Metabolomics tissue sample processing method

Abstract

The method for processing metabolomics tissue samples of the present invention comprises the following steps: cleaning the human metabolomics tissue samples with physiological saline; placing them in a first homogenization vessel, a second homogenization vessel and a third homogenization vessel, and then adding reagent 1, reagent 2 and reagent 3 respectively to the first homogenization vessel, the second homogenization vessel and the third homogenization vessel for grinding; transferring the metabolomics tissue samples in a uniform liquid state to a first collection device, a second collection device and a third collection device respectively, and immediately centrifuging the first collection device, the second collection device and the third collection device in a low-temperature centrifuge that has been balanced to 4°C to obtain supernatant, which is the three tissue metabolomics samples.

Description

Technical Field

[0001] The present invention relates to the fields of analytical chemistry and metabolomics, especially a method for processing metabolomics tissue samples. Background Art

[0002] Metabolomics is an important part of systems biology. It qualitatively identifies and quantitatively describes the expression and modification changes of small molecule metabolites in biological matrices to find the relative relationship between metabolites and physiological and pathological changes. At present, metabolomics has been widely applied in the fields of disease diagnosis and animal models, drug toxicity and mechanism research, and elucidation of gene functions. Although biological fluids such as blood and urine are easily obtained and their metabolites are related to the biochemical states at the cellular, tissue, and whole body levels, metabolic pathway changes caused by many diseases may only be detectable in the local microenvironment. Therefore, tissue research has always received much attention in metabolomics research.

[0003] In metabolomics research, the metabolite extraction rate, precision, and reproducibility of metabolomics tissue samples determine whether the metabolic changes of the body can be accurately observed. There are a wide variety of metabolites in metabolomics tissue samples, with large differences in physical properties, and the content of some metabolites is very low. The extraction process is affected by many aspects such as the extraction solvent system, extraction method, and enzyme activity. Currently, conventional extraction and processing methods are cumbersome, prone to contamination during the extraction and processing process, and prone to problems such as changes in the content of metabolites caused by the influence of biological enzymes. In addition, due to the often use of a single extraction solution, incomplete extraction of metabolites will also occur. Summary of the Invention

[0004] Aiming at the above technical problems, the object of the present invention is to provide a method for processing metabolomics tissue samples. This method has the characteristics of simple extraction process, complete extraction, strong operability, safety and environmental protection, and can be widely used in metabolomics research.

[0005] The present invention adopts the following technical solutions:

[0006] A method for processing metabolomics tissue samples of the present invention includes the following steps:

[0007] (1) Collect metabolomics tissue samples and wash the above metabolomics tissue samples clean with physiological saline;

[0008] (2) Weigh 20 - 30 mg of the washed metabolomics tissue samples respectively and place them in the first homogenization vessel, the second homogenization vessel, and the third homogenization vessel. Then add 200 - 1000 μl of reagent 1, reagent 2, and reagent 3 to the above first homogenization vessel, second homogenization vessel, and third homogenization vessel respectively, and carry out grinding until the above three portions of washed metabolomics tissue samples all become uniform liquid states;

[0009] (3) Transfer the metabolomics tissue samples in a uniform liquid state in step (2) to a first collection device, a second collection device, and a third collection device respectively;

[0010] (4) Immediately place the above-mentioned first collection device, second collection device, and third collection device in a low-temperature centrifuge that has been balanced to 4 - 8 °C for centrifugation. The centrifugation speed is 8000 - 10000 rpm, and the centrifugation time is 5 - 10 min to obtain the supernatant, which is three tissue metabolomics samples.

[0011] (5) Analyze the above three tissue metabolomics samples on the machine simultaneously;

[0012] In a method for processing a metabolomics tissue sample of the present invention, wherein: the reagent 1 is pure water containing 3 - 8% TCA; the reagent 2 is methanol; the reagent 3 is a 50% methanol aqueous solution containing 3 - 8% TCA;

[0013] In a method for processing a metabolomics tissue sample of the present invention, wherein: weigh trichloroacetic acid solid, dissolve it with pure water and 50% methanol aqueous solution respectively to obtain reagent 1 which is pure water containing 3% - 8% TCA, and reagent 3 which is a 50% methanol aqueous solution containing 3 - 8% TCA;

[0014] In a method for processing a metabolomics tissue sample of the present invention, wherein: the reagent 1 is pure water containing 5% TCA; the reagent 3 is a 50% methanol aqueous solution containing 5% TCA;

[0015] In a method for processing a metabolomics tissue sample of the present invention, wherein: the first collection device, the second collection device, and the third collection device are 2 ml plastic centrifuge tubes.

[0016] Advantages of the present invention:

[0017] The method for processing a metabolomics tissue sample of the present invention comprehensively extracts different polar metabolites, and timely terminates the enzyme reaction in the metabolomics tissue sample, so that the metabolite concentration can be maintained as close as possible to its true concentration in the body. Moreover, the processing process is simple and convenient, providing a reliable experimental basis for tissue metabolomics research. Description of the Drawings

[0018] Figure 1A For the measured concentration of arginine after treatment with reagent 1, reagent 2, and reagent 3;

[0019] Figure 1B For the measured concentration of C2 after treatment with reagent 1, reagent 2, and reagent 3;

[0020] Figure 1C For the measured concentration of C3 after treatment with reagent 1, reagent 2, and reagent 3;

[0021] Figure 1D The concentration of C4 was measured after treatment with Reagent 1, Reagent 2, and Reagent 3;

[0022] Figure 1E The concentration of C14 was measured after treatment with Reagent 1, Reagent 2, and Reagent 3;

[0023] Figure 1F The concentration of C16 was measured after treatment with Reagent 1, Reagent 2, and Reagent 3;

[0024] Figure 2A The concentration of arginine measured in the examples and comparative examples;

[0025] Figure 2B The concentration of C2 measured in the examples and comparative examples;

[0026] Figure 2C The concentration of C3 measured in the examples and comparative examples;

[0027] Figure 2D The concentration of C4 measured in the examples and comparative examples;

[0028] Figure 2E The concentration of C14 measured in the examples and comparative examples;

[0029] Figure 2F The concentration of C16 measured in the examples and comparative examples.

[0030] The present invention will be further described below in conjunction with specific examples and drawings. Detailed implementation manners

[0031] The present invention will be further explained below by examples. The examples are only for the purpose of facilitating the understanding of the present invention and are not intended to limit the present invention.

[0032] Example:

[0033] A method for processing metabolomics tissue samples, comprising the following steps:

[0034] (1) After obtaining the consent of the provider of the lung cancer metabolomics tissue sample, collect the lung cancer metabolomics tissue sample after the operation and wash it clean with physiological saline;

[0035] (2) Weigh three washed metabolomics tissue samples precisely, which are 25.90 mg, 27.93 mg, and 28.97 mg respectively, and place them in the first homogenization vessel, the second homogenization vessel, and the third homogenization vessel. Then, add 1000 μl of Reagent 1, Reagent 2, and Reagent 3 to the first homogenization vessel, the second homogenization vessel, and the third homogenization vessel respectively, and conduct grinding until the above three washed metabolomics tissue samples all become homogeneous liquid states. Among them, Reagent 1 is pure water containing 5% TCA; Reagent 2 is methanol; Reagent 3 is a 50% methanol aqueous solution containing 5% TCA. By weighing trichloroacetic acid solid and dissolving it with pure water and 50% methanol aqueous solution respectively, Reagent 1 of pure water containing 5% TCA and Reagent 3 of 50% methanol aqueous solution containing 5% TCA are obtained;

[0036] (3) Transfer the metabolomics tissue samples in the homogeneous liquid states in step (2) to the first collection device, the second collection device, and the third collection device respectively. Among them, the first collection device, the second collection device, and the third collection device are 2 ml plastic centrifuge tubes;

[0037] (4) Immediately place the above first collection device, second collection device, and third collection device in a low-temperature centrifuge that has been balanced to 4°C for centrifugation. The centrifugation speed is 10000 rpm, and the centrifugation time is 5 min. The supernatant obtained is the tissue metabolomics sample.

[0038] (5) Subject the above samples to pretreatment and then analyze them on the machine.

[0039] The experimental results are shown in Figure 1. It can be seen from Figure 1 that for amino acids and acylcarnitine metabolites with different polarities, using the method of the present invention and extracting with 3 different solvents, the extraction effects are different. Among them, arginine and acetylcarnitine have better extraction effects with pure water, propionylcarnitine and palmitoyl carnitine have better extraction effects with methanol, and butyryl carnitine and myristoyl carnitine have better extraction effects with 50% methanol aqueous solution. Therefore, it shows the necessity of using different extraction reagents during the collection process of tissue metabolomics samples.

[0040] Comparative Example

[0041] (1) Weigh the obtained lung cancer metabolomics tissue sample precisely, which is 48.26 mg, and place it in a 1 ml homogenization vessel;

[0042] (2) Accurately add 1000 μl of Reagent 3, and conduct sufficient grinding until the sample becomes a homogeneous liquid state;

[0043] (3) Transfer the homogenate to the collection device;

[0044] (4) Immediately place the above collection device into a low-temperature centrifuge pre-balanced to 4°C, centrifuge at a speed of 10,000 rpm for 5 minutes, and the supernatant obtained is the tissue metabolomics sample.

[0045] (5) Subject the above sample to pretreatment and then perform on-machine analysis.

[0046] The experimental results are shown in Figure 2. It can be seen from Figure 2 that by using the method of the present invention, not only is the required amount of metabolomics tissue sample smaller, but also the extraction effect is better, and it can better reflect its true concentration in vivo. The reason for the low measured concentration of a larger sample size may be that a larger amount of sample is difficult to fully grind in a homogenizer, or the amount of metabolite dissolved is limited in a limited solvent, etc.

[0047] In summary, a method for processing a metabolomics tissue sample provided in this embodiment is reliable in operation and has a wide range of applications. While ensuring the stability of metabolites and extraction efficiency, it simplifies the operation steps, enables the obtained sample to be qualitatively and quantitatively detected by a variety of instrument detection methods, and obtains more accurate and reliable results. The metabolomics research of tissue samples will be a good complement to the metabolomics research of biological fluids.

[0048] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for processing metabolomics tissue samples, comprising the following steps: (1) Collect metabolomics tissue samples, and wash the metabolomics tissue samples clean with physiological saline; the metabolomics tissue samples are human lung cancer metabolomics tissue samples; (2) Weigh 20 - 30 mg of the washed metabolomics tissue samples respectively, and place them in a first homogenization vessel, a second homogenization vessel, and a third homogenization vessel. Then add 200 - 1000 μL of reagent 1, reagent 2, and reagent 3 to the first homogenization vessel, the second homogenization vessel, and the third homogenization vessel respectively, and conduct grinding until the above three washed metabolomics tissue samples all become homogeneous liquid states; The reagent 1 is pure water containing 5% TCA; it is obtained by weighing trichloroacetic acid solid and dissolving it with pure water; the reagent 2 is methanol; the reagent 3 is a 50% methanol aqueous solution containing 5% TCA; it is obtained by weighing trichloroacetic acid solid and dissolving it with a 50% methanol aqueous solution; (3) Transfer the metabolomics tissue samples in a homogeneous liquid state in step (2) to a first collection device, a second collection device, and a third collection device respectively; (4) Immediately place the above first collection device, second collection device, and third collection device in a low-temperature centrifuge that has been balanced to 4 - 8 °C for centrifugation, with a centrifugation speed of 8000 - 10000 rpm and a centrifugation time of 5 - 10 min, and the obtained supernatant is three tissue metabolomics samples; (5) Analyze the above three tissue metabolomics samples on the machine simultaneously; analyze amino acids and acylcarnitine metabolites with different polarities; according to the extraction effect, select arginine and acetylcarnitine for extraction with reagent 1; select propionylcarnitine and palmitoyl carnitine for extraction with reagent 2; select butyryl carnitine and myristoyl carnitine for extraction with reagent 3.

2. The processing method according to claim 1, wherein The first collection device, the second collection device, and the third collection device are 2 ml plastic centrifuge tubes.

Images