is 1000-10000 cells/mL, the total volume is 0.5-1 mL, and the flow rate of the peristaltic pump injection is set to 0.5-10 μL/min.

Among them, the voltage applied to the ionized spray needle is a DC high voltage, which can be a positive mode or a negative mode, and the voltage can be set to 2-4kV. The applied voltage can also be pulsed high voltage, can be positive mode or negative mode, the peak value can be 2-4kV, and the frequency can be 10-10000Hz.

Among them, the preferred mass spectrometry is high-resolution mass spectrometry, with a resolution greater than 30,000, the acquisition mode is full scan, and the acquisition range is m/z 80-1200. The data acquisition time is greater than 10 minutes each time, and 10-60 cells can be collected per minute. Mass spectrometry information.

In an embodiment of the present invention, a cell dispersion sample labeled with a mass spectrometry label shown in Formula VIII at a concentration of 10,000 cells/mL is analyzed, crystal violet is used as an internal standard, and the injection flow rate is 1 μL/min, and the cells are dispersed and arranged. The device is a 5-turn spiral microchannel with a channel cross-section of 70 μm wide and 50 μm high, and the distance between two adjacent circles is 100 μm. The outlet of the channel is connected with the injection needle of the quartz glass capillary through a capillary tube with an inner diameter of 50 μm. The tip of the injection needle has an inner diameter of 30 μm and an outer diameter of 50 μm, and a +3kV DC high voltage is applied to the inside of the spray needle. Orbitrap mass spectrometer was used for detection, the resolution was set to 35000, the acquisition range was m/z 80-1200, and the acquisition time was 10 minutes. In the obtained mass spectrum, the mass-nucleus ratio produced by the mass spectrum label shown in formula VIII is m/z=628.3693 (z=2), the mass-nucleus ratio produced by the internal standard crystal violet is m/z=372.2340, in addition to A large number of intracellular metabolites with a mass-nucleus ratio m/z in the range of 80-1200.

The above step 3) is data analysis. In the obtained total ion current chromatogram, a single cell mass spectrum is obtained at the highest point of each single cell signal peak. The single cell mass spectrum data processing specifically includes:

3-1) According to the theory of mass labels mass spectrum of nuclear extract than the number of signal strength of the tag _{T 1, T 2, ...,} T a, a target protein, nuclear mass tolerable error ratio is within 5ppm;

3-2) According to the theoretical mass-nucleus ratio of the metabolite, extract the signal intensity of the corresponding metabolite in the spectrum. M ₁ , M ₂ ,..., M _b , b is the number of metabolites identified, and the tolerance of the mass-nucleus ratio is 5 ppm Within

3-3) Extract the internal standard signal intensity S in the spectrum according to the theoretical mass-nucleus ratio of the internal standard molecule, and the tolerance of the mass-nucleus ratio is within 5 ppm. Use the internal standard signal intensity S as the reference to determine the signal intensity of the mass spectrum label and the signal intensity of metabolites Carry out normalization, the normalized intensity is T ₁ /S, T ₂ /S,..., T _a /S, M ₁ /S, M ₂ /S,..., M _b /S, recorded as each Mass spectrometry detection results of each cell sample: C _x ((T ₁ /S) _x , (T ₂ /S) _x ,..., (T _a /S) _x , (M ₁ /S) _x , (M ₁ /S ) _x , (M ₂ /S) _x ,..., (M _b /S) _x ) are _{all the information of C x} cells detected in mass spectrometry (mass spec label and metabolites), and x represents the serial number of the cell sample;

3-4) The mass spectrum detection results of multiple cells are summarized into a data matrix, with the mass spectrum label and metabolite information in each cell as variables:

3-5) Use the intensity values of the variables detected in the single cell (that is, each variable in the above data matrix), use data analysis algorithms (such as principal component analysis, hierarchical clustering, etc.) to type and identify cells, and Differential analysis and other related applications of mass spectrometry flow cytometry.

The organic mass spectrometry flow cytometry analysis technology provided by the present invention is a single cell analysis technology with high universality, high throughput and suitable for simultaneous detection of multiple targets. Different target proteins can be simultaneously labeled and detected using flow mass spectrometry probes with different mass spectrometry tags. Moreover, due to the high mass resolution of mass spectrometry, signals with small differences in mass-to-nucleus ratio can be easily separated in the mass spectrum, thus ensuring that the organic mass spectrometry flow analysis method of the present invention can simultaneously detect multiple protein markers and multiple metabolites In the application. The organic mass spectrometry flow cytometry analysis technology of the present invention can be used for cell identification, cell typing, and research on characteristic difference substances in cells, and has broad application prospects in the fields of stem cell analysis, tumor diagnosis, and systems biology research.

The flow-type mass spectrometry probe of the present invention is a highly sensitive protein labeling and detection tool. A variety of signal amplification strategies are applied to the probe to make the sensitivity reach the level of single-cell analysis. By detecting mass spectrometry tags with good mass spectrometry responsiveness to replace the target protein, the problem of low ionization efficiency and low mass spectrometry response of protein molecules in the mass spectrometry detection process is solved; the protein molecules are labeled by flow mass spectrometry probes, so that a large number of mass spectrometry tags are marked on A protein molecule amplifies the detection signal in quantity.

The flow-type mass spectrometer probe of the present invention is a probe capable of realizing high-efficiency online dissociation through M-S bond breakage under electrospray conditions. Under the voltage condition of 2-4kV, a large number of tag molecules can be dissociated from the surface of nanoparticles quickly and online, which provides a guarantee for the realization of high-throughput flow cytometry analysis.

The device for cell dispersion and ionization in the present invention is a simple structure, low cost, multifunctional and highly integrated flow cell processing device, which is equipped with cell sampling, cell dispersion and arrangement, cell label label dissociation, and cell The functions of metabolite release and sample ionization are integrated, which can realize the analysis of 10-60 cells per minute. Moreover, this part of the device is versatile and can be adapted to a variety of different types of mass spectrometer mass analyzers.

Description of the drawings

Fig. 1 is a schematic diagram of labeling cells by a flow cytometry mass spectrometry probe of the present invention.

Fig. 2 is a schematic flow chart of the organic mass spectrometry flow cytometry technique used in the embodiment of the present invention.

Figure 3 is a photo of a microchannel chip used in an embodiment of the present invention.

Figure 4 is a scanning electron microscope image of a flow mass spectrometer probe prepared in an embodiment of the present invention.

Figure 5 is a total ion current chromatogram and a single cell mass spectrum obtained from the detection of MCF-7 cells and MDA-MB-231 cells in an embodiment of the present invention.

Fig. 6 is a diagram showing the results of principal component analysis of MCF-7 cells and MDA-MB-231 cells according to the signal intensity normalized by metabolites and labels in the embodiment of the present invention.

Fig. 7 is a volcano graph of MCF-7 cells and MDA-MB-231 cells based on metabolites and label differences in an embodiment of the present invention.

Detailed ways

The technical solutions of the present invention will be further illustrated by embodiments in conjunction with the drawings below, but the protection scope of the present invention is not limited by the specific conditions of these embodiments.

Example: Use the organic mass spectrometry flow cytometry technology of the present invention to analyze MCF-7 cells (C ₁ ) and MDA-MB-231 cells (C ₂ ), and use six cell surface antigens: CA125, CEA, EpCAM, CD24, CD44 and CD133 are target proteins, and the flow analysis process of organic mass spectrometry is shown in Figure 2.

Specific steps are as follows:

(1) Use 5 circles of PDMS microchannel chip (Figure 3), the channel width is 70μm, the height is 50μm, the distance between two adjacent circles is 100μm, connect the peristaltic pump injection tube and the microchannel chip inlet and the microchannel chip outlet Connected to the quartz glass capillary nozzle, the tip of the nozzle has an inner diameter of 30 μm and an outer diameter of 50 μm. Install the connected device before the entrance of the orbitrap mass spectrometer.

(2) MCF-7 cells and MDA-MB-231 cells cultured adherently in a 6cm cell culture dish were used as the analysis objects. When the cell growth is in good condition, digest with Accutase cell digestion enzyme, collect the suspended cells, count them with a cell counter, and dilute the cell concentration to 10 ⁴ /mL with PBS.

Add antibodies (CA125 antibody, CEA antibody, EpCAM antibody, CD24 antibody, CD44 antibody or CD133 antibody) and PEG-SH link arms 4, 7, 10, 13, 16, 19, 22, 25, 32 respectively in the 10mM HEPES solution ,35,38,41,44,47,50,53-hexadecanoxa-28,29-dithiapentahexadecanedioic acid di-N-succinimidyl ester (NHS-PEG-SS- PEG-NHS, MW=1109.26) (antibody: link arm=2:1, molar ratio), stir at room temperature for 12 hours, and fix the antibody on both ends of the link arm. Subsequently, 30 μL of the linked antibody (antibody concentration of 120 μg/mL) was added to 1 mL of the gold nanoparticle solution with a concentration of 1 nM, and potassium carbonate was added (to make the final concentration of potassium carbonate 1.8 mM) and stirring was continued for 12 hours. Then add 90 μL of mass spectrometry label (formula XII: RMT331, formula IX: RMT387, formula X: RMT415, formula VIII: RMT443, formula XI: RMT467 or formula XIII: RMT491) with a concentration of 100 μM to the mixed solution and continue the reaction for 12 hours. Centrifuge at 9000 rpm for 15 minutes, discard the supernatant, and then disperse in water to wash twice by centrifugation. Finally, each tube is dispersed in 200 μL PBS buffer solution to obtain gold nanoprobes corresponding to six target proteins (GNPs-anti-CD24/RMT331, GNPs-anti-EpCAM/RMT387, GNPs-anti-CEA/RMT415, GNPs-anti-CA125/RMT443, GNPs-anti-CD133/RMT467 and GNPs-anti-CD44/RMT491), the six gold nanoprobes are of equal volume The projection electron micrograph of the prepared gold nanoprobe is shown in Figure 4 after mixing.

Add 100 μL of six gold nanoprobes mixed in equal proportions to 1 mL of cell suspension with a cell concentration of 10 ⁴ /mL, incubate at 37°C for 20 minutes in the dark, wash the cells 3 times with PBS, centrifuge and re-disperse after each wash. It was washed again with water, centrifuged (1800 rpm, 2 min) and dispersed in 1 mL of 60% cold methanol dispersion.

(3) Take 1mL of labeled and dispersed cell suspension and immediately perform organic mass spectrometry flow analysis. The parameters of mass spectrometry flow analysis are set as follows: vortex rotation speed 400rpm, injection flow rate 1μL/min, voltage value set to +3kV, The mass spectrum resolution is set to 35000, the acquisition mode is full scan mode, the acquisition range is m/z 80-1200, and the acquisition time is 10 minutes.

(4) The mass spectrum of a single cell is extracted from the collected mass spectrum ion current chromatogram (for example, Figure 5), a total of 145 mass spectra of MCF-7 cells and 121 mass spectra of MDA-MB-231 cells, Extract 84 kinds of metabolites in the mass spectrum, such as m/z 116.0706, 732.5538, 760.5851, etc., the dissociated mass spectrum label m/z 516.2441, 572.3067, 600.3380, 628.3693, 652.3693, 676.3693) and the crystal violet internal standard m/z 372.2434, the mass-nucleus ratio error tolerance is within 5ppm of the corresponding intensity of the mass spectrum signal: M ₁ , M ₂ ,..., M ₈₄ , T ₁ , T ₂ ,..., T ₆ and S. The intensities of the metabolites extracted from each mass spectrum are normalized based on the intensities of the internal standards, and the following data matrix is obtained:

Based on the normalized signal intensity of each cell metabolite and label as a variable, use principal component analysis to perform cell typing (Figure 6), calculate the multiple of change and P value, draw a volcano graph and compare the difference analysis between the two cells before (Figure 7).

Claims (15)

An organic mass spectrometry flow device for single cell analysis, including a peristaltic pump, a cell dispersion and arrangement device, an ionization spray needle, and a mass detector, wherein the peristaltic pump, the cell dispersion and arrangement device and the ionization spray needle pass between The pipelines are connected in sequence, and the outlet of the ionization nozzle is aligned with the inlet of the mass spectrometer; the cell sample with the mass spectrometer label is mixed and enters the cell dispersion and arrangement device under the action of the peristaltic pump to obtain a monodisperse and orderly arranged cell effluent into the ion The chemical spray needle realizes the online dissociation of the cell mass spectrum label and the online release of the cell content. After ionization, it is detected by the mass detector. The organic mass spectrometry flow device according to claim 1, wherein the peristaltic pump is a low-flow peristaltic pump with an adjustable flow rate ranging from 0.1 μL/min to 50 μL/min. The organic mass spectrometry flow device according to claim 1, wherein the cell dispersing and arranging device is a microchannel chip, which is composed of a multi-turn spiral channel with a rectangular cross section, and the cross section is 20-40 μm in height and 30 in width. -80μm rectangle, the number of spiral turns is 3-10. The organic mass spectrometry flow device according to claim 1, wherein the ionized spray needle is a hollow tube made of glass or metal, and the overall inner diameter is less than 100 μm, one end is a tip, and the inner diameter of the tip is 20-30 μm. The diameter is less than 100 μm, the other end is non-tip, and the non-tip is connected with the outlet end of the cell dispersing and arranging device. An organic mass spectrometry flow analysis method for single cell analysis, including the following steps: 1) Incubate the cell suspension samples C ₁ , C ₂ ,..., C _{x with a} mixture of multiple flow mass spectrometry probes G ₁ , G ₂ ,..., Ga to label the cell target protein, where x represents The serial number of the cell sample, a represents the number of the flow cytometry mass spectrometry probe, which includes a precious metal nanoparticle core and an antibody and/or nucleic acid aptamer self-assembled on the surface of the precious metal nanoparticle through the formation of an MS bond between the sulfhydryl group and the precious metal. Mass spectrum label, where M stands for precious metal element; 2) Using the organic mass spectrometry flow cytometer according to any one of claims 1 to 4 to perform mass spectrometry detection on the labeled cell sample in step 1); 3) Data analysis: Extract multiple single cell mass spectra from the mass spectrometry detection results of step 2), and normalize the mass spectrometry signal according to the internal standard signal intensity in each mass spectrum to obtain the markers and metabolites in each cell The normalized intensity signals of the signals are used as the basis for cell typing, cell identification, and cell difference analysis. The organic mass spectrometry flow analysis method according to claim 5, characterized in that, in step 1) adding a flow mass spectrometry probe to the cell suspension after incubation and washing the cells to remove unidentified components and remaining flow mass spectrometry probes, The cells are then dispersed in a dispersion suitable for mass spectrometry flow analysis to obtain a sample of cells to be tested with a concentration of 10-1,000,000 cells/mL. The organic mass spectrometry flow analysis method of claim 6, wherein the dispersion liquid used to disperse the cells in step 1) is an aqueous solution containing volatile salts with an isotonic concentration of the cells, or pure water and has a cell fixation effect. The organic solution is mixed according to a certain ratio, and a certain amount of internal standard substance used for internal standard method quantification is added to the dispersion liquid. The organic mass spectrometry flow analysis method according to claim 5, wherein in step 1), the flow mass spectrometry probe is prepared by self-assembly: adding thiolated antibody and/ Or nucleic acid aptamer solution, and react for a period of time in the dark at room temperature; then add the mass spectrum label solution to the system, and react for a period of time in the dark at room temperature; centrifuge and wash to remove unbound antibody and/or nucleic acid aptamer and mass spec label ; Finally, the nanoparticles are dispersed in a buffer solution. 8. The organic mass spectrometry flow analysis method according to claim 8, wherein when preparing the flow mass spectrometry probe, the molar ratio of precious metal nanoparticles to antibody and/or nucleic acid aptamer is 1:10-1: 1000, the molar ratio of the precious metal nanoparticles to the mass spectrum tag is 1:1000-1:10000; the particle size of the precious metal nanoparticles is 15nm-25nm. The organic mass spectrometry flow analysis method according to claim 5, wherein the molecular weight of the mass spectrometry tag in the flow mass spectrometry probe in step 1) is within 1500 Da, and its general structural formula is as shown in formula I:

In formula I, n is an integer of 6-15, m is an integer of 0-8, and R is a group with mass spectrometry sensitizing ability and a molecular weight of 50-1000 Da. The organic mass spectrometry flow analysis method according to claim 10, wherein, in the mass spectrometer label shown in formula I, the R group is one of the following groups a) to f): a) The structure shown in formula II:

In formula II, x is an integer of ₁ to 5, and R 1, R ₂ , and R _{3 are the} same or different, and are a short chain alkyl group of C1 to C4; b) The structure shown in formula III:

In formula III, R ₁ , R ₂ , and R _{3 are the} same or different, and are C1-C4 short-chain alkyl groups; R ₄ represents one or more substituents on the phenyl group, and is hydrogen or C1-C4 short-chain alkyl groups ; The connection position of the polyethylene glycol chain of formula I and formula III is in the ortho, meta or para position of the phenyl group. c) The structure shown in formula IV:

In formula IV, R ₁ represents one or more substituents on the pyridyl group, which is hydrogen or a C1-C4 short-chain alkyl group; the linking position of the polyethylene glycol chain in formula I and formula IV is 1N of the pyridyl group Atom, or attached to 2C, 3C, 4C, 5C or 6C; d) The structure shown in formula V:

In formula V, R ₁ represents one or more substituents on the quinolinyl group, which is hydrogen or a C1-C4 short-chain alkyl group; the linking position of the polyethylene glycol chain in the formula I and the formula V is at the quinolinyl group On the 1N atom of, or connected to 2C, 3C, 4C, 5C, 6C, 7C or 8C; e) The structure shown in formula VI:

In formula VI, R ₁ and R _{2 are the} same or different, and are hydrogen or C1-C4 short-chain alkyl; R ₃ represents one or more substituents on the phenyl group, which is hydrogen or C1-C4 short-chain alkyl ; The connection position of the polyethylene glycol chain in formula I and formula VI is in the ortho, meta or para position of the phenyl group; f) The structure shown in formula VII:

In formula VII, R ₁ , R ₂ , R ₃ , and R _{4 are the} same or different, and are hydrogen or a short-chain C1-C4 alkyl group; R ₅ represents one or more substituents at positions 1C, 3C and/or 4C , Is hydrogen or a C1-C4 short-chain alkyl group; R ₆ represents one or more substituents at positions 5C, 7C and/or 8C, and is hydrogen or a C1-C4 short-chain alkyl group; R ₇ represents 9C, One or more substituents at positions 10C, 11C, 12C or 13C are hydrogen or C1-C4 short-chain alkyl groups; wherein, R ₁ and R ₂ , R ₃ and R ₄ , R ₁ and 1C position, R ₂ and 3C positions, R ₃ and 5C positions, R ₄ and 7C positions are independent of each other or form a ring; the connection position of the polyethylene glycol chain in formula I and formula VII is at 9C, 10C, 11C, 12C or 13C of the phenyl group Bit up. The organic mass spectrometry flow analysis method according to claim 11, wherein the structure of the mass spectrometer tag is as shown in formula VIII, formula IX, formula X, formula XI, formula XII or formula XIII:

The organic mass spectrometry flow analysis method according to claim 5, wherein in step 2), the labeled cell sample in step 1) is vortexed and mixed, and the input injection flow rate is set to 0.5-10 μL/min. The peristaltic pump enters the cell dispersion and arrangement device under the action of the peristaltic pump to obtain the monodisperse and orderly arranged cell effluent and flow into the ionized spray needle, and apply 2-4kV DC high voltage or the peak value of 2-4kV to the ionized spray needle. The pulse-type high voltage with a frequency of 10-10000 Hz realizes the online dissociation of the cell mass spectrum label and the online release of the cell content, which is detected by a mass spectrometer. The organic mass spectrometry flow analysis method according to claim 5, characterized in that, in step 3) in the total ion current chromatogram obtained, the mass spectrum of the single cell is obtained at the highest point of the peak of each single cell signal. The cell mass spectrum is processed as follows: _{3-1) Extract the signal intensity T 1} , T ₂ ,..., T _a , and a of the target protein in the spectrum according to the theoretical mass-nucleus ratio of the mass-nucleus ratio, and the tolerance of the mass-nucleus ratio is within 5 ppm; 3-2) According to the theoretical mass-nucleus ratio of the metabolite, extract the signal intensity of the corresponding metabolite in the spectrum. M ₁ , M ₂ ,..., M _b , b is the number of metabolites identified, and the tolerance of the mass-nucleus ratio is 5 ppm Within 3-3) Extract the internal standard signal intensity S in the spectrum according to the theoretical mass-nucleus ratio of the internal standard molecule, and the tolerance of the mass-nucleus ratio is within 5 ppm. Use the internal standard signal intensity S as the reference to determine the signal intensity of the mass spectrum label and the signal intensity of metabolites Carry out normalization, the normalized intensity is T ₁ /S, T ₂ /S,..., T _a /S, M ₁ /S, M ₂ /S,..., M _b /S, recorded as each Mass spectrometry results of each cell: C _x ((T ₁ /S) _x , (T ₂ /S) _x ,..., (T _a /S) _x , (M ₁ /S) _x , (M ₁ /S) _x , (M ₂ /S) _x ,..., (M _b /S) _x ), x represents the cell sample number. The organic mass spectrometry flow analysis method according to claim 14, wherein the mass spectrometry detection results of a plurality of cells obtained in step 3-3) are summarized into a data matrix:

Based on the variables in the data matrix, different cell types can be identified, typed or differentiated, and other applications of mass spectrometry flow cytometry.

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