What is mass cytometry(MC)?


mass cytometry

Mass cytometry(MC) is the application of inductively coupled plasma mass spectrometry (ICP-MS) to the analysis of single cells. The principle is to label antibodies with purified single element isotopes, and then use ICP-MS to detect the element ions. peak. The technology combines flow cytometry and ICP-MS. The operation process is simple. First, the cells are labeled with specific antibodies with metal elements (usually lanthanide metals) 3-4, and then sent to the mass flow cytometer. The cells are atomized in a single cell droplet state and then enter a high temperature. Plasma generates free atoms, and the quadrupole selects only ions within the mass range of lanthanide metals, removes the remaining ions, and uses TOF mass spectrometry to detect the relative signal intensity of lanthanide metal ions. Mass cytometry was first developed by researchers at the University of Toronto. The first commercial mass cytometer was named Cytometry by Time-Of-Flight (CyTOF), and the related antibody reagents were produced and sold by DVS Sciences.

Traditional flow cytometry uses fluorophores as reporter molecules to determine the expression level of target molecules by quantifying the emission intensity, but the emission spectra of fluorophores often overlap, especially when multi-parameter measurements are performed in the same experiment , it is difficult to distinguish multiple fluorophores. Mass cytometry uses stable lanthanide metals of a single molecular weight instead of fluorescent groups as reporter molecules. The elemental mass spectrometer can accurately distinguish different atomic masses by molecular weight, and there is no signal overlap between different lanthanide metal masses, which increases the accuracy of quantification. .

At present, mass cytometry can simultaneously detect 51 target proteins, detect 1000 cells per second, and an average of 100 samples per day, with a minimum detection limit of 100 copies of molecules, and the validated lanthanide metal-labeled antibodies More than 400 species; in addition to conventional proteins, mass cytometry can also be used for protein post-translational modifications1, protein degradation products5, detection of cell viability, cell size, mRNA transcript expression6, DNA synthesis rate7 and protease Determination of activity 8 etc.

Just like label-free quantification in proteomics, the quantification accuracy of mass cytometry for targets is also affected by differences in ionization efficiency of different instruments, instrument status, etc. At the same time, traditional mass cytometry only Can detect one sample with low throughput. Therefore, mass-tag cellular barcoding (MCB) came into being.

After the cells are treated with drugs, etc., and fixed with paraformaldehyde, MCB reagents are used to barcode the cells of different samples, and then the different samples are mixed for pretreatment and detection by conventional mass spectrometry flow cytometry. Finally, the cell samples are classified by detecting the element ions corresponding to the barcodes.

Differently treated samples were fixed with formaldehyde, labeled with different mDOTA molecules, and then subjected to conventional mass spectrometry flow cytometry pretreatment and detection.

The mDOTA molecule used here is a bifunctional compound, one end can chelate metal ions, and the other end can be combined with the free sulfhydryl group on the protein in the cell in the form of a covalent bond. One mDOTA molecule can chelate a lanthanide metal atom, then Why is it called barcode? Barcode means that a variety of mDOTA molecules chelated by lanthanide metal elements can be added at the same time. After the covalent bond of CyTOF is broken, the peak of each lanthanide metal element can be formed with or without two cases. The lanthanum used The more types of mDOTA reagents chelated by metal elements, the more types of barcodes that can be formed in the end. Using 7 kinds of lanthanide metal elements chelated mDOTA reagents can form 2 to the 7th power barcode, that is, 128 barcodes can be marked at the same time. sample. The use of MCB enables the simultaneous quantitative analysis of single cells in multiple samples, which increases the analytical throughput of mass cytometry and increases the accuracy of relative quantification.

Cells are covalently labeled with the bifunctional molecule mDOTA (maleimido-mono-amide-DOTA). One end of mDOTA can chelate rare earth elements, and the other end can react with sulfhydryl groups of intracellular proteins.

However, MCB has certain defects. First, after each instrument calibration, the ion detection sensitivity of the mass cytometer will change. Although it can be calibrated with an internal reference, the detection after mixing the samples will reduce the degree of variation between samples; secondly , the lanthanide metal elements used by MCB and the lanthanide metal coupled with the antibody used later cannot be the same, so the use of MCB narrows the choice of subsequent labeled antibodies. Therefore, MCB reagents based on palladium were further developed. The construction of MCB molecules using the 6 isotopic atoms of palladium has the following two advantages: First, because palladium is incompatible with the reagent for labeling antibodies (Diethylene triamine pentaacetic acid (DTPA)-based polymer), palladium will not be used for antibody labeling. Therefore, the use of palladium in the MCB molecule will not interfere with the selection of subsequent labeled antibodies; second, Pd has 6 stable isotopes, 102, 104, 105, 106, 108 and 110 amu, with high purity, 91%, 96 %, 98%, 99%, 99% and 99%, these isotopes are far from other lanthanide metal elements in mass and will not affect the subsequent detection of antibody-coupled lanthanide metal elements.

Seven kinds of lanthanide metal elements are used to chelate mDOTA reagents, and mDOTA reagents are added to form 2 to the 7th power barcode, that is, 128 samples can be marked simultaneously.

Advantages and disadvantages of mass cytometry

The advantage is that there are many channels that can be detected at the same time, and now 51 target proteins can be detected at the same time; the operability is strong, and the reagents are all commercialized; although the speed is not comparable to the flow cytometer (10,000 cells per second), but It is not slow, and can detect 1000 cells per second; the cost is low, the average measurement cost per cell is only 0.005 cents, compared with the average of 22 US dollars per cell for single-cell sequencing technology, it can be said to be quite cheap.

Of course, there are still many shortcomings in this technology. First, the cells are nebulized and ionized during the analysis, and the intact living cell state cannot be retained after the analysis; Second, the sensitivity is lower than that of the fluorophore, and the molecules expressed at very low levels cannot be detected, and the dynamic range of the detection can only reach at most 3-4 times, and the detection range of fluorophores can reach about 50 times; third, the ionization efficiency between different instruments is different, and it is necessary to correct the signal intensity by incorporating polystyrene beads to achieve the same instrument. The comparison of the data from the previous studies; Fourth, like fluorophore-based flow cytometry, mass cytometry still cannot detect small molecule metabolites and cannot perform continuous dynamic monitoring of a cell; Finally, and most importantly One point, not all proteins in the proteome can be monitored, only specific target proteins can be detected, so a more rigorous experimental design is required.

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