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Single-cell single-tube sample preparation and single-cell proteomics analysis method

A proteomics and sample preparation technology, applied in the field of single-cell single-tube sample processing and single-cell proteomics analysis, which can solve problems such as low enzymatic hydrolysis efficiency, reduced enzymatic hydrolysis efficiency, and protein loss.

Inactive Publication Date: 2021-05-25
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the fact that proteins do not have the characteristics of direct amplification and the sensitivity of proteomics analysis is limited, the detection of proteins in a small amount or even a single cell is technically very challenging.
The biggest difficulty in single-cell proteomics analysis is that the amount of sample is very small, and the protein of a single cell (such as a single human cell) is about 100-200pg, and the protein of the cell is lost due to adsorption on the inner wall of the test tube during the processing, giving Sample preparation poses great difficulties
Conventional proteome sample pretreatment methods are only suitable for a large number of cell samples, and the enzymatic hydrolysis efficiency is low when the conventional reaction system is applied. When the sample concentration is very low (single-cell samples), the enzymatic hydrolysis efficiency will also decrease at this time, so There is an urgent need to develop a series of innovative technologies to solve this problem
At present, there is no mature single-cell proteomics analysis method. The proteomics sample processing method based on single-tube processing proposed by us is simple, easy to operate, strong universality, and good practicability

Method used

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  • Single-cell single-tube sample preparation and single-cell proteomics analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: DDM pretreatment research

[0054] (1): Multicellular research

[0055] To achieve precise quantification of the proteome of individual cells, we systematically assessed sample recovery using a homogeneous protein sample (i.e., cell lysate) in a single PCR tube.

[0056] Cell lysis: MCF cells in the growth phase were washed twice with ice-cold PBS solution, and 1ml of ice-cold PBS solution (containing 1% phosphatase inhibitor cocktail (Pierce), 10mM NaF (Sigma)) was added to collect the cells in a PCR tube. The cells were centrifuged at 1500rpm at 4°C for 10min, and the excess PBS solution was discarded. Then add cell lysis buffer (250mM HEPES, 8M urea, 150Mm Nacl, 1% Triton X-100, pH 6.0) to resuspend the cell pellet, the ratio of lysis buffer to cell pellet is 3:1. Centrifuge at 14000rpm at 4°C for 10min. Reserve Soluble protein component. Protein concentration was determined by BCA method.

[0057] Add 1 μg of cell lysate (approximately equal to 1000MC...

Embodiment 2

[0063] according to figure 2 Schematic diagram of the single-cell proteomics study on the human lung adenocarcinoma cell line Hcc827:

[0064] 1. Cell culture

[0065] Take out the cryopreservation tube containing the human lung adenocarcinoma cell line Hcc827, put it in a 37°C water bath, and shake it constantly to make it melt quickly; after the cells are thawed, take it out of the water bath, and centrifuge at 1000rpm for 5min; Remove the supernatant, add 1ml of fresh medium (1640+10% fetal bovine serum+1% sodium glutamate+1% sodium pyruvate) to resuspend; transfer to a culture bottle for static culture; cell culture at 37°C, 5% CO2 constant temperature and humidity incubator.

[0066] 2. Cell picking

[0067] Add 0.1% DDM to the PCR tube, fill it up completely (completely immerse the inner wall of the PCR tube), let stand at room temperature overnight (at least 12h), suck out the DDM solution, and air-dry the PCR tube naturally in a fume hood.

[0068] Select cancer c...

Embodiment 3

[0085]Compared with Example 2, the only difference is that during the cell picking process, 1, 5, 10, and 100H1975 cell samples were picked respectively for proteomics research (that is, the difference is only that the number of cells picked Different, other steps are with embodiment 2). For example, during the cell picking process, 1, 5, 10, and 100H1975 cells were picked from the fresh cell suspension using the serial dilution method and put into a single DDM-pretreated PCR tube. The sample in the sample was digested by enzyme, and the PCR tube was capped and inserted into a 2ml sample bottle for LC-MS / MS analysis.

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Abstract

The invention belongs to the technical field of cell proteomic analysis, and particularly relates to a single-tube treatment-based single-cell proteomic analysis sample preparation method, which comprises the following steps: pretreating the inner wall of a test tube by adopting DDM; adding a single cell to be determined into the pretreated test tube; using a single test tube (such as a PCR (Polymerase Chain Reaction) tube) to carry out cracking, protein denaturation, reductive alkylation, enzymolysis and the like on single cells, and obtaining a to-be-tested polypeptide solution for single cell proteome analysis in the test tube. The invention also comprises a method for directly injecting the obtained sample to be detected and carrying out liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. The problem of single-cell protein loss can be effectively solved, and the effect of single-cell proteomics analysis can be effectively improved. Besides, the method is simple, convenient and efficient, does not need additional instruments and other reagents, can be easily implemented in a common proteomics laboratory, and provides a new analysis tool for the development of single-cell proteomics.

Description

technical field [0001] The invention belongs to the field of protein analysis, in particular to a single-cell single-tube sample processing and a single-cell proteomics analysis method. Background technique [0002] Proteomics is one of the post-genome projects, and it is also a hot research field in recent years. Compared with genes, proteins, which are the direct executors of functions in the living body, have greater significance in revealing the mechanism of life development and the occurrence and development of diseases. [0003] In recent years, proteomics research based on cell populations has become increasingly difficult to meet the needs of more in-depth exploration of life functions because it will inevitably average the information in a large number of cells. Understanding the characteristics of cells and their interactions at the single-cell level can provide more valuable information on the heterogeneity among cells in biological systems, so there is an increa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/88
CPCG01N30/02G01N30/06G01N30/72G01N30/88G01N2030/067G01N2030/8831G01N2570/00
Inventor 张鹏飞贺毅
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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