CN114459872A - Automatic processing method and device for conducting proteomics analysis on FFPE sample - Google Patents
Automatic processing method and device for conducting proteomics analysis on FFPE sample Download PDFInfo
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Abstract
The invention discloses an automatic processing method and device for carrying out proteomics analysis on an FFPE sample. The automatic processing method comprises the following steps: s1, the first moving module places the sample tube with the FFPE sample in a first preset position; s2, the identification unit identifies the sample tube to obtain the identification information of the sample tube; s3, selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample; and S4, processing the FFPE sample based on the target production mode to obtain a processing result. By applying the technical scheme of the invention, the automatic processing method can realize the purpose of automatically processing the FFPE sample, achieve the technical effect of improving the sample processing efficiency, and further solve the technical problem of low efficiency caused by the manual participation completion of the FFPE sample proteomics pretreatment in the related technology.
Description
Technical Field
The invention relates to the field of biotechnology automation, in particular to an automatic processing method and device for carrying out proteomics analysis on an FFPE sample.
Background
In biopsy, paraffin sections (FFPE) have been used to preserve the histological and morphological structure of tissues for the histopathological diagnosis of disease. The FFPE tissue is highly stable and can be stored at room temperature for a long time. Thus, a large number of normal and pathological FFPE samples are produced worldwide that contain information related to diagnosis, treatment and outcome. Thus, the FFPE tissue bank is now a potentially valuable resource for retrospective studies of biomarker discovery and validation for cancer and other important diseases. Combining high throughput proteomic analysis techniques with FFPE will reveal a large amount of disease-related information, playing an important role in disease marker discovery, disease diagnosis, prognosis, monitoring and treatment-related aspects.
The pretreatment process for carrying out proteomics analysis on the FFPE sample comprises the following steps: dewaxing, hydrating, protein extracting, protein quantifying, enzymolysis, desalting, freeze-drying and quantifying. These treatments are currently performed mainly manually by highly qualified professionals. The pretreatment process of the FFPE sample is complicated and needs high-intensity manual operation. And the manual operation flux is low, the labor cost is high, and the production efficiency in the whole industry is low. In addition, human error and subjective judgments can lead to product quality variations. In addition, a toxic reagent (xylene) can be contacted in the sample treatment process, so that great potential safety hazard exists.
Disclosure of Invention
The invention aims to provide an automatic processing method and device for carrying out proteomics analysis on an FFPE sample, and aims to solve the technical problems that in the prior art, a paraffin section sample is difficult to extract, the extraction efficiency is low, and the experiment period is long.
To achieve the above objects, according to one aspect of the present invention, there is provided an automated processing method for proteomic analysis of FFPE samples. The automatic processing method comprises the following steps: s1, the first moving module places the sample tube with the FFPE sample in a first preset position; s2, the identification unit identifies the sample tube to obtain the identification information of the sample tube; s3, selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample; s4, processing the FFPE sample based on the target production mode to obtain a processing result; wherein, processing the sample based on the target production mode comprises: the second moving module moves the sample tube to an automatic cover opening and closing machine through a mechanical arm to execute cover opening operation; the second moving module carries the sample tube to an automatic workstation through a mechanical arm so as to add a dewaxing reagent to the sample tube through the automatic workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment to obtain a pretreated sample; the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the sample to an automatic workstation to remove supernatant, and then adds a dewaxing reagent into the automatic workstation to repeat dewaxing treatment once to obtain a dewaxed sample; then sequentially adding hydrating reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain hydrated samples, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain crude protein samples; adding acetone into the crude protein sample through an automatic workstation for protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate; and the scanning module scans and records the plate number of the protein sample plate and puts the protein sample plate into the protein library position.
Further, the automatic processing method further comprises the following steps: generating a detection sheet after obtaining the protein sample; and sending the detection sheet to an intelligent production line so as to perform protein quantification on the protein sample by using the intelligent production line.
Further, will detect the list and send intelligent production line to utilize intelligent production line to carry out protein detection to the protein sample, include: quantifying the protein sample by an enzyme-labeling instrument to obtain the protein concentration; taking a protein sample to perform SDS-PAGE gel running analysis so as to judge whether the protein sample meets the subsequent polypeptide processing requirement or not and obtain a judgment result; executing a polypeptide processing flow when the judgment result shows that the protein sample meets the conditions; and re-extracting the protein sample when the judgment result shows that the protein sample does not meet the polypeptide treatment condition.
Further, executing the polypeptide processing flow comprises: and carrying out enzymolysis, positive pressure desalination, quantification and plate rotation on the protein sample meeting the polypeptide treatment conditions.
Further, the automatic processing method further comprises the following steps: after ordering of a client is received, under the condition that polypeptide processing is determined to be needed, automatically performing order splicing and typesetting on polypeptides of different projects, scanning to confirm the serial number of a new sample plate, and then adding an enzymolysis reagent for enzymolysis; then adding an acidifying reagent, shaking, uniformly mixing and then centrifuging at a high speed; then taking the sample supernatant to carry out positive pressure desalination, and purifying the polypeptide sample; freeze-drying the desalted polypeptide solution; redissolving the freeze-dried polypeptide, and taking a small amount of polypeptide solution for quantification; then diluting the polypeptide based on the quantitative result; and then centrifuging at a high speed, transferring the polypeptide sample to a 96-well plate on a machine, recording the plate number to obtain the final polypeptide sample to be machined, and arranging the machine list on the system.
According to another aspect of the present invention, an automated processing device for proteomic analysis of FFPE samples is provided. The automated processing device includes: the first moving module is used for placing the sample tube with the FFPE sample in a first preset position; the identification unit is used for identifying the sample tube to obtain the identification information of the sample tube; the selection unit is arranged for selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample; the first processing unit is used for processing the FFPE sample based on the target production mode to obtain a processing result; wherein, the first processing unit includes: the automatic cover opening and closing module is used for opening the cover of the sample tube; a second moving module configured to transport the sample tube between the modules in the first processing unit by the robot arm; the automatic workstation module is used for adding various reagents into the sample tube, and taking or removing supernatant; the automatic oscillator module is used for oscillating the sample in the sample tube; a high-speed centrifuge module configured to centrifuge a sample in a sample tube; the scanning module is used for scanning and recording the plate number of the protein sample plate; the control module is used for controlling the second moving module to move the sample tube to the automatic cap opening and closing machine through the mechanical arm to execute cap opening operation; the second moving module carries the sample tube to an automatic workstation through a mechanical arm so as to add a dewaxing reagent to the sample tube through the automatic workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment to obtain a pretreated sample; the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the sample to an automatic workstation to remove supernatant, and then adds a dewaxing reagent into the automatic workstation to repeat dewaxing treatment once to obtain a dewaxed sample; then sequentially adding hydrating reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain hydrated samples, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain crude protein samples; adding acetone into the crude protein sample through an automatic workstation for protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate; the scanning module scans and records the plate number of the protein sample plate, and the second moving module places the protein sample plate into the protein library position.
Further, the automated processing unit further comprises: the generating module is used for generating a detection sheet after the protein sample is obtained; and the detection module is used for sending the detection sheet to the intelligent production line so as to detect the protein of the protein sample by utilizing the intelligent production line.
Further, a detection module comprising: the processing submodule is used for sampling the protein sample to obtain a target sample; the code scanning submodule is used for carrying out centrifugal code scanning operation on the target sample to obtain a code-scanned target sample; the judgment submodule is set to judge whether the target sample meets the polypeptide processing condition or not according to the result to obtain a judgment result; the first determining submodule is arranged for executing the polypeptide processing flow when the judgment result shows that the sample meets the polypeptide processing condition; and a second determining submodule configured to reacquire the sample when the determination result indicates that the sample does not meet the polypeptide processing condition.
Further, the first determination submodule includes: the sequencing submodule is used for sequencing the samples meeting the polypeptide processing conditions and performing plate combination on the re-sequenced samples; and the fragmentation submodule is used for carrying out whole-plate sampling and plate number recording on the sample subjected to plate processing, and carrying out automatic polypeptide processing operation on the sample of the plate number recording to obtain a polypeptide sample to be processed on the computer.
Further, the control module is also configured to control the automatic workstation module to add an enzymolysis reagent to the protein sample meeting the polypeptide processing conditions, perform enzymolysis treatment on the sample to which the enzymolysis reagent is added, add an acidification reagent to the sample after the enzymolysis treatment, and obtain the polypeptide sample after the acidification after the high-speed centrifugation; the automatic processing device also comprises a purification module which is used for desalting the acidified polypeptide sample and freeze-drying the sample after the desalting; optionally, the automated processing unit further comprises: the automatic detection module is used for automatically detecting the polypeptide sample after desalination and purification; optionally, the automated processing unit further comprises: an acquisition unit configured to acquire a placing order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal process on the sample; and the plate rotating unit is used for rotating the sample after the oscillation centrifugation.
According to still another aspect of the present invention, there is provided a computer-readable storage medium. The computer readable storage medium includes a stored program that when executed performs any of the automated processing methods described above for proteomic analysis of FFPE samples.
According to yet another aspect of the present invention, an automated sample processing system is provided. The automated processing system comprises a memory and a processor, wherein the memory is stored with a computer program, and the processor is configured to execute any one of the automated processing methods for proteomic analysis of FFPE samples through the computer program.
By applying the technical scheme of the invention, the automatic processing method can realize the purpose of automatically processing the FFPE sample, achieve the technical effect of improving the sample processing efficiency, and further solve the technical problem of low efficiency caused by the manual participation completion of the FFPE sample proteomics pretreatment in the related technology.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows a flow diagram of a method of automated processing of a sample according to an embodiment of the invention;
FIG. 2 shows a flow diagram of automated extraction of FFPE sample protein according to an embodiment of the invention;
FIG. 3 shows a flow chart of protein quality control according to an embodiment of the present invention;
FIG. 4 shows a polypeptide processing flow diagram according to an embodiment of the invention; and
FIG. 5 shows a schematic diagram of an automated processing unit for samples according to an embodiment of the invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
According to an exemplary embodiment of the present invention, an automated processing method for proteomic analysis of FFPE samples is provided. The automatic processing method comprises the following steps: s1, the first moving module places the sample tube with the FFPE sample in a first preset position; s2, the identification unit identifies the sample tube to obtain the identification information of the sample tube; s3, selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample; s4, processing the FFPE sample based on the target production mode to obtain a processing result; wherein, processing the sample based on the target production mode comprises: the second moving module moves the sample tube to an automatic cover opening and closing machine through a mechanical arm to execute cover opening operation; the second moving module carries the sample tube to an automatic workstation through a mechanical arm so as to add a dewaxing reagent to the sample tube through the automatic workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment to obtain a pretreated sample; the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the sample to an automatic workstation to remove supernatant, and then adds a dewaxing reagent into the automatic workstation to repeat dewaxing treatment once to obtain a dewaxed sample; then sequentially adding hydrating reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain hydrated samples, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain crude protein samples; adding acetone into the crude protein sample through an automatic workstation for protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate; and the scanning module scans and records the plate number of the protein sample plate and puts the protein sample plate into the protein library position.
By applying the technical scheme of the invention, the automatic processing method can realize the purpose of automatically processing the FFPE sample, achieve the technical effect of improving the sample processing efficiency, and further solve the technical problem of low efficiency caused by the manual participation completion of the FFPE sample proteomics pretreatment in the related technology.
Wherein "shake high temperature incubation" refers to the general meaning in the art, such as: incubate at 95 ℃ with shaking at 1000 rpm. Among them, crosslinking is released and reductive alkylation is carried out at 95 ℃, and crosslinking between proteins and nucleic acids and between proteins is generated after formaldehyde treatment, which is not beneficial to subsequent treatment and is required to be released.
In order to improve the automation level, the automation processing method further comprises the following steps: generating a detection sheet after obtaining the protein sample; and sending the detection sheet to an intelligent production line so as to perform protein quantification on the protein sample by using the intelligent production line.
In an exemplary embodiment of the present invention, a test order is sent to an intelligent production line for protein testing of a protein sample using the intelligent production line, comprising: quantifying the protein sample by an enzyme-labeling instrument to obtain the protein concentration; taking a protein sample to perform SDS-PAGE gel running analysis so as to judge whether the protein sample meets the subsequent polypeptide processing requirement or not and obtain a judgment result; executing a polypeptide processing flow when the judgment result shows that the protein sample meets the conditions; and re-extracting the protein sample when the judgment result shows that the protein sample does not meet the polypeptide treatment condition.
To further increase the level of automation, a polypeptide processing scheme is performed comprising: and carrying out enzymolysis, positive pressure desalination, quantification and plate rotation on the protein sample meeting the polypeptide treatment conditions.
Preferably, the automated processing method further comprises: after ordering of a client is received, under the condition that polypeptide processing is determined to be needed, automatically performing order splicing and typesetting on polypeptides of different projects, scanning to confirm the serial number of a new sample plate, and then adding an enzymolysis reagent for enzymolysis; then adding an acidifying reagent, shaking, uniformly mixing and then centrifuging at a high speed; then taking the sample supernatant to carry out positive pressure desalination, and purifying the polypeptide sample; freeze-drying the desalted polypeptide solution; redissolving the freeze-dried polypeptide, and taking a small amount of polypeptide solution for quantification; then diluting the polypeptide based on the quantitative result; and then centrifuging at a high speed, transferring the polypeptide sample to a 96-well plate on a machine, recording the plate number to obtain the final polypeptide sample to be machined, and arranging the machine list on the system.
According to an exemplary embodiment of the present invention, an automated processing device for proteomic analysis of FFPE samples is provided. The device includes: the first moving module is used for placing the sample tube with the FFPE sample in a first preset position; the identification unit is used for identifying the sample tube to obtain the identification information of the sample tube; the selection unit is arranged for selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample; the first processing unit is used for processing the FFPE sample based on the target production mode to obtain a processing result; wherein, the first processing unit includes: the automatic cover opening and closing module is used for opening the cover of the sample tube; a second moving module configured to transport the sample tube between the modules in the first processing unit by the robot arm; the automatic workstation module is used for adding various reagents into the sample tube and taking or removing supernatant; the automatic oscillator module is used for oscillating the sample in the sample tube; a high-speed centrifuge module configured to centrifuge a sample in a sample tube; the scanning module is used for scanning and recording the plate number of the protein sample plate; the control module is used for controlling the second moving module to move the sample tube to the automatic cap opening and closing machine through the mechanical arm to execute cap opening operation; the second moving module carries the sample tube to an automatic workstation through a mechanical arm so as to add a dewaxing reagent to the sample tube through the automatic workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment to obtain a pretreated sample; the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the sample to an automatic workstation to remove supernatant, and then adds a dewaxing reagent into the automatic workstation to repeat dewaxing treatment once to obtain a dewaxed sample; then sequentially adding hydrating reagents with different proportions through an automatic workstation, vibrating, centrifuging and removing the supernatant to obtain hydrated samples, adding protein lysate through the automatic workstation, vibrating, incubating at high temperature, centrifuging and taking the supernatant to obtain crude protein samples; adding acetone into the crude protein sample through an automatic workstation for protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate; the scanning module scans and records the plate number of the protein sample plate, and the second moving module places the protein sample plate into the protein library position.
Preferably, the automated processing apparatus further comprises: the generation module is used for generating a detection sheet after the protein sample is obtained; and the detection module is used for sending the detection sheet to the intelligent production line so as to detect the protein of the protein sample by utilizing the intelligent production line.
In an exemplary embodiment of the invention, a detection module includes: the processing submodule is used for sampling the protein sample to obtain a target sample; the code scanning submodule is used for carrying out centrifugal code scanning operation on the target sample to obtain a code-scanned target sample; the judgment submodule is set to judge whether the target sample meets the polypeptide processing condition or not according to the result to obtain a judgment result; the first determining submodule is arranged for executing the polypeptide processing flow when the judgment result shows that the sample meets the polypeptide processing condition; and a second determining submodule configured to reacquire the sample when the determination result indicates that the sample does not meet the polypeptide processing condition.
Typically, the first determination submodule includes: the sequencing submodule is used for sequencing the samples meeting the polypeptide processing conditions and performing plate combination on the re-sequenced samples; and the fragmentation submodule is used for carrying out whole-plate sampling and plate number recording on the sample subjected to plate processing, and carrying out automatic polypeptide processing operation on the sample of the plate number recording to obtain a polypeptide sample to be processed on the computer.
Preferably, the control module is further configured to control the automatic workstation module to add an enzymolysis reagent to the protein sample meeting the polypeptide processing conditions, perform enzymolysis on the sample to which the enzymolysis reagent is added, add an acidification reagent to the sample after the enzymolysis, and obtain the polypeptide sample after acidification after high-speed centrifugation; the automatic processing device also comprises a purification module which is used for desalting the acidified polypeptide sample and freeze-drying the desalted sample; optionally, the automated processing unit further comprises: the automatic detection module is used for automatically detecting the polypeptide sample after desalination and purification; optionally, the automated processing unit further comprises: an acquisition unit configured to acquire a placing order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal process on the sample; and the plate rotating unit is used for rotating the sample after the oscillation centrifugation.
According to an exemplary embodiment of the present invention, a computer-readable storage medium is provided, which comprises a stored program that, when executed, performs any of the automated methods for proteomic analysis of FFPE samples described above.
According to an exemplary embodiment of the present invention, an automated processing system for samples is provided, which comprises a memory and a processor, wherein the memory stores a computer program, and the processor is configured to execute any one of the automated processing methods for proteomic analysis of FFPE samples by the computer program.
The beneficial effects of the present invention will be further described with reference to the following examples, wherein the reagents used in the following examples are conventional in the art.
Example 1
Sample preparation: human tissue paraffin section
Reagent:
dewaxing reagent: xylene
Hydration reagent: 100% ethanol, 75% ethanol, 50% ethanol and PBS
The protein lysate comprises: 4% SDS, 100mM Tris, 10mM TCEP, 40mM CAA and 100mM NaCl.
Acetone (II)
The DB redissolution comprises: 8M Urea and 100mM TEAB.
Setting relevant experiment parameters: adding dewaxing reagent, shaking and mixing for 30s, incubating at room temperature for 10min, centrifuging at 6000g for 2min, removing supernatant, and repeating the operation once; adding a hydration reagent in turn: adding 100% ethanol, 75% ethanol, 50% ethanol and PBS, adding hydration reagent each time, shaking and mixing for 30s, incubating at room temperature for 5min, centrifuging at 6000g for 2min, and removing supernatant; adding protein lysate, shaking and mixing uniformly for 30s, and shaking and incubating at 95 ℃ and 1000rpm for 60 min; cooling to room temperature, adding tetraploid cold acetone, precipitating for 2h, centrifuging at 6000g for 10min, removing supernatant, adding cold acetone, shaking and mixing for 30s, centrifuging at 6000g for 10min, removing supernatant, and cleaning repeatedly. Adding the DB compound solution, shaking and mixing uniformly for 30s to dissolve the protein again.
Adding 5 times volume of enzymolysis buffer (50mM ammonium bicarbonate) and pancreatin, shaking and mixing uniformly for 30s, and incubating for 4h at 37 ℃. Adding formic acid water to make the final concentration of formic acid be 0.1% -1%; and (3) centrifuging at 6000g for 10min, taking the supernatant, slowly passing through a C18 desalting column, continuously washing for 3 times by using a washing solution (0.1% formic acid and 3% acetonitrile), adding a proper amount of eluent (0.1% formic acid and 70% acetonitrile), collecting the filtrate, and freeze-drying. And (5) redissolving and quantifying.
The automated steps and procedures used are shown in fig. 1-4:
s1, the first moving module places the sample tube with the FFPE sample in a first preset position;
s2, the identification unit identifies the sample tube to obtain the identification information of the sample tube;
s3, selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample;
s4, processing the FFPE sample based on the target production mode to obtain a processing result;
wherein, processing the sample based on the target production mode comprises: the second moving module moves the sample tube to an automatic cover opening and closing machine through a mechanical arm to execute cover opening operation; the second moving module carries the sample tube to an automatic workstation through a mechanical arm so as to add a dewaxing reagent to the sample tube through the automatic workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment to obtain a pretreated sample; the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the sample to an automatic workstation to remove supernatant, and then adds a dewaxing reagent into the automatic workstation to repeat dewaxing treatment once to obtain a dewaxed sample; then sequentially adding hydrating reagents with different proportions through an automatic workstation, vibrating, centrifuging and removing the supernatant to obtain hydrated samples, adding protein lysate through the automatic workstation, vibrating, incubating at high temperature, centrifuging and taking the supernatant to obtain crude protein samples; adding acetone into the crude protein sample through an automatic workstation for protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate; and the scanning module scans and records the plate number of the protein sample plate and puts the protein sample plate into the protein library position.
In this embodiment, the automated processing method further includes: generating a detection sheet after obtaining the protein sample; and sending the detection sheet to an intelligent production line so as to perform protein quantification on the protein sample by using the intelligent production line. The method specifically comprises the following steps: quantifying the protein sample by using an enzyme-labeling instrument to obtain the protein concentration; taking a protein sample to perform SDS-PAGE gel running analysis so as to judge whether the protein sample meets the subsequent polypeptide processing requirement or not and obtain a judgment result; executing a polypeptide processing flow when the judgment result shows that the protein sample meets the conditions; and re-extracting the protein sample when the judgment result shows that the protein sample does not meet the polypeptide treatment condition.
In this embodiment, the executing the polypeptide processing procedure comprises: and carrying out enzymolysis, positive pressure desalination, quantification and plate rotation on the protein sample meeting the polypeptide treatment conditions.
In this embodiment, the automated processing method further includes: after ordering of a client is received, under the condition that polypeptide processing is determined to be needed, automatically performing order splicing and typesetting on polypeptides of different projects, scanning to confirm the serial number of a new sample plate, and then adding an enzymolysis reagent for enzymolysis; then adding an acidifying reagent, shaking, uniformly mixing and then centrifuging at a high speed; then taking the sample supernatant to carry out positive pressure desalination, and purifying the polypeptide sample; freeze-drying the desalted polypeptide solution; redissolving the freeze-dried polypeptide, and taking a small amount of polypeptide solution for quantification; then diluting the polypeptide based on the quantitative result; and then centrifuging at a high speed, transferring the polypeptide sample to a 96-well plate on a machine, recording the plate number to obtain the final polypeptide sample to be machined, and arranging the machine list on the system.
Accordingly, the above-mentioned automated method employs the following automated apparatus, see fig. 5, including:
the first moving module is used for placing the sample tube with the FFPE sample in a first preset position;
the identification unit is used for identifying the sample tube to obtain the identification information of the sample tube;
the selection unit is arranged for selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample;
the first processing unit is used for processing the FFPE sample based on the target production mode to obtain a processing result; wherein, the first processing unit includes:
the automatic cover opening and closing module is used for opening the cover of the sample tube;
a second moving module configured to transport the sample tube between the modules in the first processing unit by the robot arm;
the automatic workstation module is used for adding various reagents into the sample tube, and taking or removing supernatant;
the automatic oscillator module is used for oscillating the sample in the sample tube;
a high-speed centrifuge module configured to centrifuge a sample in a sample tube;
the scanning module is used for scanning and recording the plate number of the protein sample plate; and
the control module is used for controlling the second moving module to move the sample tube to the automatic cap opening and closing machine through the mechanical arm to execute cap opening operation; the second moving module carries the sample tube to an automatic workstation through a mechanical arm so as to add a dewaxing reagent to the sample tube through the automatic workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment to obtain a pretreated sample; the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the sample to an automatic workstation to remove supernatant, and then adds a dewaxing reagent into the automatic workstation to repeat dewaxing treatment once to obtain a dewaxed sample; then sequentially adding hydrating reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain hydrated samples, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain crude protein samples; adding acetone into the crude protein sample through an automatic workstation for protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate; the scanning module scans and records the plate number of the protein sample plate, and the second moving module places the protein sample plate into the protein library position.
The automated processing unit further comprises: the generating module is used for generating a detection sheet after the protein sample is obtained; and the detection module is used for sending the detection sheet to the intelligent production line so as to detect the protein of the protein sample by utilizing the intelligent production line.
The detection module includes: the processing submodule is used for sampling the protein sample to obtain a target sample; the code scanning submodule is used for carrying out centrifugal code scanning operation on the target sample to obtain a code-scanned target sample; the judgment submodule is set to judge whether the target sample meets the polypeptide processing condition or not according to the result to obtain a judgment result; the first determining submodule is arranged for executing the polypeptide processing flow when the judgment result shows that the sample meets the polypeptide processing condition; and a second determining submodule configured to reacquire the sample when the determination result indicates that the sample does not meet the polypeptide processing condition.
The first determination submodule includes: the sequencing submodule is used for sequencing the samples meeting the polypeptide processing conditions and performing plate combination on the re-sequenced samples; and the fragmentation submodule is used for carrying out whole-plate sampling and plate number recording on the sample subjected to plate processing, and carrying out automatic polypeptide processing operation on the sample of the plate number recording to obtain a polypeptide sample to be processed on the computer.
The control module is also configured to control the automatic workstation module to add an enzymolysis reagent to the protein sample meeting the polypeptide processing conditions, perform enzymolysis processing on the sample to which the enzymolysis reagent is added, add an acidification reagent to the sample after the enzymolysis processing, and obtain the polypeptide sample after acidification after high-speed centrifugation; the automatic processing device also comprises a purification module which is used for desalting the acidified polypeptide sample and freeze-drying the sample after the desalting; optionally, the automated processing unit further comprises: the automatic detection module is used for automatically detecting the polypeptide sample after desalination and purification; optionally, the automated processing unit further comprises: an acquisition unit configured to acquire a placing order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal process on the sample; and the plate rotating unit is used for rotating the sample after the oscillation centrifugation.
After the mass spectrometer was operated, the number of identified proteins was obtained by searching the data, and the results are shown in table 1.
TABLE 1
| Sample(s) | Number of proteins | Sample(s) | Number of proteins | Sample(s) | Number of proteins | Sample(s) | Number of proteins |
| 1 | 3711 | 11 | 3645 | 21 | 3759 | 31 | 3735 |
| 2 | 3794 | 12 | 3757 | 22 | 3764 | 32 | 3704 |
| 3 | 3663 | 13 | 2523 | 23 | 3771 | 33 | 3798 |
| 4 | 3625 | 14 | 3565 | 24 | 3789 | 34 | 3756 |
| 5 | 2191 | 15 | 3625 | 25 | 3587 | 35 | 3592 |
| 6 | 2682 | 16 | 3666 | 26 | 3750 | 36 | 3678 |
| 7 | 3469 | 17 | 2388 | 27 | 3717 | 37 | 3807 |
| 8 | 3316 | 18 | 1985 | 28 | 3729 | ||
| 9 | 2133 | 19 | 3785 | 29 | 3824 | ||
| 10 | 2727 | 20 | 3753 | 30 | 3815 |
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the invention adopts an automatic method to carry out the pretreatment of the sample, can realize high efficiency and high repeatability, processes the sample without being influenced by human, reduces the contact between human and toxic reagents, and is carried out in a workbench in the whole process.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. An automated processing method for proteomics analysis of FFPE samples, comprising the steps of:
s1, the first moving module places the sample tube with the FFPE sample in a first preset position;
s2, the identification unit identifies the sample tube to obtain the identification information of the sample tube;
s3, selecting a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample;
s4, processing the FFPE sample based on the target production mode to obtain a processing result; wherein processing the sample based on the target production mode comprises:
the second moving module moves the sample tube to an automatic cover opening and closing machine through a mechanical arm to execute a cover opening operation;
the second moving module carries the sample tube to an automated workstation through a mechanical arm to add a dewaxing reagent to the sample tube through the automated workstation;
the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment to obtain a pretreated sample;
the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the pretreated sample to the automatic workstation to remove supernatant, and then adds the dewaxing reagent into the automatic workstation to repeat dewaxing treatment once to obtain a dewaxed sample;
then sequentially adding hydrating reagents with different proportions into the automatic workstation, vibrating and centrifuging, removing the supernatant to obtain hydrated samples, adding protein lysate into the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking the supernatant to obtain crude protein samples;
adding acetone into the crude protein sample through the automatic workstation to perform protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate;
and the scanning module scans and records the plate number of the protein sample plate and puts the protein sample plate into a protein library position.
2. The automated processing method of claim 1, further comprising: generating a detection sheet after obtaining the protein sample; and sending the detection sheet to an intelligent production line so as to perform protein quantification on the protein sample by using the intelligent production line.
3. The automated processing method of claim 2, wherein sending the test order to an intelligent production line for protein testing of the protein sample using the intelligent production line comprises:
quantifying the protein sample by using an enzyme-labeling instrument to obtain the protein concentration;
performing SDS-PAGE gel running analysis on a protein sample to judge whether the protein sample meets the subsequent polypeptide processing requirement or not to obtain a judgment result;
executing a polypeptide processing flow when the judgment result shows that the protein sample meets the conditions;
and re-extracting the protein sample when the judgment result shows that the protein sample does not meet the polypeptide treatment condition.
4. The automated processing method of claim 3, wherein the executing a polypeptide processing flow comprises: and carrying out enzymolysis, positive pressure desalination, quantification and plate rotation on the protein sample meeting the polypeptide treatment conditions.
5. The automated processing method of claim 4, further comprising: after ordering of a client is received, under the condition that polypeptide processing is determined to be needed, automatically performing order splicing and typesetting on polypeptides of different projects, scanning to confirm the serial number of a new sample plate, and then adding an enzymolysis reagent for enzymolysis; then adding an acidifying reagent, shaking, uniformly mixing and then centrifuging at a high speed; then taking the sample supernatant to carry out positive pressure desalination, and purifying the polypeptide sample; freeze-drying the desalted polypeptide solution; redissolving the freeze-dried polypeptide, and taking a small amount of polypeptide solution for quantification; then diluting the polypeptide based on the quantitative result; and then centrifuging at a high speed, transferring the polypeptide sample to a 96-well plate on a machine, recording the plate number to obtain the final polypeptide sample to be machined, and arranging the machine list on the system.
6. An automated processing device for proteomic analysis of FFPE samples, comprising:
the first moving module is used for placing the sample tube with the FFPE sample in a first preset position;
the identification unit is used for identifying the sample tube to obtain the identification information of the sample tube;
a selecting unit configured to select a target production mode based on the identification information, wherein the target production mode is a mode for processing the FFPE sample;
the first processing unit is used for processing the FFPE sample based on the target production mode to obtain a processing result; wherein the first processing unit comprises:
an automatic lid opening and closing module configured to open the sample tube;
a second movement module configured to transport the sample tube between the modules in the first processing unit by a robot arm;
an automated workstation module configured to add various reagents to the sample tube, take a supernatant, or remove a supernatant for processing;
an automated oscillator module configured to perform oscillation processing on the sample in the sample tube;
a high speed centrifuge module configured to centrifuge the sample in the sample tube;
the scanning module is used for scanning and recording the plate number of the protein sample plate; and
the control module is used for controlling the second moving module to move the sample tube to the automatic cover opening and closing machine through the mechanical arm to execute the cover opening operation; the second moving module carries the sample tube to an automated workstation through a mechanical arm to add a dewaxing reagent to the sample tube through the automated workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator to carry out oscillation dewaxing treatment, so as to obtain a pretreated sample; the second moving module transfers the pretreated sample to a high-speed centrifuge for centrifugal treatment, then transfers the pretreated sample to the automatic workstation to remove supernatant, and then adds the dewaxing reagent into the automatic workstation to repeat dewaxing treatment for one time to obtain a dewaxed sample; then sequentially adding hydrating reagents with different proportions into the automatic workstation, vibrating and centrifuging, removing the supernatant to obtain hydrated samples, adding protein lysate into the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking the supernatant to obtain crude protein samples;
adding acetone into the crude protein sample through the automatic workstation to perform protein precipitation, centrifuging to remove supernatant, cleaning twice with acetone, redissolving the purified protein sample, and transferring to a protein sample plate; and the scanning module scans and records the plate number of the protein sample plate, and the second moving module places the protein sample plate into a protein library position.
7. The automated processing device of claim 6, further comprising:
a generation module configured to generate a test ticket after obtaining the protein sample;
and the detection module is used for sending the detection sheet to an intelligent production line so as to carry out protein detection on the protein sample by utilizing the intelligent production line.
8. The automated processing apparatus of claim 7, wherein the detection module comprises:
the processing submodule is used for sampling the protein sample to obtain a target sample;
the code scanning submodule is used for carrying out centrifugal code scanning operation on the target sample to obtain a code-scanned target sample;
the judgment submodule is set to judge whether the target sample meets the polypeptide processing condition or not according to the result to obtain a judgment result;
a first determining submodule configured to execute a polypeptide processing procedure when the judgment result indicates that the sample meets the polypeptide processing condition;
a second determining submodule configured to reacquire the sample if the determination indicates that the sample does not meet the polypeptide processing condition.
9. The automated processing apparatus of claim 8, wherein the first determination submodule comprises:
the sequencing submodule is used for sequencing the samples meeting the polypeptide processing conditions and performing plate combination on the re-sequenced samples;
and the fragmentation submodule is used for carrying out whole-plate sampling and plate number recording on the sample subjected to plate processing, and carrying out automatic polypeptide processing operation on the sample of the plate number recording to obtain a polypeptide sample to be processed on the computer.
10. The automated processing device according to claim 9, wherein the control module is further configured to control the automated workstation module to add an enzymatic reagent to the protein sample meeting the polypeptide processing conditions, perform enzymatic treatment on the sample to which the enzymatic reagent is added, add an acidifying reagent to the sample after the enzymatic treatment, and obtain the polypeptide sample after the acidification after the high-speed centrifugation;
the automatic processing device also comprises a purification module which is used for desalting the acidified polypeptide sample and freeze-drying the desalted sample;
optionally, the automated processing apparatus further comprises: the automatic detection module is used for automatically detecting the polypeptide sample after desalination and purification;
optionally, the automated processing apparatus further comprises: an acquisition unit configured to acquire a placing order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal process on the sample; and the plate rotating unit is used for rotating the sample after the oscillation centrifugation.
11. A computer-readable storage medium comprising a stored program, wherein the program when executed performs the automated processing method for proteomic analysis of FFPE samples of any one of claims 1 to 5.
12. An automated processing system for samples comprising a memory and a processor, the memory having stored therein a computer program, characterized in that the processor is arranged to execute the automated processing method for proteomic analysis of FFPE samples as claimed in any one of claims 1 to 5 by means of the computer program.
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