Method for processing extremely small tissue samples in one step

Abstract

The application belongs to the technical field of proteomics, and particularly relates to a one-step method for processing trace tissue samples. The method comprises the following steps: 1, preparing a sample; 2, obtaining a peptide segment: adding a Mix Buffer into an ep tube, non-contact ultrasonic incubation, finally adding an acid solution to terminate enzyme cutting, and centrifuging to obtain a supernatant; the Mix Buffer is composed of rapid trypsin, CAA, TECP, DDM and rapid trypsin buffer. The application combines all steps of lysis-reduction-alkylation-enzyme cutting into one step by optimizing the Mix buffer, the step is simple and the cost is low, the time for sample preparation is greatly saved, and a good proteomics test effect is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of proteomics, and particularly relates to a method for processing trace tissue samples in one step. BACKGROUND

[0002] The sample pretreatment process is a very important step in proteomics research. Before mass spectrometry analysis, different types of samples need to be subjected to protein cleavage, reduction alkylation, protease digestion, and desalination to obtain peptide samples suitable for on-machine collection. However, in the sample preparation process, multiple steps are required and the sample often needs to be transferred multiple times. For trace samples, due to the limitation of sample amount, the conventional preparation method results in a large amount of sample loss. Therefore, it is of great significance to develop a sample processing technology with simple steps and shortened time for the treatment of trace samples.

[0003] Currently, researchers have developed various methods for processing trace samples. For example, the FASP (Filter-Aided Sample Preparation) method uses a molecular weight cutoff (MWCO) filter device to centrifuge the protein and retain it in the device, and completes enzyme digestion in the device to obtain polypeptides that meet the on-machine requirements. This method is relatively stable and compatible with a wide range of cleavage solutions, 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 combine with the free amine or carboxyl groups of the protein under the induction of organic solvents, and then performs the enzyme digestion step. The SP3 method reduces sample loss by adsorbing protein on magnetic beads through chemical binding and is suitable for different starting amounts of samples. However, the aggregation of magnetic beads in the sample preparation process of the SP3 method hinders the adaptation of high-throughput experimental systems. The SISPROT (Simple and Intergrated Spintip-based Proteomics Technology) method integrates all sample preparation on a Tip column, shortening the sample preparation time and loss. However, this method requires multiple elution treatments to obtain polypeptide samples that meet the on-sample conditions and cannot be compatible with high-salt concentration solutions, resulting in high experimental costs. Commonly used methods include the IST (In-Stage Tip digestion) method, which is similar to the FASP method, 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 for protein processing, and the obtained polypeptides need to be further spin-dried and reconstituted, which is time-consuming and complex to operate, inevitably causing sample loss during operation. SUMMARY

[0005] To address the problems existing in the prior art, this invention aims to provide a simple and rapid one-step method for preparing trace amounts of proteomics samples. The entire pretreatment process, including protein lysis and enzymatic digestion, is completed in one step. After pretreatment, the sample can be directly loaded after acidification and centrifugation. This method is rapid and stable, making it particularly suitable for the pretreatment of extremely small amounts of samples.

[0006] This invention provides a one-step method for processing extremely small amounts of tissue samples, comprising the following steps:

[0007] 1. Sample preparation: Take a small amount of sample and place it in an EP tube. Perform the corresponding preparation work according to the sample type.

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

[0009] 1.2 For FFPE samples: Add dewaxing agent to the sample tube to dewax, rehydrate using a gradient of different concentrations of alcohol, discard the supernatant, and then open the cap to dry;

[0010] 2. Obtaining peptides: Add Mix Buffer to the ep tube, incubate by non-contact sonication, and finally add acidic solution to terminate enzyme digestion. Centrifuge and collect the supernatant.

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

[0012] For fresh tissue and dewaxed FFPE tissue samples, this invention optimizes the Mix buffer composition, adjusts the enzyme composition, and adds reducing and alkylating agents, combining all steps of lysis-reduction-alkylation-enzymatic digestion into one step. The process is simple and low-cost, eliminating the time-consuming multi-step desalting operation, as well as the time-consuming rotary drying and reconstitution operations, further reducing sample loss and greatly saving sample preparation time.

[0013] Rapid trypsin can be any commercially available rapid trypsin. For the rapid trypsin buffer, choose a compatible buffer recommended by the manufacturer.

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

[0015] In one embodiment of the present invention, for an FFPE sample, step 1.2 includes:

[0016] 1) Incubate the FFPE wax roll sections at 65℃ 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 and 37°C for 10 min; centrifuge at 16000 rcf for 3 min, and discard the supernatant;

[0019] 4) Repeat this process two or more times, such as 2-3 times, 3-5 times, etc., and finally remove all the liquid on the last attempt;

[0020] 5) Add 1 mL of anhydrous ethanol, shake at 800 rpm and 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 and 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 and 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 and 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 two, 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 two, 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 two, the incubation conditions are 70°C and 500 rpm for 1-2 hours.

[0028] In one embodiment of the present invention, in step two, the centrifugation conditions are 14000g for 10min.

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

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

[0031] In one embodiment of the present invention, the trace sample in step one is a tissue sample with an initial amount ≤1mg, which may be a tissue sample from various organs such as the brain, heart, liver, and lungs.

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

[0033] For fresh tissue and dewaxed FFPE tissue samples, this invention optimizes the mix buffer, combining all steps of lysis-reduction-alkylation-enzymatic digestion into one step. The process is simple and low-cost, eliminating the time-consuming multiple steps of desalting, as well as the time-consuming rotary drying and reconstitution operations, further reducing sample loss and significantly saving sample preparation time (from the conventional 24 hours to 2.5 hours). It also achieves better proteomics testing results and is particularly suitable for extremely small amounts of tissue samples with an initial volume of less than 1 mg. Attached Figure Description

[0034] Figure 1 Comparison of protein and peptide identification in different tissues and with literature;

[0035] Figure 2 For FFPE sample identification quantity;

[0036] Figure 3 To compare different sample preparation methods. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The processes, conditions, reagents, experimental methods, etc. of implementing the present invention are all common knowledge and general knowledge in the field, except for the contents specifically mentioned below. The present invention does not have any special limitations.

[0038] Example 1: Mix buffer composition optimization

[0039] This embodiment tested the effect of different Mix buffer components on identification under the same other conditions, including adjusting the amount of enzyme, trying to add or subtract reducing agents and alkylating agents, and using different buffer solutions for sample preparation to compare identification levels.

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

[0041] After trying different mix buffers and comparing the results, the optimal identification was found under the conditions of 0.03 μg / μL rapid trypsin + 0.4% DDM + 1 mM TECP + 4 mM CAA + rapid buffer, with a protein count of 7487 and a peptide count of 112593. This condition was used as the mix buffer formulation for subsequent experiments.

[0042] Table 1 Comparison of different experimental conditions

[0043]

[0044] Example 2: Processing of Fresh Tissue Samples

[0045] Microsample processing was performed using different types of fresh frozen mouse tissue samples. The specific steps are as follows:

[0046] 1) Weigh 2mg of different tissue samples using sterilized scissors and tweezers, place the tissue samples in a culture dish, divide the tissue samples into 6 equal parts, and put all the tissue fragments into 1.5mL centrifuge tubes.

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

[0048] 3) Repeat each sample 6 times. Add 30 μL of mix buffer (the optimal formula in Example 1) to each sample tube, add a small amount of washed tissue fragments the size of a millet grain, extract the protein using non-contact sonication, and then incubate at 70°C and 500 rpm for 1-2 h. Add 10% FA acidification to terminate the enzyme digestion, centrifuge at 14000g for 10 min and remove the supernatant.

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

[0050] Processed samples were collected using a Thermo U3000 HPLC system coupled with Bruker Tims-Tof, with 300 ng collected per sample over 120 min in DIA mode. The collected data were analyzed using a Spectronaut 18 with Direct DIA database search. The search results were compiled and analyzed, and compared with publicly available literature data. Figure 1 As shown.

[0051] Samples were prepared using the method of this invention for different tissues. The average protein identification value for kidney tissue was 8200, and the average peptide identification value was 122760; the average protein identification value for spleen tissue was 8297, and the average peptide identification value was 115923; the average protein identification value for lung tissue was 8500, and the average peptide identification value was 136216. Referring to the library construction data in the literature by Tian Lu et al., the average protein identification value for kidney tissue was 6911, and the average peptide identification value was 55840; the average protein identification value for spleen tissue was 7472, and the average peptide identification value was 65700; the average protein identification value for lung tissue was 6064, and the average peptide identification value was 48210. The amounts of proteins and peptides identified using the method of this invention are greater than the reference data in the literature, indicating that this method is applicable to the preparation of samples from different tissue types and the results are relatively stable.

[0052] Example 3: FFPE wax roll treatment

[0053] The following are the specific steps for processing small samples using FFPE wax rolls from different tissues:

[0054] 1) Incubate the wax roll in a 65℃ oven 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 and 37°C for 10 min; centrifuge at 16000 rcf for 3 min, and discard the supernatant.

[0057] 4) Repeat 2-3 times, and on the last time, remove all the liquid. You can wash several times.

[0058] 5) Add 1 mL of anhydrous ethanol, shake at 800 rpm and 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 and 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 and 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 and 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 optimal formulation in Example 1), sonicate non-contactly for 5 min, 20 s off, 20 s on, 85%, and incubate at 70°C for 2 h. Add 10% FA acidification to terminate the enzyme digestion.

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

[0065] Processed samples were collected using a Thermo U3000 liquid chromatography system coupled with Bruker Tims-Tof, with 300 ng collected per sample over 120 minutes in DIA mode. The collected data were analyzed using Spectronaut 18 with Direct DIA database search.

[0066] The search results show ( Figure 2 The protein identification levels of brain, heart, liver, and lung samples were above 6000. Among them, liver and lung samples showed better identification levels compared with the laboratory multi-step FFPE preparation method, indicating that this invention patent is applicable to FFPE sample preparation.

[0067] Example 4: Comparison of different treatment methods

[0068] Fresh rat liver tissue samples and FFPE (waxed roll) tissues from Examples 2 and 3 were compared with conventional multi-step sample preparation methods and PCT (pressure cycling technology). The results are as follows: Figure 3 As shown.

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

[0070] PCT is a rapid pretreatment technique that uses specific temperatures and time intervals to subject trace samples to a programmed, periodic alternation between normal and ultra-high pressure. This process disrupts the physical structure of the biological sample, thereby achieving precise pretreatment. This method can extract peptides from tissues that can be used for product analysis within 3 hours.

[0071] The processing method of this invention combines all steps into one step, which is simple and low-cost. It eliminates the time-consuming multi-step desalting operation, as well as the time-consuming rotary evaporation and reconstitution operations, further reducing sample loss and saving sample preparation time (from the conventional approximately 24 hours to 2.5 hours). The results show that the method of this invention performs well in the identification of both proteins and peptides.

[0072] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A one-step method for processing extremely small amounts of tissue samples, characterized in that, Includes the following steps: (a) Sample preparation: Take a very small amount of sample and place it in an EP tube. Perform the corresponding preparation work according to the sample type; the very small amount of sample is a tissue sample with an initial amount ≤1 mg. (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, discard the supernatant, and repeat three times; (1.2) For FFPE samples: Add dewaxing agent to the ep tube to dewax, use different concentrations of alcohol gradient to rehydrate, discard the supernatant, and then open the cap to dry; (ii) Obtaining peptides: Add Mix Buffer to the ep tube, incubate by non-contact sonication, and finally add acidic solution to terminate enzyme digestion. Centrifuge and collect the supernatant. The Mix Buffer consists of rapid trypsin, CAA, TECP, DDM and rapid trypsin buffer, wherein 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%.

2. The method according to claim 1, characterized in that, In step 1.1, the amount of PBS buffer containing protease inhibitor added is 500 μL, shaken in a metal bath at 4 °C for 2 min, followed by centrifugation at 1500 g for 3 min, and repeated three times.

3. The method according to claim 1, characterized in that, Step 1.2 includes: 1) Incubate FFPE wax roll slices at 65℃ 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 and 37°C for 10 min; centrifuge at 16000 rcf for 3 min, and discard the supernatant; 4) Repeat at least twice, and on the last time, remove all the liquid. 5) Add 1 mL of anhydrous ethanol, shake at 800 rpm and 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 and 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 and 37°C for 5 min; centrifuge at 17000 rcf for 3 min, and discard the supernatant; 8) Add 200 μL of water, 800 rpm, 37℃, shake 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, In step two, the non-contact ultrasound conditions are 20s off and 20s on, with an ultrasound power of 85% and an ultrasound time of 5 minutes.

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

6. The method according to claim 1, characterized in that, In step two, the centrifugation conditions are 14000 g for 10 min.

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

8. The method according to any one of claims 1-7, characterized in that, It also includes step three, preparation for the instrument: using Nanodrop to detect peptide concentration, prepare a peptide sample of 100 ng / μL that meets the requirements for instrument use.

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