Method for treating extremely micro tissue sample by one-step method

Abstract

The invention belongs to the technical field of proteomics, and particularly relates to a method for treating an extremely-micro tissue sample through a one-step method. The method comprises the following steps: 1, preparing a sample; 2, obtaining a peptide fragment: adding Mix Buffer into an ep tube, incubating after non-contact ultrasonic treatment, finally adding an acid solution to terminate enzyme digestion, and centrifuging to take a supernatant; the Mix Buffer is composed of rapid trypsin, CAA, TECP, DDM and a rapid trypsin buffer solution. According to the method, the Mix buffer is optimized, all steps of cracking, reduction, alkylation and enzyme digestion are combined into one step, the steps are simple, the cost is low, the sample preparation time is greatly saved, and a better proteomics test effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of proteomics, and in particular relates to a one-step method for processing extremely trace tissue samples. Background Art

[0002] Sample pretreatment is an extremely important step in proteomics research. Before mass spectrometry analysis, different types of samples need to be subjected to protein cleavage, reduction alkylation, protease hydrolysis, and desalting to obtain peptide samples that are suitable for machine collection. However, in the process of sample preparation, there are multiple steps and the sample usually needs to be transferred multiple times. For trace samples, due to the limitation of sample size, conventional preparation methods lead to high sample loss. Therefore, it is of great significance to develop a sample processing technology with simple steps and shortened time for the processing of trace samples.

[0003] At present, researchers have developed a variety of methods for processing trace samples, such as the FASP (Filter-Aided Sample Preparation) method, which retains proteins in the device by centrifugation using a molecular weight cutoff (MWCO) ultrafiltration device, and completes enzymatic digestion in the device to obtain peptides that meet the needs of the machine. This method is relatively stable and compatible with a wide range of lysates, but the sample loss is large when the sample amount is low; the SP3 (Single-Pot, Solid Phase-enhanced Sample-Preparation) method uses the hydrophilic carboxyl or amine coating on the surface of magnetic beads to complete the binding with the free amine or carboxyl groups of proteins under the induction of organic solvents, and further performs the enzymatic digestion step. The SP3 method adsorbs proteins on magnetic beads through chemical binding to reduce sample loss and is suitable for samples of different starting amounts. However, the aggregation of magnetic beads during the sample preparation process of the SP3 method will hinder the adaptation of high-throughput experimental systems; SISPROT (Simple and Intergrated Spintip-based Proteomics The In-Stage Tip Digestion (IST) method integrates all sample preparation on a Tip column, shortening the time and loss of sample preparation. However, this method requires multiple elution treatments to obtain peptide samples that meet the loading conditions and is not compatible with high salt concentration solutions, resulting in high experimental costs. Another common method is the In-Stage Tip digestion (IST), which is similar to FASP and uses a C18 membrane as a physical barrier to block insoluble substances and macromolecules, and elutes peptides through solid phase extraction.

[0004] The above methods all require a protein extraction step and then protein treatment when used, and the obtained polypeptide needs to be further spin-dried and then redissolved, which is time-consuming and complicated to operate, and inevitably causes sample loss during the operation. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention aims to provide a simple and rapid one-step preparation method for extremely small amounts of proteomics samples. The entire pre-treatment process of protein cleavage and enzymatic hydrolysis of the micro-sample is completed in one step. After the pre-treated sample is acidified and centrifuged, it can be directly loaded. The method of the present invention is rapid and stable and is particularly suitable for the pre-treatment of extremely small amounts of samples.

[0006] The present invention provides a one-step method for processing an extremely small amount of tissue sample, comprising the following steps:

[0007] 1. Prepare the sample: Take a small amount of sample and place it in an EP tube, and perform corresponding preparations according to the sample type;

[0008] 1.1 For fresh tissue samples: add PBS buffer containing protease inhibitors to the ep tube, shake in a metal bath at 4°C, then centrifuge and discard the supernatant. Repeat three times;

[0009] 1.2 For FFPE samples: add dewaxing agent to the sample tube for dewaxing, use different concentrations of alcohol gradient to rehydrate, discard the supernatant, and then open the lid to dry;

[0010] 2. Obtain peptides: Add Mix Buffer to the ep tube, incubate after non-contact ultrasound, and finally add acidic solution to terminate enzyme cleavage, and centrifuge to obtain the supernatant;

[0011] The Mix Buffer consists of rapid trypsin, CAA, TECP, DDM and rapid trypsin buffer.

[0012] For fresh tissue and FFPE tissue samples after dewaxing treatment, the present invention optimizes the Mix buffer components to adjust the enzyme components, adds a reducing agent and an alkylating agent, and combines all the steps of cleavage-reduction-alkylation-enzyme cleavage into one step. The steps are simple and low in cost, and the multi-step time-consuming operation of desalting and the time-consuming spin drying and re-dissolution operations are omitted, thereby further reducing the loss of samples and greatly saving the time of sample preparation.

[0013] The fast trypsin can be a commonly used commercially available fast trypsin. The fast trypsin buffer can be a buffer that matches it and is recommended by the merchant.

[0014] In one embodiment of the present invention, for fresh tissue samples, in step 1.1, the amount of PBS buffer containing protease inhibitors added is 500 μL, and the sample is shaken in a metal bath at 4° C. for 2 min, followed by centrifugation at 1500 g for 3 min.

[0015] In one embodiment of the present invention, for FFPE samples, step 1.2 comprises:

[0016] 1) Incubate the FFPE wax roll sections at 65°C for 60 min;

[0017] 2) Centrifuge the incubated slices to the bottom of the tube;

[0018] 3) Add 1 mL of wax melting reagent, shake at 800 rpm, 37°C for 10 min; centrifuge at 16000 rcf for 3 min, and discard the supernatant;

[0019] 4) Repeat 2 or more times, such as 2 to 3 times, 3 to 5 times, etc., and absorb the liquid completely the last time;

[0020] 5) Add 1 mL of anhydrous ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant;

[0021] 6) Add 1 mL of 90% ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant;

[0022] 7) Add 1 mL of 75% ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant;

[0023] 8) Add 200 μL of water, shake at 800 rpm, 37°C for 2 min; centrifuge at 17000 rcf for 5 min, and discard the supernatant;

[0024] 9) Open the lid and put it in the oven to dry.

[0025] In one embodiment of the present invention, in step 2, the concentration of rapid trypsin in the Mix Buffer is 0.02-0.05 μg / μL, the concentration of CAA is 4 mM, the concentration of TECP is 1 mM, and the concentration of DDM is 0.4%. Preferably, the concentration of rapid trypsin in the Mix Buffer is 0.03 μg / μL, the concentration of CAA is 4 mM, the concentration of TECP is 1 mM, and the concentration of DDM is 0.4%.

[0026] In one embodiment of the present invention, in step 2, the non-contact ultrasonic conditions are 20s off, 20son, ultrasonic power 85%, and ultrasonic time 5min.

[0027] In one embodiment of the present invention, in step 2, the incubation conditions are 70° C. and 500 rpm for 1-2 h.

[0028] In one embodiment of the present invention, in step 2, the centrifugation condition is 14000g for 10 min.

[0029] In one embodiment of the present invention, in the step 2, the acidic solution is 10% FA.

[0030] In one embodiment of the present invention, step three is also included: preparation for the machine: using Nano drop to detect the peptide concentration, and preparing a peptide sample of 100 ng / μL that meets the machine requirements.

[0031] In one embodiment of the present invention, the trace sample in step 1 is a tissue sample with a starting amount of ≤1 mg, which can be a tissue sample of the brain, heart, liver, lung, or other organs.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] For fresh tissue and FFPE tissue samples after dewaxing treatment, the present invention combines all the steps of lysis-reduction-alkylation-enzyme cleavage into one step by optimizing the Mix buffer. The step is simple and the cost is low. The multi-step time-consuming operation of desalting and the time-consuming spin drying and re-dissolution operations are omitted, which further reduces the loss of samples and greatly saves the time of sample preparation (from the conventional about 24 hours to 2.5 hours). It also achieves better proteomics test results and is particularly suitable for extremely trace tissue samples with a starting amount of less than 1 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 To compare protein identification and peptide identification of different tissues and with the literature;

[0035] Figure 2 Identification quantity for FFPE samples;

[0036] Figure 3 Comparison of different sample preparation methods. DETAILED DESCRIPTION

[0037] The present invention is further described in detail in conjunction with the following specific embodiments and drawings. The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited in the present invention.

[0038] Example 1 Mix buffer composition optimization

[0039] This example tests the effect of different Mix buffer components on identification under the same other conditions, wherein the amount of enzyme used is adjusted, reducing agents and alkylating agents are added or subtracted, and different buffers are used for sample preparation to compare the identification amount.

[0040] It can be seen that the identification results of rapid trypsin in aqueous solution are the worst; it is improved in rapid trypsin buffer. The addition of DDM and CAA must be combined with appropriate rapid trypsin concentrations to achieve better identification results.

[0041] After trying different Mix buffers and comparing the results, we found that the best identification condition was 0.03μg / μL rapid trypsin+0.4%DDM+1mMTECP+4mM CAA+rapid buffer, with 7487 proteins and 112593 peptides identified. This condition was used as the Mix buffer formula for subsequent experiments.

[0042] Table 1 Comparison of different experimental conditions

[0043]

[0044] Example 2 Fresh tissue sample processing

[0045] Different types of fresh frozen tissue samples from mice were used for microsample processing. The specific steps are as follows:

[0046] 1) Use sterilized scissors and forceps to weigh 2 mg of different tissue samples, place the tissue samples in a culture dish, divide the tissue samples into 6 equal parts, and place all tissue fragments in a 1.5 mL centrifuge tube.

[0047] 2) Add 500 μL of PBS containing protease inhibitors to the centrifuge tube, shake in a metal bath at 4°C for 2 min, then centrifuge at 1500 g for 3 min, and discard the supernatant.

[0048] 3) Repeat 6 times for each sample, add 30 μL mix buffer (the formula is the optimal formula in Example 1) to each sample tube, add millet-sized washed tissue fragments, use non-contact ultrasound to extract protein, then incubate at 70°C, 500 rpm for 1-2 h, add 10% FA to acidify to terminate enzyme cleavage, and centrifuge at 14000g for 10 min to remove the supernatant.

[0049] 4) Use nanodrop to measure peptide concentration, and prepare each sample at 100 ng / μL for loading and testing.

[0050] The processed samples were collected by Thermo U3000 liquid chromatography coupled with Bruker Tims-Tof. 300 ng of each sample was collected for 120 min in DIA mode. The collected data were analyzed by Direct DIA using spectronaut 18. The search results were collated and analyzed, and compared with the published literature data. Figure 1 shown.

[0051] Different tissues were prepared using the method of the embodiment of the present invention. The mean protein identification of kidney tissue was 8200, and the mean peptide identification was 122760; the mean protein identification of spleen tissue was 8297, and the mean peptide identification was 115923; the mean protein identification of lung tissue was 8500, and the mean peptide identification was 136216. Referring to the library data in the literature of Tian Lu et al., kidney tissue protein identification was 6911, peptide identification was 55840; spleen tissue protein identification was 7472, peptide identification was 65700; lung tissue protein identification was 6064, and peptide identification was 48210; after comparison, the amount of protein and peptide identified by the method of the present invention was greater than the reference data in the literature, indicating that this method is suitable for sample preparation of different tissue types, and the results are relatively stable.

[0052] Example 3 FFPE wax roll processing

[0053] Use FFPE wax rolls of different tissues to process trace samples. The specific steps are as follows:

[0054] 1) Place the wax roll in an oven at 65°C and incubate for 60 minutes.

[0055] 2) Centrifuge the slices to the bottom of the tube.

[0056] 3) Add 1 mL of wax melting reagent, shake at 800 rpm, 37°C for 10 min; centrifuge at 16000 rcf for 3 min, and discard the supernatant.

[0057] 4) Repeat 2-3 times, absorb the liquid cleanly the last time, and wash several times.

[0058] 5) Add 1 mL of anhydrous ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant.

[0059] 6) Add 1 mL of 90% ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant.

[0060] 7) Add 1 mL of 75% ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant.

[0061] 8) Add 200 μL of water, shake at 800 rpm, 37°C for 2 min; centrifuge at 17000 rcf for 5 min, and discard the supernatant.

[0062] 9) Open the lid and put it in the oven to dry for 5 minutes.

[0063] 10) Add to a centrifuge tube containing 15 μL of mix buffer (the formula is the optimal formula in Example 1), non-contact ultrasound for 5 min, 20 s off, 20 s on, 85%, incubate at 70°C for 2 h. Add 10% FA to acidify and terminate the enzyme digestion.

[0064] 11) Take out the sample, centrifuge at 14000g for 10 min, take the supernatant to measure the concentration, use nanodrop to measure the peptide concentration, and prepare each sample at 100ng / μL for sample loading and detection.

[0065] The processed samples were collected by Thermo U3000 liquid chromatography coupled with Bruker Tims-Tof, with 300 ng of each sample collected for 120 min in DIA mode. The collected data were analyzed using Spectronaut 18 with Direct DIA library search.

[0066] The search results show ( Figure 2 ), the protein identification amount of brain, heart, liver and lung samples was above 6000, among which liver and lung samples obtained better identification amount compared with the laboratory multi-step FFPE preparation method, indicating that the patent of the present invention is suitable for FFPE sample preparation.

[0067] Example 4 Comparison of different treatment methods

[0068] The fresh tissue samples of rat liver and FFPE (wax roll) tissues in Examples 2 and 3 were compared with the conventional multi-step sample preparation method and PCT (pressure cycling technology) technology. The results are as follows: Figure 3 shown.

[0069] Conventional multi-step sample preparation requires multiple steps such as protein extraction, peptide enzymatic cleavage, desalting, and thermal evaporation. The entire process usually takes 2 days.

[0070] PCT is a rapid pre-treatment technology that uses specific temperatures and time intervals to allow trace samples to undergo programmed, periodic cycles between normal pressure and ultra-high pressure, thereby destroying the physical structure of biological samples and achieving the purpose of precise advance. This method can extract peptides that can be used for product analysis from tissues within 3 hours.

[0071] The processing method of the embodiment of the present invention combines all the steps into one step, which is simple and low-cost, and eliminates the multi-step time-consuming operation of desalting and the time-consuming spin drying and re-dissolution operations, further reducing the loss of samples and saving sample preparation time (from the conventional approximately 24 hours to 2.5 hours). The results show that the method of the present invention has good performance in both protein and peptide identification.

[0072] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A one-step method for processing extremely small amounts of tissue samples, characterized in that: The following steps are involved:

1. Prepare the sample: Take a small amount of sample and place it in an EP tube, and perform corresponding preparations according to the sample type; 1.1 For fresh tissue samples: add PBS buffer containing protease inhibitors to the ep tube, shake in a metal bath at 4°C, then centrifuge and discard the supernatant. Repeat three times; 1.2 For FFPE samples: add dewaxing agent to the sample tube for dewaxing, use different concentrations of alcohol gradient to rehydrate, discard the supernatant, and then open the lid to dry; 2. Obtain peptides: Add Mix Buffer to the ep tube, incubate after non-contact ultrasound, and finally add acidic solution to terminate enzyme cleavage, and centrifuge to obtain the supernatant; The Mix Buffer consists of fast trypsin, CAA, TECP, DDM and fast trypsin buffer.

2. The method according to claim 1, characterized in that In the step 1.1, 500 μL of PBS buffer containing protease inhibitors was added, shaken in a metal bath at 4° C. for 2 min, and then centrifuged at 1500 g for 3 min, which was repeated three times.

3. The method according to claim 1, characterized in that The step 1.2 comprises: 1) Incubate the FFPE wax roll sections at 65°C for 60 min; 2) Centrifuge the incubated slices to the bottom of the tube; 3) Add 1 mL of wax melting reagent, shake at 800 rpm, 37°C for 10 min; centrifuge at 16000 rcf for 3 min, and discard the supernatant; 4) Repeat at least 2 times, and absorb all the liquid in the last step; 5) Add 1 mL of anhydrous ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant; 6) Add 1 mL of 90% ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant; 7) Add 1 mL of 75% ethanol, shake at 800 rpm, 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant; 8) Add 200 μL of water, shake at 800 rpm, 37°C for 2 min; centrifuge at 17000 rcf for 5 min, and discard the supernatant; 9) Open the lid and put it in the oven to dry.

4. The method according to claim 1, characterized in that: The concentration of fast trypsin in the Mix Buffer is 0.02-0.05 μg / μL, the concentration of CAA is 4 mM, the concentration of TECP is 1 mM, and the concentration of DDM is 0.4%.

5. The method according to claim 1, characterized in that In the step 2, the non-contact ultrasonic conditions are 20 s off, 20 s on, ultrasonic power is 85%, and ultrasonic time is 5 min.

6. The method according to claim 1, characterized in that In the step 2, the incubation conditions are 70° C. and 500 rpm for 1-2 h.

7. The method according to claim 1, characterized in that In the step 2, the centrifugation condition is 14000g for 10 minutes.

8. The method according to claim 1, characterized in that In the step 2, the acidic solution is 10% FA.

9. The method according to any one of claims 1 to 8, characterized in that: It also includes step three, preparation for the machine: use Nano drop to detect the peptide concentration and prepare a peptide sample of 100 ng / μL that meets the machine requirements.

10. The method according to any one of claims 1 to 9, characterized in that: The trace sample in step 1 is a tissue sample with a starting amount of ≤1 mg.

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