CN113075114A - Organic mass spectrometry flow cytometry analysis technology - Google Patents
Organic mass spectrometry flow cytometry analysis technology Download PDFInfo
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Abstract
The invention discloses an organic mass spectrum flow cytometry analysis technology, which comprises the following steps: the preparation method of the protein labeling flow mass spectrum probe and the cell labeling method, the construction of an organic mass spectrum flow analysis device, the mass spectrum flow detection of a cell suspension sample, and the extraction and analysis of the information of a protein label and a metabolite in a single-cell mass spectrogram. The technology has strong universality and simple device, and can realize rapid, high-efficiency, high-throughput and high-sensitivity sample analysis. The flow-type mass spectrometry probe marks a large number of organic labels on target proteins, so that the detection sensitivity reaches the single cell level, qualitative and quantitative analysis of various target proteins and a large number of metabolites at the single cell level can be realized, and a large number of single cell level information can be obtained. The technology has wide application prospect in the fields of cell identification, cell typing, research on characteristic difference substances in cells, tumor diagnosis, systematic biology research and the like.
Description
Technical Field
The invention relates to a flow analysis technology based on organic mass spectrometry with single cells as analysis objects, in particular to a universal preparation method of a protein labeling flow mass spectrometry probe, a cell labeling method, a cell monodispersion and arrangement technology, a single cell mass spectrometry detection ionization interface design, a single cell level protein and metabolite detection and quantification method based on mass spectrometry signals and application of the single cell level protein and metabolite detection and quantification method in single cell identification and typing.
Background
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 cellular level. Single cell analysis refers to the monitoring of vital activities at an even smaller level in a single cell, and the main objects of analysis are the content and dynamic changes of important biomolecules, such as DNA, RNA, proteins and small molecule metabolites, in a single cell. Due to the existence of cell heterogeneity, many conventional analysis methods use a large number of cells as a whole as an analysis object, and obtain only an average result, so that important information of the cell heterogeneity is lost, while cell heterogeneity research has important biological life significance, especially in the research fields of system biology, tumor cells, stem cells, cell drug resistance and the like, so that the single cell analysis method provides an important analysis means for the cell heterogeneity and many important biological problems. But due to the characteristics of single cells: the analysis method for really realizing single cell analysis and obtaining as much information as possible from genome to metabolome at the single cell level needs to be based on high sensitivity and simultaneously meets the requirements of wide dynamic range, high analysis speed, high analysis flux and the like, thereby undoubtedly providing great challenges for the analysis method.
The cell flow analysis based on fluorescence detection is a representative high-throughput single cell analysis method, and obtains content information of target substances such as protein, nucleic acid and the like in a single cell by performing fluorescence labeling on the target substances mainly comprising protein or nucleic acid in the cell and performing fluorescence detection. Due to the problem of overlapping bandwidths of fluorescent signals, the number of targets that can be simultaneously detected by such flow cytometry techniques is greatly limited, and unlabeled small molecule metabolite information cannot be detected. The flow cytometry analysis based on the inorganic mass spectrum greatly exerts the advantage of high resolution of the mass spectrum, replaces the traditional fluorescent label with a heavy metal element label for labeling the surface of a cell or a target object in the cell, has the capability of simultaneously detecting 40 parameters in a single cell, and successfully realizes the problems of cell typing, cell diversity analysis, cell behavior analysis and the like. However, heavy metal tags are expensive, separation and preparation are difficult, a single ionization mode is also a major development bottleneck of an inorganic mass spectrometry flow cytometry, and meanwhile, the inorganic mass spectrometry-based method also has inherent limitations, namely a compound structure cannot be obtained, and small molecule metabolites in cells cannot be directly detected.
The organic mass spectrometry plays an increasingly important role in single cell detection by virtue of the capabilities of high sensitivity, qualitative and quantitative detection, simultaneous detection of multiple substances and capability of providing a large amount of molecular structure information, and particularly, the organic mass spectrometry can be used for detecting a large amount of unlabelled small molecule metabolite information at the single cell level. The method combines the conventional labeling technology in the traditional flow cytometry with organic mass spectrometry to develop the organic mass spectrometry cell flow analysis, can greatly exert the advantages of the flow cytometry on the analysis flux and the macromolecule detection, and the advantages of the organic mass spectrometry on the multi-target analysis and the small molecule detection on the single cell level, thereby obtaining a large amount of comprehensive substance information of single cell level from the large molecule to the small molecule.
The key points of the organic mass spectrum cell flow analysis technology are that a high-sensitivity mass spectrum label is designed, and a cell analysis ionization interface capable of realizing online label dissociation and online cell metabolite release is designed. The ideal cell flow label needs to have the functions of specific identification, efficient on-line dissociation, signal amplification and the like, and needs to have economy, expansibility and universality, and can be adapted to high-throughput cell analysis. Typical organic mass spectrometry tags capable of labeling macromolecules in cells are reported in documents (Y.Wang, R.Du, Qiao and B.Liu.Chem.Commun, 2018,54, 9659-. Typical ionization methods and design formats of ionization devices capable of achieving release of single-cell contents are reported in the documents (G.Li, S.Yuan, S.ZHEN, Y.Liu and G.Huangang.Anal.Chem., 2018,90, 3409-. Ionization interfaces capable of preliminary flow analysis, capable of detecting metabolite information but incapable of simultaneous detection of proteins are reported in the literature (q.huang, s.mao, m.khan, l.zhou and j.lin.chem.commu., 2018,54, 2595-.
Disclosure of Invention
The invention aims to provide an organic mass spectrometry flow analysis method which has high throughput, high sensitivity and strong applicability and can obtain a large amount of information in single cells, and can carry out qualitative and quantitative analysis on the content of a large amount of single cells from micromolecular metabolites to macromolecular proteins in cell dispersion liquid, in order to realize high-throughput cell analysis by an organic mass spectrometry flow cytometry technology, simultaneously detect the function of single cell level from macromolecules to micromolecular information, and make up the problems that the sensitivity of a marker is insufficient, online dissociation cannot be realized, the analysis throughput of an ionization method is low, and simultaneous detection of macromolecules and metabolites cannot be realized in the prior art.
In a first aspect of the invention, an organic mass spectrometry flow device for single cell analysis is provided, which comprises a peristaltic pump, a cell dispersing and arranging device, an ionization spray needle and a mass spectrometry detector, wherein the peristaltic pump, the cell dispersing and arranging device and the ionization spray needle are sequentially connected through a pipeline, and an outlet of the ionization spray needle is aligned with an inlet of the mass spectrometry detector; after being uniformly mixed, the cell sample with the mass spectrum label enters a cell dispersing and arranging device under the action of a peristaltic pump, the obtained monodisperse ordered-arranged cell effluent flows into an ionization spray needle, the online dissociation of the cell mass spectrum label and the online release of the cell content are realized, and the cell sample is detected by a mass spectrum detector after ionization.
In the organic mass flow device for single cell analysis, the peristaltic pump is preferably a low-flow-rate peristaltic pump with the flow rate in an adjustable range of 0.1-50 muL/min. The cell sample was mixed well under low speed vortex and entered the peristaltic pump. Low speed swirling may be achieved by a vortex mixer.
The cell dispersing and arranging device can be a micro-channel chip or other single cell dispersing devices, the micro-channel chip can be a multi-turn spiral channel with a rectangular section, the section is a rectangle with the height of 20-40 mu m and the width of 30-80 mu m, the number of turns of the spiral is 3-10, and the material of the channel is polydimethylsiloxane preferably.
The ionization spray needle is a hollow tube made of glass or metal, the whole inner diameter of the ionization spray needle is smaller than 100 micrometers, one end of the ionization spray needle is a tip, the inner diameter of the tip is 20-30 micrometers, the outer diameter of the tip is smaller than 100 micrometers, the other end of the ionization spray needle is a non-tip, and the non-tip is connected with the outlet end of the cell dispersing and arranging device. The ionization spray needle can also be a cell dispersion and arrangement device integrated spray needle, extends outwards along the outlet end of the cell dispersion and arrangement device, is tapered at the foremost end, has an inner diameter of 20-30 μm, and has an outer diameter smaller than 100 μm.
The mass spectrometer detector is suitable for ion trap mass spectrometry, quadrupole mass spectrometry, triple tandem quadrupole mass spectrometry, time-of-flight mass spectrometry, electrostatic field orbitrap mass spectrometry and/or Fourier transform ion cyclotron resonance mass spectrometry and the like. In the second aspect of the present invention, an organic mass spectrometry flow analysis method for single cell analysis is provided, which includes steps of labeling target proteins such as surface antigens in cells by using a flow mass spectrometry probe, sample introduction and arrangement control of cells, ionization and mass spectrometry detection of cells, data processing, and the like. The method comprises the following specific steps:
1) sample C of cell suspension1，C2，…，CxWith multiple flow mass spectrometry probes G1，G2，…，GaThe mixed solution is incubated together to mark cell target protein, wherein x is a natural number representing the serial number of a cell sample, a is a natural number representing the serial number of a flow mass spectrum probe, and the flow mass spectrum probe comprises a noble metal nanoparticle inner core and a self-body mass spectrum probe which forms an M-S bond (M ═ Au, Ag and other noble metals) with noble metals through sulfydrylThe antibody/aptamer and the mass spectrum label are assembled on the surface of the noble metal nanoparticle;
2) carrying out mass spectrum detection on the cell sample marked in the step 1) by using the organic mass spectrum flow device;
3) and (3) data analysis: extracting a plurality of single-cell mass spectrograms from the mass spectrometric detection result in the step 2), and normalizing mass spectrometric signals in each mass spectrogram according to the internal standard signal intensity to obtain normalized intensity signals of the marker and metabolite signals in each cell. The intensity signals of the markers and metabolites are used as the basis for cell typing, cell identification and cell differential analysis.
The step 1) is to realize the labeling of target protein on the cell surface and/or in the cell, firstly, a certain amount of certain types of flow mass spectrometry probes are added into a cell suspension with a certain concentration, and the cell suspension is incubated for a period of time (usually 10min-30min) at a certain temperature (such as 37 ℃); after incubation, the cells are washed to remove unidentified components, typically using phosphate buffered saline, and the remaining flow mass spectrometry probe is removed by multiple centrifugations to discard the supernatant and resuspend the cells. And after washing, dispersing the cells in a dispersion liquid suitable for mass flow analysis by using a centrifugal resuspension mode again to obtain a cell sample to be detected.
Preferably, the concentration of the test cell sample is 10 to 1000000 cells/mL, and the test cell sample may be a cell of a single cell line, a cell of a mixed multiple cell lines, an unknown cell suspension sample, or the like.
Wherein the type of the flow mass spectrum probe incubated with the cell is the same as the type of the target protein of the concerned cell, and the quantity of each probe is added according to the ratio of the number of the probes to the number of the cells of 1:100000-1: 1000000. The flow mass spectrometry probe used is a high-sensitivity universal multifunctional nanoprobe, which comprises a noble metal nanoparticle inner core, and an antibody/aptamer and a mass spectrometry label which are self-assembled on the surface of the noble metal nanoparticle through a thiol and noble metal forming an M-S bond (M ═ Au, Ag and other noble metals) (see FIG. 1).
The invention also provides a preparation method of the flow mass spectrometry probe. The flow type mass spectrum probe is prepared by adopting a one-pot reaction method, and the sulfhydrylation antibody/aptamer and a mass spectrum label are combined on the noble metal nano particle in a self-assembly mode, and the method comprises the following specific steps: adding a thiolated antibody/aptamer solution into an aqueous solution of the noble metal nanoparticles, and reacting for a period of time (such as 10-16h) at room temperature in a dark place; adding mass spectrum label solution (mass spectrum label molecule is dissolved in aprotic solvent such as acetonitrile in advance) into the system, and reacting for a period of time (such as 10-16h) at room temperature in a dark place; centrifuging and washing for 3-5 times to remove unbound antibody/aptamer and mass spectrum tag; the nanoparticles are finally dispersed in a buffer solution (usually phosphate buffer). Wherein, the molar ratio of the noble metal nano-particles to the antibody/aptamer can be selected to be 1:10-1: 1000; the molar ratio of the noble metal nanoparticles to the mass spectrometry tags can be 1:1000-1: 10000.
In the preparation method of the flow mass spectrometry probe, the noble metal nanoparticles are preferably gold nanoparticles, silver nanoparticles and the like. The gold nanoparticles can be prepared by a method of reducing chloroauric acid by trisodium citrate, and the silver nanoparticles can be prepared by a method of reducing silver nitrate by trisodium citrate and sodium borohydride. The particle size of the noble metal nanoparticles is preferably 15nm to 25 nm.
The antibody/aptamer is preferably thiol-modified, and is selected to specifically bind to the target protein. The antibody may be subjected to a thiol modification at the amino terminus of the protein by reacting the antibody with a linker having a thiol group at one terminus and an N-hydroxysuccinimide at the other terminus (e.g., 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50, 53-hexadecaneoxa-28, 29-dithiapentahexadecanedioic acid di-N-succinimidyl ester) at a molar ratio of 2:1 in N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) sodium salt buffer solution at room temperature for 1 to 12 hours in the absence of light. The aptamer used is preferably a 5 'or 3' HS- (CH)2)6-a thiol-modified aptamer which can be a deoxyribonucleic acid aptamer or a ribonucleic acid aptamer.
The used mass spectrum label is a small organic molecule which can realize on-line dissociation and has good mass spectrum response, the molecular weight is within 1500Da, and the molecular structure general formula is shown as formula I:
in the formula I, n is an integer of 6-15, m is an integer of 0-8, R is a group with mass spectrum sensitizing capability and molecular weight of 50-1000Da, wherein the group contains but is not limited to one or more of quaternary ammonium group, pyridyl group, quinolyl group, amino group, alkyl substituted amino group (such as dimethylamino group and diethylamino group) and other electropositive groups or strong proton affinity groups, and is preferably a molecular group with an aromatic ring conjugated structure.
The mass spectrometry signature shown in formula I has three components: one end of the structure is sulfydryl, and the sulfydryl can be self-assembled on the surface of the noble metal nanoparticle through an M-S (M ═ Au, Ag and other noble metals) bond; the chain structure consisting of a rigid alkyl chain and a flexible polyethylene glycol chain is connected behind the sulfydryl, so that the self-assembly efficiency of the mass spectrum label on the surface of the nano particle can be improved, and the combination quantity of the mass spectrum label on the surface of the nano particle is increased; the other end of the structure is an R-group structure.
Preferably, the R group in formula I may be a structure having formula II as a main body:
in the formula II, x is an integer of 1-5, R1、R2、R3The same or different, and is C1-C4 short chain alkyl (such as methyl, ethyl, propyl, etc.).
Preferably, the R group in formula I may also be a structure based on formula III:
in the formula III, R1、R2、R3The same or different, is C1-C4 short chain alkyl (such as methyl, ethyl, propyl, etc.), R4Represents on phenylOne or more substituents are hydrogen or C1-C4 lower alkyl (such as methyl, ethyl, propyl, etc.). The connecting position of the polyethylene glycol chain in the formula I and the formula III can be in the ortho-position, the meta-position or the para-position of the phenyl; r4The attachment position of (a) may be at other unsubstituted sites on the phenyl.
Preferably, the R group in formula I may also be a structure having formula IV as a main moiety:
in the formula IV, R1Represents one or more substituents on the pyridyl and is hydrogen or C1-C4 short-chain alkyl (such as methyl, ethyl, propyl and the like). The position of the polyethylene glycol chain in formula I attached to formula IV may be at the 1N atom of the pyridyl group, or at the 2C, 3C, 4C, 5C or 6C position, R1The attachment position of (b) may be on an unsubstituted N atom or other unsubstituted C atom on the pyridyl group.
Preferably, the R group in formula I may also be a structure having formula V as a main moiety:
in the formula V, R1Represents one or more substituents on the quinolyl and is hydrogen or C1-C4 short-chain alkyl (such as methyl, ethyl, propyl and the like). The position of attachment of the polyethylene glycol chain in formula I to formula V may be at the 1N atom of the quinolinyl group, or at the 2C, 3C, 4C, 5C, 6C, 7C or 8C position. R1The attachment position of (b) may be on an unsubstituted N atom or other unsubstituted C atom on the quinolinyl group.
Preferably, the R group in formula I may also be a structure having formula VI as the main moiety:
in the formula VI, R1、R2The same or different, are hydrogen or C1-C4Lower alkyl groups (e.g., methyl, ethyl, propyl, etc.); r3Represents one or more substituents on the phenyl and is hydrogen or C1-C4 short-chain alkyl. The position of the polyethylene glycol chain in formula I attached to formula VI may be ortho, meta or para to the phenyl group, R3The attachment position of (a) may be at other unsubstituted sites on the phenyl.
Preferably, the R group in formula I may also be a structure based on formula VII:
in the formula VII, R1、R2、R3、R4The same or different, is hydrogen or C1-C4 short chain alkyl (such as methyl, ethyl, propyl, etc.); r5Represents one or more substituents at the 1C, 3C and/or 4C position, and is hydrogen or C1-C4 short chain alkyl; r6Represents one or more substituents at the 5C, 7C and/or 8C position, and is hydrogen or C1-C4 short chain alkyl; r7Represents one or more substituents at the 9C, 10C, 11C, 12C or 13C position, and is hydrogen or C1-C4 short-chain alkyl; wherein R is1And R2、R3And R4、R1And 1C position, R2And 3C position, R3And 5C position, R4And the 7C position may form a ring. The position of attachment of the polyethylene glycol chain in formula I to formula VII may be at the 9C, 10C, 11C, 12C or 13C position of the phenyl group.
The partial structure of the base R of the mass spectrum label has the characteristics of high ionization efficiency and high mass spectrum response in electrospray mass spectrometry detection, and a large number of mass spectrum labels are self-assembled on the noble metal nanoparticles and are marked on each target protein, so that the mass spectrum label has a signal amplification function.
The mass spectrum labels can be efficiently dissociated on line in the electrospray process, the break of an M-S bond is realized, and two mass spectrum labels are generated and connected through a disulfide bond. During the preparation process, the molar ratio of the nanoparticles to the mass spectrum label can be selected to be 1:1000-1: 10000.
The mass spectrum label used in the invention has strong expansibility, and by using the mass spectrum label shown in formula I, a series of mass spectrum labels with similar response capability but different m/z in mass spectrum detection can be generated by changing the short chain length of the short chain alkyl group in the R group, so that the mass spectrum label can be used for simultaneously labeling and detecting a plurality of protein target substances. Generally, the mass spectrometry tag mass-to-nuclear ratios bound to the surfaces of different flow mass spectrometry probes for different target proteins differ by more than 1.
In some embodiments of the invention, a 20nm diameter gold nanoparticle is used as the inner core, a 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50, 53-hexadecane oxa-28, 29-dithiapentahexadecane diacid di-N-succinimidyl ester linked CA125 antibody is selected, and the structure of the mass spectrum tag is shown as formula VIII; the molar ratio of the gold nanoparticles to the antibody to the mass spectrometry label is 1:24: 9000.
The mass spectrum labeled probe used in the step 1) can be prepared in advance and stored at the temperature of 4-8 ℃ for later use.
The dispersion finally used for dispersing the cells in the step 1) can be an aqueous solution containing volatile salts (such as ammonium acetate) with cell isotonic concentration or a solution obtained by mixing pure water and organic solution (such as methanol and the like) with cell fixing effect according to a certain ratio (such as 1:1, 2: 3). And a certain amount of internal standard substance is also added into the dispersion liquid for internal standard method quantification. The selectable internal standard substance is a compound which has a structure similar to that of the mass spectrum label but generates a mass-to-nucleus ratio obviously different from that of the mass spectrum label in mass spectrum detection, for example, when the molecule of the formula I with the R group having the structure shown in the formula VII is used as the mass spectrum label, the selectable internal standard substance is a compound such as crystal violet, methyl violet and the like, and the concentration of the internal standard substance in the spraying solvent can be selected from 0.05-0.5 mu mol/L. In the obtained mass spectrogram, the intensity of other mass spectrum signals is normalized by the intensity of an internal standard signal.
And step 2) realizing mass spectrum detection of the marked cell sample, wherein the adopted organic mass spectrum flow device is as described above. The specific analysis process of the cell sample by using the device is as follows: placing the sample tube filled with the cell dispersion liquid on a vortex mixer to enable the cells to be in a low-speed vortex state (such as 400rpm), inserting a peristaltic pump tube into the cell sample tube, setting the flow rate of the peristaltic pump, and injecting the sample by using the peristaltic pump; and opening mass spectrum scanning, applying a certain voltage to the ionization spray needle, and collecting mass spectrum data. The cell sample is firstly pumped into the cell dispersing and arranging device by a peristaltic pump to realize the monodispersion of the cells and make the cells sequentially flow into the ionization spray needle in a more ordered arrangement state, the dissociation of the mass spectrum label and the release of the metabolite are realized under the condition of electrospray, the mass spectrum detection of the label and the metabolite is realized, and the mass spectrum ion flow graph changing along with the time and the mass spectrogram acquired at each moment are recorded.
Preferably, the concentration of the cell sample analyzed in each time is 1000-10000/mL, the total volume is 0.5-1mL, and the flow rate of the sample injection by the peristaltic pump is set to be 0.5-10 muL/min.
The voltage applied to the ionization spray needle is direct-current high voltage, can be in a positive mode or a negative mode, and can be set to be 2-4 kV. The applied voltage may also be a pulsed high voltage, either positive or negative, with a peak of 2-4kV and a frequency of 10-10000 Hz.
The preferred mass spectrum is a high-resolution mass spectrum, the resolution is more than 30000, the acquisition mode is full scanning, the acquisition range is m/z 80-1200, the data acquisition time is more than 10min each time, and the mass spectrum information of 10-60 cells can be acquired every minute.
In one embodiment of the invention, 10000 per mL of cell dispersion samples marked by mass spectrum labels shown in formula VIII are analyzed, crystal violet is used as an internal standard, the sampling flow rate is 1 muL/min, the cell dispersing and arranging device is a 5-turn spiral micro-channel, the cross section of the micro-channel is 70 muM in width and 50 muM in height, and the distance between every two adjacent turns is 100 muM. The outlet of the channel is connected with a quartz glass capillary nozzle needle through a capillary tube with the inner diameter of 50 mu m, the inner diameter of the tip of the nozzle needle is 30 mu m, the outer diameter of the nozzle needle is 50 mu m, and +3kV direct-current high voltage is applied to the interior of the nozzle needle. And an Orbitrap mass spectrometer is adopted for detection, the resolution is set to be 35000, the acquisition range is m/z 80-1200, and the acquisition time is 10 min. In the mass spectra obtained, the mass spectrum tag of formula VIII gave a mass/z ratio of 628.3693(z 2), the internal standard crystal violet gave a mass/z ratio of 372.2340, and there were a number of intracellular metabolites with a mass/z ratio m/z in the range of 80-1200.
The step 3) is data analysis, and in the obtained total ion current chromatogram, a single cell mass spectrum is obtained at the peak of each single cell signal, and the single cell mass spectrum data processing specifically comprises:
3-1) extracting the signal intensity T of the label in the spectrogram according to the theoretical mass-nuclear ratio of the mass spectrum label1，T2，…，TaA is the quantity of the target protein, and the tolerance error of the nucleo-cytoplasmic ratio is within 5 ppm;
3-2) extracting the signal intensity M of the corresponding metabolite in the spectrogram according to the theoretical mass-nuclear ratio of the metabolite1，M2，…，MbB is the number of identified metabolites, the tolerance error of the proton to nuclear ratio is within 5 ppm;
3-3) extracting the internal standard signal intensity S in the spectrogram according to the theoretical mass-nuclear ratio of the internal standard molecules, wherein the tolerance error of the mass-nuclear ratio is within 5ppm, the mass spectrum label signal intensity and the metabolite signal intensity are normalized by taking the internal standard signal intensity S as a reference, and the normalized intensities are respectively T1/S，T2/S，…，Ta/S，M1/S，M2/S，…，Mb(S), recorded as mass spectrometric measurements for each cell sample: cx((T1/S)x，(T2/S)x，…，(Ta/S)x，(M1/S)x，(M1/S)x，(M2/S)x，…，(Mb/S)x) Is C detected in mass spectrumxAll information on the cells (mass spectrometry tags and metabolites), x represents the cell sample number;
3-4) summarizing the mass spectrum detection results of a plurality of cells into a data matrix, and taking the mass spectrum label and metabolite information in each cell as variables:
3-5) using the intensity values of the detected variables in single cells (i.e., the variables in the data matrix), using data analysis algorithms (e.g., principal component analysis, systematic clustering, etc.) for cell typing, identification, and differential analysis, among other related applications of mass cytometry.
The organic mass spectrum flow cytometry analysis technology provided by the invention is a single cell analysis technology which has high universality and high throughput and is suitable for multi-target simultaneous detection. Different target proteins can be labeled and detected simultaneously by using flow mass spectrometry probes with different mass spectrometry labels. In addition, due to the high mass resolution of mass spectrum, signals with small differences of mass-to-nuclear ratio can be easily separated in a mass spectrogram, so that the application of the organic mass spectrum flow analysis method in the simultaneous detection of multiple protein markers and multiple metabolites is ensured. The organic mass spectrometry flow cytometry technology can be used for cell identification, cell typing and research of characteristic difference substances in cells, and has wide application prospects in the fields of stem cell analysis, tumor diagnosis, system biology research and the like.
The flow mass spectrometry probe is a high-sensitivity protein labeling and detecting tool. The probe applies a plurality of signal amplification strategies to ensure that the sensitivity reaches the single cell analysis level. The problems of low ionization efficiency and low mass spectrum response of protein molecules in the mass spectrum detection process are solved by detecting mass spectrum labels with good mass spectrum responsiveness to replace target proteins; protein molecules are marked by a flow mass spectrometry probe, so that a large number of mass spectrometry labels are marked on one protein molecule, and detection signals are quantitatively amplified.
The flow mass spectrometry probe provided by the invention is a probe capable of realizing efficient online dissociation through M-S bond breakage under an electrospray condition. Under the voltage condition of 2-4kV, a large number of label molecules are rapidly dissociated from the surface of the nano particle on line, and the guarantee is provided for realizing high-flux flow cytometry analysis.
The device for cell dispersion and ionization in the invention is a flow cytometry device with simple structure, low cost, multifunction and high integration, has a plurality of functions of cell sample introduction, cell dispersion and arrangement, cell label dissociation, cell metabolite release and sample ionization, and can realize sample introduction analysis of 10-60 cells per minute. And the part of the device has universality and can be adapted to mass spectrum mass analyzers of various types.
Drawings
FIG. 1 is a schematic diagram of the labeling of cells by flow mass spectrometry probe cells of the present invention.
FIG. 2 is a schematic flow chart of an organic mass cytometry technique used in embodiments of the present invention.
FIG. 3 is a photograph of a microchannel chip used in an embodiment of the invention.
FIG. 4 is a scanning electron microscope photograph of a flow mass spectrometer probe prepared in an example of the present invention.
FIG. 5 is a total ion flow chromatogram and a single cell mass chromatogram obtained by detecting MCF-7 cells and MDA-MB-231 cells in the example of the present invention.
FIG. 6 is a graph showing the results of principal component analysis of MCF-7 cells and MDA-MB-231 cells according to the normalized signal intensities of metabolites and tags in the examples of the present invention.
FIG. 7 is a volcano plot of MCF-7 cells and MDA-MB-231 cells analyzed according to metabolite and tag differences in examples of the present invention.
Detailed Description
The technical solutions of the present invention are further illustrated by the following embodiments with reference to the drawings, but the protection scope of the present invention is not limited by the specific conditions of these embodiments.
Example (b): MCF-7 cells (C) were analyzed by the organic mass cytometry method of the present invention1) And MDA-MB-231 cells (C)2) And six cell surface antigens: CA125, CEA, EpCAM, CD24, CD44 and CD133 are target proteins, and the flow of organic mass spectrometry flow analysis is shown in FIG. 2.
The method comprises the following specific steps:
(1) a PDMS micro-channel chip (figure 3) with 5 circles is used, the channel width is 70 μm, the channel height is 50 μm, the distance between two adjacent circles is 100 μm, a peristaltic pump sample inlet pipe and a micro-channel chip inlet are connected, a micro-channel chip outlet is connected with a quartz glass capillary spray needle, the inner diameter of the tip of the spray needle is 30 μm, the outer diameter is 50 μm, and the connected device is arranged in front of an orbitrap mass spectrum inlet.
(2) MCF-7 cells and MDA-MB-231 cells which are cultured in a 6cm cell culture dish in an adherent way are taken as analysis objects. In the case of good cell growth state, the cells were digested with Accutase cell digesting enzyme, suspended cells were collected and counted by a cell counter, and the cell concentration was diluted to 10 with PBS4/mL。
Antibodies (CA125 antibody, CEA antibody, EpCAM antibody, CD24 antibody, CD44 antibody or CD133 antibody) and PEG- SH linker arm 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50, 53-hexadecane oxa-28, 29-dithiapentahexadecane diacid di-N-succinimidyl ester (NHS-PEG-S-PEG-NHS, MW 1109.26) (antibody: linker arm 2:1, molar ratio) were added to 10mM HEPES solution, respectively, and the antibodies were fixed at both ends of the linker arm by stirring at room temperature for 12 hours. Subsequently, 30. mu.L of the linked antibody (antibody concentration: 120. mu.g/mL) was added to 1mL of a 1nM gold nanoparticle solution, and potassium carbonate (to make the final concentration of potassium carbonate 1.8mM) was added and stirring was continued for 12 hours. Then, 90. mu.L of a mass spectrometric tag (formula XII: RMT331, formula IX: RMT387, formula X: RMT415, formula VIII: RMT443, formula XI: RMT467 or formula XIII: RMT491) with a concentration of 100. mu.M was added to the mixture to continue the reaction for 12 hours, after the reaction, centrifugation was carried out at 9000rpm for 15 minutes, the supernatant was discarded, and the mixture was centrifuged and washed twice in water, and finally each tube was dispersed in 200. mu.L of 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), and the six gold nanoprobes were mixed in equal volumes to prepare a projection electron microscope photograph of the obtained gold nanoprobes as shown in FIG. 4.
Cell concentration at 1mL of 104Adding 100 mu L of six gold nanoprobes mixed in equal proportion into/mL of cell suspension, incubating for 20min at 37 ℃ in the dark, washing the cells for 3 times by PBS, centrifuging and redispersing after each washing, washing for 1 time by water, centrifuging (1800rpm, 2min) and redispersing in 1mL of 60% cold methanol dispersion.
(3) Taking 1mL of marked and dispersed cell suspension, and immediately carrying out organic mass spectrometry, wherein the parameters of the mass spectrometry are set as follows: the vortex rotation speed is 400rpm, the sample injection flow rate is 1 mu L/min, the voltage value is set to be +3kV, the mass spectrum resolution is set to be 35000, the acquisition mode is a full scanning mode, the acquisition range is m/z 80-1200, and the acquisition time is 10 min.
(4) Extracting a mass spectrum of a single cell (for example, fig. 5), a mass spectrum of 145 MCF-7 cells and a mass spectrum of 121 MDA-MB-231 cells in total from the acquired mass spectrum ion current chromatogram, extracting 84 metabolites such as m/z 116.0706, 732.5538, 760.5851 and the like in the mass spectrum, and obtaining the mass spectrum labels m/z 516.2441, 572.3067, 600.3380, 628.3693, 652.3693, 676.3693) and the crystal violet internal standard m/z 372.2434 after dissociation, wherein the mass spectrum signal has an intensity corresponding to a mass spectrum signal with a mass-nuclear ratio error tolerance of less than 5 ppm: m1，M2，…，M84，T1，T2，…，T6And S. Normalizing the metabolite intensity extracted from each mass spectrogram by using the internal standard intensity as a reference to obtain the following data matrix:
based on the normalized signal intensity of each cell metabolite and label as variables, cell typing was performed by principal component analysis (fig. 6), fold change and P value were calculated, and differential analysis before comparing two cells with a volcanic chart was plotted (fig. 7).
Claims (15)
1. An organic mass spectrometry flow device for single cell analysis comprises a peristaltic pump, a cell dispersing and arranging device, an ionization spray needle and a mass spectrometry detector, wherein the peristaltic pump, the cell dispersing and arranging device and the ionization spray needle are sequentially connected through pipelines, and an outlet of the ionization spray needle is aligned to an inlet of the mass spectrometry detector; after being uniformly mixed, the cell sample with the mass spectrum label enters a cell dispersing and arranging device under the action of a peristaltic pump, the obtained monodisperse ordered-arranged cell effluent flows into an ionization spray needle, the online dissociation of the cell mass spectrum label and the online release of the cell content are realized, and the cell sample is detected by a mass spectrum detector after ionization.
2. The organic mass flow device of claim 1, wherein the peristaltic pump is a low flow rate peristaltic pump having a flow rate in the adjustable range of 0.1 μ L/min to 50 μ L/min.
3. The organic mass flow device of claim 1, wherein the cell dispersion and arrangement device is a microchannel chip comprising a plurality of turns of spiral channels of rectangular cross-section, the cross-section being rectangular with a height of 20-40 μm and a width of 30-80 μm, the number of turns of the spiral being 3-10.
4. The organic mass spectrometer flow device of claim 1, wherein the ionization needle is a hollow tube of glass or metal having an overall inner diameter of less than 100 μm, a tip at one end having an inner diameter of 20-30 μm and an outer diameter of less than 100 μm, and a non-tip at the other end connected to the outlet end of the cell dispersion and alignment device.
5. An organic mass spectrometry flow analysis method for single cell analysis, comprising the steps of:
1) sample C of cell suspension1，C2，…，CxWith multiple flow mass spectrometry probes G1，G2，…，GaThe mixture of (a) and (b) is incubated together to label the target protein of the cell, wherein x representsThe cell sample serial number, a represents the serial number of a flow mass spectrum probe, the flow mass spectrum probe comprises a noble metal nanoparticle inner core, an antibody and/or a nucleic acid aptamer and a mass spectrum label, wherein the antibody and/or the nucleic acid aptamer and the mass spectrum label are self-assembled on the surface of the noble metal nanoparticle through M-S bonds formed by sulfydryl and noble metal, and M represents a noble metal element;
2) performing mass spectrometric detection on the cell sample marked in step 1) by using the organic mass spectrometer of any one of claims 1 to 4;
3) and (3) data analysis: extracting a plurality of single-cell mass spectrograms from the mass spectrometric detection result in the step 2), normalizing mass spectrometric signals in each mass spectrogram according to the internal standard signal intensity to obtain normalized intensity signals of the marker and metabolite signals in each cell, and taking the intensity signals as the basis for cell typing, cell identification and cell differential analysis.
6. The method of claim 5, wherein step 1) comprises adding the flow mass spectrometry probe to the cell suspension, incubating the cell suspension, washing the cells to remove the unidentified components and the remaining flow mass spectrometry probe, and dispersing the cells in a dispersion suitable for mass spectrometry flow analysis to obtain a sample of the test cells having a concentration of 10-1000000 cells/mL.
7. The method for mass spectrometry flow analysis according to claim 6, wherein the dispersion used for dispersing the cells in step 1) is an aqueous solution containing volatile salts with cell isotonic concentration, or a solution obtained by mixing pure water and an organic solution with cell immobilization in a certain ratio, and a certain amount of an internal standard substance for internal standard method quantification is added to the dispersion.
8. The method of organic mass spectrometry flow analysis of claim 5, wherein the flow mass spectrometry probe is prepared by self-assembly in step 1): adding a thiolated antibody and/or a nucleic acid aptamer solution into an aqueous solution of the noble metal nanoparticles, and reacting for a period of time at room temperature in a dark place; adding a mass spectrum label solution into the system, and reacting for a period of time at room temperature in a dark place; centrifuging and washing to remove unbound antibodies and/or aptamers and mass spectrometry tags; finally, the nanoparticles are dispersed in a buffer solution.
9. The method for mass spectrometry flow analysis of claim 8, wherein in preparing the mass spectrometry probe, the molar ratio of the noble metal nanoparticles to the antibody and/or aptamer is 1:10 to 1:1000, and the molar ratio of the noble metal nanoparticles to the mass spectrometry tag is 1:1000 to 1: 10000; the particle size of the noble metal nano particles is 15nm-25 nm.
10. The method for mass spectrometry flow analysis of claim 5, wherein the mass spectrometry tag in the mass spectrometry probe in step 1) has a molecular weight within 1500Da and a general structural formula shown in formula I:
in the formula I, n is an integer of 6-15, m is an integer of 0-8, and R is a group with mass spectrum sensitizing capability and a molecular weight of 50-1000 Da.
11. The method of mass spectrometry flow analysis of claim 10, wherein in the mass spectrometry tag of formula I, the R group is one of the following groups a) to f):
a) a structure of formula II:
in the formula II, x is an integer of 1-5, R1、R2、R3Identical or different, is C1-C4 short chain alkyl;
b) a structure of formula III:
in the formula III, R1、R2、R3The same or different, is C1-C4 short chain alkyl; r4Represents one or more substituents on the phenyl and is hydrogen or C1-C4 short-chain alkyl; the connecting position of the polyethylene glycol chain and the formula III in the formula I is ortho-position, meta-position or para-position of the phenyl.
c) A structure of formula IV:
in the formula IV, R1Represents one or more substituents on the pyridyl and is hydrogen or C1-C4 short-chain alkyl; the connecting position of the polyethylene glycol chain in the formula I and the formula IV is on 1N atom of pyridyl, or on 2C, 3C, 4C, 5C or 6C position;
d) a structure represented by formula V:
in the formula V, R1Represents one or more substituents on the quinolyl and is hydrogen or C1-C4 short-chain alkyl; the position of the connection of the polyethylene glycol chain in the formula I and the formula V is on the 1N atom of the quinolyl group, or on the 2C, 3C, 4C, 5C, 6C, 7C or 8C position;
e) a structure of formula VI:
in the formula VI, R1、R2The same or different, is hydrogen or C1-C4 short chain alkyl; r3Represents one or more substituents on the phenyl and is hydrogen or C1-C4 short-chain alkyl; the connecting position of the polyethylene glycol chain in the formula I and the connecting position of the polyethylene glycol chain in the formula VI is ortho, meta or para of the phenyl;
f) a structure of formula VII:
in the formula VII, R1、R2、R3、R4The same or different, is hydrogen or C1-C4 short chain alkyl; r5Represents one or more substituents at the 1C, 3C and/or 4C position, and is hydrogen or C1-C4 short chain alkyl; r6Represents one or more substituents at the 5C, 7C and/or 8C position, and is hydrogen or C1-C4 short chain alkyl; r7Represents one or more substituents at the 9C, 10C, 11C, 12C or 13C position, and is hydrogen or C1-C4 short-chain alkyl; wherein R is1And R2、R3And R4、R1And 1C position, R2And 3C position, R3And 5C position, R4And 7C phase are mutually independent or form a ring; the position of attachment of the polyethylene glycol chain in formula I to formula VII is at the 9C, 10C, 11C, 12C or 13C position of the phenyl group.
12. The method of mass organic mass spectrometry of claim 11, wherein the mass spectrometry tag has a structure according to formula VIII, formula IX, formula X, formula XI, formula XII, or formula XIII:
13. the organic mass spectrometry flow analysis method of claim 5, wherein in step 2), the cell sample marked in step 1) is vortexed and mixed uniformly, then is input into a peristaltic pump with a sample injection flow rate set to be 0.5-10 μ L/min, enters a cell dispersing and arranging device under the action of the peristaltic pump, cell effluent liquid with monodisperse ordered arrangement is obtained and flows into an ionization spray needle, 2-4kV direct current high voltage or pulse type high voltage with a peak value of 2-4kV and a frequency of 10-10000Hz is applied to the ionization spray needle, online dissociation of a cell mass spectrometry label and online release of cell contents are realized, and detection is performed by a mass spectrometry detector.
14. The method of organic mass spectrometry flow analysis of claim 5, wherein step 3) obtains a single cell mass spectrum at the peak of each single cell signal in the obtained total ion current chromatogram, and performs the following data processing on the single cell mass spectrum:
3-1) extracting the signal intensity T of the mass spectrum label in the spectrogram according to the theoretical mass-nuclear ratio of the mass spectrum label1，T2，…，TaA is the quantity of the target protein, and the tolerance error of the nucleo-cytoplasmic ratio is within 5 ppm;
3-2) extracting the signal intensity M of the corresponding metabolite in the spectrogram according to the theoretical mass-nuclear ratio of the metabolite1，M2，…，MbB is the number of identified metabolites, the tolerance error of the proton to nuclear ratio is within 5 ppm;
3-3) extracting the internal standard signal intensity S in the spectrogram according to the theoretical mass-nuclear ratio of the internal standard molecules, wherein the tolerance error of the mass-nuclear ratio is within 5ppm, the mass spectrum label signal intensity and the metabolite signal intensity are normalized by taking the internal standard signal intensity S as a reference, and the normalized intensities are respectively T1/S，T2/S，…，Ta/S，M1/S，M2/S，…，Mb(S), recorded as mass spectrometric detection of each cell: cx((T1/S)x，(T2/S)x，…，(Ta/S)x，(M1/S)x，(M1/S)x，(M2/S)x，…，(Mb/S)x) And x represents the cell sample number.
15. The method of claim 14, wherein the mass spectrometric measurements of the plurality of cells obtained in step 3-3) are combined into a data matrix:
identification, typing or differential analysis of different cell types and other applications of mass flow based on individual variables in the data matrix.
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