CN119125395A - Method for treating trace tissue sample by one-step method - Google Patents

Abstract

The invention belongs to the technical field of proteomics, and particularly relates to a method for treating a trace tissue sample by a one-step method. The method comprises the steps of preparing a sample, and obtaining peptide segments, wherein Mix Buffer is added into an ep tube, incubated after non-contact ultrasonic treatment, finally acid solution is added to terminate enzyme digestion, supernatant is centrifugally taken, and the Mix Buffer consists of fast trypsin, CAA, TECP, DDM and fast trypsin Buffer. According to the invention, by optimizing Mix buffer, all the steps of cleavage, reduction, alkylation and enzyme digestion are combined into one step, the steps are simple, the cost is low, the time for preparing a sample is greatly saved, and a better proteomics test effect is achieved.

Description

Method for treating trace tissue sample by one-step method

Technical Field

The invention belongs to the technical field of proteomics, and particularly relates to a method for treating a trace tissue sample by a one-step method.

Background

The pretreatment process of the sample in the proteomics research is an important step, and different types of samples need to be subjected to protein cleavage, reductive alkylation, protease enzymolysis and desalination before mass spectrometry analysis, so that the peptide fragment sample with the collection of the machine is obtained. However, in the process of sample preparation, the sample is transferred in multiple steps and often multiple times are required. For micro samples, conventional preparation methods result in more sample loss due to the limitation of the sample size. Therefore, developing sample processing technology that is simple in steps and can shorten the time duration is of great significance to the processing of micro samples.

At present, researchers develop various methods for processing micro samples, such as FASP (Filter-AIDED SAMPLE Preparation) method, which is to use a molecular weight cut-off (MWCO) ultrafiltration device to centrifuge and retain proteins In the device, and finish enzyme digestion In the device to obtain polypeptides meeting the requirement of the on-line machine, the method is relatively stable and compatible with extensive lysate, but the loss of samples is larger when the sample size is lower, SP3 (Single-post, solid Phase-ENHANCED SAMPLE-Preparation) method utilizes carboxyl or amino coating with hydrophilic surface of magnetic beads, the combination with free amino or carboxyl of proteins is finished under the induction of organic solvent, enzyme digestion step is further carried out, the SP3 method adsorbs proteins on the magnetic beads through chemical combination to reduce the loss of the samples and is applicable to samples with different initial weights, the SP3 method can block the adaptation of a high-flux experiment system due to the aggregation of the magnetic beads, SISPROT (SIMPLE AND INTERGRATED SPINTIP-based Proteomics Technology) method integrates all sample Preparation on a Tip column, the time and loss of Preparation are shortened, the method is nevertheless carried out to meet the conditions of high-elution time and high-loss of samples, the elution requirement of the samples can not meet the requirement of the In-Phase-current experiment system, and the sample concentration is also compatible with the Solid-Phase-separation method is more than that the Solid Phase-separation solution is more than normal, and the sample concentration of the peptides can not be eluted by using a Solid Phase-separation method (6218) or a similar method is more than the solution, and the sample is more compatible with the high-soluble Phase-Phase sample-soluble substance can be obtained.

The method needs the steps of protein extraction and protein treatment when in use, the obtained polypeptide needs to be re-dissolved after further spin-drying, the time is long, the operation is complex, and the loss of samples is unavoidable in operation.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a simple, convenient and rapid one-step preparation method of a proteomics trace sample. By completing the whole pretreatment process of protein cleavage and enzymolysis of the trace sample in one step. The pretreated sample can be directly loaded after acidification and centrifugation are completed. The method of the invention is fast and stable and is particularly suitable for the pretreatment of very small amounts of samples.

The invention provides a method for treating a trace tissue sample by a one-step method, which comprises the following steps:

1. Preparing a sample, namely placing a trace sample into an ep tube, and performing corresponding preparation work according to the type of the sample;

1.1 for fresh tissue samples, adding PBS buffer containing protease inhibitor to ep tube, shaking at 4 ℃ in metal bath, centrifuging, discarding supernatant, repeating for three times;

1.2, for FFPE samples, dewaxing by adding a dewaxing agent into a sample tube, using alcohol gradient rehydration with different concentrations, discarding supernatant, and then uncovering and drying;

2. Obtaining peptide segments, namely adding Mix Buffer into an ep tube, incubating after non-contact ultrasonic treatment, adding an acidic solution to terminate enzyme digestion, and centrifuging to obtain supernatant;

the Mix Buffer consisted of fast trypsin (RAPID TRYPSIN), CAA, TECP, DDM and fast trypsin Buffer (RAPID TRYPSIN Buffer).

Aiming at fresh tissues and FFPE tissue samples subjected to dewaxing treatment, the invention adjusts enzyme components by optimizing Mix buffer components, adds a reducing agent and an alkylating agent, combines all steps of cracking, reduction, alkylation and enzyme digestion into one step, has simple steps and low cost, omits multi-step time-consuming operation of desalting and time-consuming spin-drying operation and re-dissolving operation, further reduces the loss of samples, and greatly saves the time for preparing the samples.

The rapid trypsin may be a commercially available rapid trypsin commonly used. The fast trypsin buffer is selected to be matched with the fast trypsin buffer, and the buffer recommended by a merchant is selected.

In one embodiment of the invention, for fresh tissue samples, the amount of protease inhibitor-containing PBS buffer added in step 1.1 is 500. Mu.L, shaken for 2min at 4℃in a metal bath, and centrifuged for 3min at 1500 g.

In one embodiment of the present invention, for FFPE samples, the step 1.2 includes:

1) Incubating the FFPE wax roll slices at 65 ℃ for 60min;

2) Centrifuging the incubated slice to the bottom of the tube;

3) Adding 1mL of wax melting reagent, 800rpm,37 ℃, vibrating for 10min, 16000rcf, centrifuging for 3min, and discarding the supernatant;

4) Repeating for more than 2 times, such as 2-3 times, 3-5 times, and the like, and finally sucking the liquid for one time;

5) Adding 1mL of absolute ethanol, 800rpm,37 ℃, shaking for 5min, 17000rcf, centrifuging for 3min, and discarding the supernatant;

6) Adding 1mL of 90% ethanol, 800rpm,37 ℃, shaking for 5min, centrifuging for 3min, and discarding the supernatant;

7) Adding 1mL of 75% ethanol, 800rpm,37 ℃, shaking for 5min, centrifuging for 3min, and discarding the supernatant;

8) 200. Mu.L of water was added, 800rpm,37℃and shaking for 2min, 17000rcf, centrifugation for 5min, and the supernatant was discarded;

9) And (5) opening the cover and putting the cover into a baking oven for baking.

In one embodiment of the present invention, in the second step, RAPID TRYPSIN concentration in the Mix Buffer is 0.02-0.05. Mu.g/. Mu.L, CAA concentration is 4mM, TECP concentration is 1mM, and DDM concentration is 0.4%. Preferably, the concentration of RAPID TRYPSIN in the Mix Buffer is 0.03. Mu.g/. Mu.L, the concentration of CAA is 4mM, the concentration of TECP is 1mM, and the concentration of DDM is 0.4%.

In one embodiment of the present invention, in the second step, the non-contact ultrasonic condition is 20s off, 20s on, the ultrasonic power is 85%, and the ultrasonic time is 5min.

In one embodiment of the invention, in the second step, the incubation conditions are 70℃and 500rpm for 1-2 hours.

In one embodiment of the present invention, in the second step, the centrifugation condition is 14000g for 10min.

In one embodiment of the invention, in the second step, the acidic solution is 10% fa.

In one embodiment of the invention, the method further comprises the step of preparing on-line, namely detecting the concentration of the peptide fragments by using Nano drop, and preparing 100 ng/. Mu.L of polypeptide sample meeting the on-line requirement.

In one embodiment of the present invention, the trace sample in the first step is a tissue sample with an initial amount of 1mg or less, and may be a tissue sample of each organ such as brain, heart, liver, lung, etc.

Compared with the prior art, the invention has the following beneficial effects:

Aiming at fresh tissues and FFPE tissue samples subjected to dewaxing treatment, all the steps of cracking, reduction, alkylation and enzyme digestion are combined into one step by optimizing Mix buffer, the steps are simple and low in cost, the multi-step time-consuming operation of desalting and the time-consuming spin drying operation and re-dissolving operation are omitted, the loss of the samples is further reduced, the time for preparing the samples is greatly saved (the time is shortened to 2.5 hours from about 24 hours in the conventional process), the better proteomics test effect is achieved, and the method is particularly suitable for very small amount of tissue samples with the initial amount of 1 mg.

Drawings

FIG. 1 shows the identification of different histones and peptide fragments in comparison with the literature;

FIG. 2 is a sample identification amount of FFPE;

FIG. 3 is a comparison of different sample preparation methods.

Detailed Description

The process, conditions, reagents, experimental methods, etc. for carrying out the present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the details except for the following specific details, which are set forth in the following description.

Example 1Mix buffer component optimization

This example tested the effect of different Mix buffer components on the assay under the same other conditions, wherein sample preparation was performed using different buffers to compare the amounts of the assays by adjusting the amount of enzyme, attempting to add and subtract reducing agent, alkylating agent.

As can be seen, RAPID TRYPSIN has the worst identification result in the aqueous solution, and the better identification result can be achieved by adding DDM and CAA with proper RAPID TRYPSIN concentration after improving in RAPID TRYPSIN buffer.

Comparison of the results after different Mix buffers were tried shows that the conditions of 0.03 mug/mu L RAPID TRYPSIN +0.4%DDM+1mMTECP+4mM CAA+rapid buffer were optimal for identification, protein 7487 and peptide fragment identification 112593, and the conditions were used as Mix buffer formula in the subsequent experiments.

Table 1 comparison of different experimental conditions

EXAMPLE 2 fresh tissue sample treatment

The method adopts fresh frozen tissue samples of different types of mice to process micro samples, and comprises the following specific steps:

1) 2mg of different tissue samples were weighed with sterilized scissors forceps, placed in a petri dish, 6 aliquots of tissue samples were aliquoted, and all tissue fragments were placed into 1.5mL centrifuge tubes.

2) 500. Mu.L of PBS containing protease inhibitors was added to the centrifuge tube, shaken for 2min at 4℃in a metal bath, then centrifuged for 3min at 1500g, and the supernatant was discarded.

3) Each sample was repeated 6 times, 30 μl mix Buffer (formulation optimal for example 1) was added to each sample tube, small grain size washed tissue fragments were added, proteins were extracted using non-contact ultrasound, followed by incubation at 70 ℃ for 1-2h at 500rpm, acidification termination with 10% fa was added, and centrifugation at 14000g for 10min to remove supernatant.

4) The peptide concentration was measured using nanodrop and each sample was formulated for 100 ng/. Mu.L of the ready-to-load assay.

The treated samples were collected using a Thermo U3000 liquid chromatograph combined Bruker Tims-Tof combination with 300ng each sample, 120min acquisition time, and acquisition mode DIA. The collected data was analyzed using spectronaut a by DIRECT DIA search of the library. And (3) sorting and analyzing the library searching result, and comparing the library searching result with published literature data, wherein the result is shown in figure 1.

Different tissues are subjected to sample preparation by using the method provided by the embodiment of the invention, namely, a kidney tissue protein identification mean value 8200, a peptide fragment identification mean value 122760, a spleen tissue protein identification mean value 8297, a peptide fragment identification mean value 115923 and a lung tissue protein identification mean value 8500 and a peptide fragment identification mean value 136216. Referring to database building data in Tian Lu et al, kidney tissue protein identification 6911, peptide fragment identification 55840, spleen tissue protein identification 7472, peptide fragment identification 65700, lung tissue protein identification 6064 and peptide fragment identification 48210, the comparison of the amounts of protein and peptide fragment identified by the method of the invention is larger than that of reference data of the literature, so that the method is applicable to sample preparation of different tissue types, and the result is stable.

Example 3FFPE wax roll treatment

The FFPE wax rolls of different tissues were used for micro sample processing as follows:

1) The wax rolls were placed in a 65 ℃ oven for 60min incubation.

2) The sections were centrifuged to the bottom of the tube.

3) 1ML of wax melting reagent was added, 800rpm,37 ℃, shaking 10min, 16000rcf, centrifuging for 3min, and discarding the supernatant.

4) Repeating for 2-3 times, and finally sucking the liquid completely, and washing for several times.

5) 1ML of absolute ethanol was added, 800rpm,37℃and shaking for 5min, 17000rcf, centrifugation for 3min, and the supernatant was discarded.

6) 1ML of 90% ethanol was added, 800rpm,37℃and shaking for 5min, 17000rcf, centrifugation for 3min, and the supernatant was discarded.

7) 1ML of 75% ethanol was added, 800rpm,37℃and shaking for 5min, 17000rcf, centrifugation for 3min, and the supernatant was discarded.

8) 200. Mu.L of water was added, 800rpm,37℃and 2min of shaking, 17000rcf, centrifuged for 5min, and the supernatant was discarded.

9) And (5) opening the cover, and putting the cover into a baking oven to be dried for 5min.

10 Add to centrifuge tube containing 15 μl mix buffer (formulation is optimal for example 1), sonicate 5min,20s off,20s on,85% without contact, incubate for 2h at 70 temperature. Acidification with 10% FA stops cleavage.

11 Samples were removed, centrifuged at 14000g for 10min to obtain supernatant for concentration, and nanodrop were used to measure peptide concentration, each sample was formulated at 100 ng/. Mu.L for loading detection.

The treated samples were collected using a Thermo U3000 liquid chromatograph combined Bruker Tims-Tof combination with 300ng each sample, 120min acquisition time, and acquisition mode DIA. The collected data was analyzed using Spectronaut a by DIRECT DIA search of the library.

The results of searching the library show (figure 2) that the protein identification amount of brain, heart, liver and lung samples is above 6000, wherein the liver samples and the lung samples are better identified by comparing with the FFPE preparation method of a laboratory multi-step method, which shows that the invention is suitable for FFPE sample preparation.

Example 4 comparison of different treatments

The results of taking fresh tissue samples of murine livers and FFPE (wax roll) tissues of examples 2 and 3 versus conventional multi-step sample preparation methods, PCT (pressure cycling technique) techniques are shown in fig. 3.

Conventional multi-step sample preparation requires multiple steps of protein extraction, peptide fragment cleavage, desalination, heat evaporation and the like, and the whole process generally takes 2 days.

PCT is a rapid pretreatment technique, and by using specific temperature and time interval, the micro sample is subjected to procedural, periodic and alternate cycling between normal pressure and ultrahigh pressure, and the physical structure of the biological sample is destroyed from beating, so as to achieve the purpose of precise advance.

The treatment method of the embodiment of the invention combines all the steps into one step, has simple steps and low cost, omits multi-step time-consuming operation of desalination and time-consuming spin-drying operation and re-dissolving operation, further reduces the loss of samples, and saves the sample preparation time (from conventional about 24 hours to 2.5 hours). The results show that the method of the invention has better performance on the identification quantity of the protein and the peptide fragment.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. A one-step method for processing a very small amount of tissue samples, characterized in that it comprises the following steps: 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 step 1.1, the amount of PBS buffer containing protease inhibitors added is 500 μL, shaken in a metal bath at 4°C for 2 minutes, and then centrifuged at 1500g for 3 minutes, and 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 20s off, 20s on, ultrasonic power 85%, and ultrasonic time 5min. 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 hours. 7. The method according to claim 1, characterized in that in the step 2, the centrifugation condition is 14000g for 10 min. 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, further comprising step 3, 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. 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 an initial amount of ≤1 mg.