CN114459872B - Automated processing method and device for performing proteomics analysis on FFPE sample - Google Patents
Automated processing method and device for performing proteomics analysis on FFPE sample Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses an automated processing method and device for performing proteomics analysis on FFPE samples. The automatic processing method comprises the following steps: s1, a first moving module places a sample tube with FFPE samples placed at a first preset position; s2, the identification unit identifies the sample tube to obtain 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. By applying the technical scheme of the invention, the purpose of automatically processing the FFPE sample can be realized by the automatic processing method, the technical effect of improving the sample processing efficiency is achieved, and the technical problem that the pretreatment of the FFPE sample proteomics needs to be completed manually in the related technology and has lower efficiency is solved.
Description
Technical Field
The invention relates to the field of biotechnology automation, in particular to an automated processing method and device for performing proteomics analysis on FFPE samples.
Background
In vivo tissue testing, paraffin sections (FFPE) have been used to preserve the histological and morphological structure of tissue for histopathological diagnosis of disease. The FFPE tissue is highly stable and can be stored for a long period of time at room temperature. Thus, a large number of normal and pathological FFPE samples containing information related to diagnosis, treatment and outcome are produced worldwide. Thus, FFPE tissue libraries are now a potential valuable resource for retrospective research in biomarker discovery and validation of cancer and other important diseases. Combining high throughput proteomic analysis techniques with FFPE will reveal a large amount of information related to disease, playing an important role in disease marker discovery, disease diagnosis, prognosis, monitoring and therapy related aspects.
The pretreatment flow of FFPE samples for proteomics analysis comprises the following steps: dewaxing, hydrating, extracting protein, quantifying protein, enzymolysis, desalting, freeze drying and quantifying. Currently these treatments are done mainly manually by high quality professionals. The FFPE sample pretreatment process is complicated and requires high-strength manual operation. 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 judgment can lead to uneven product quality. In addition, toxic reagents (xylenes) can be contacted during sample processing, presenting a significant safety hazard.
Disclosure of Invention
The invention aims to provide an automated processing method and device for performing proteomics analysis on an FFPE sample, and aims to solve the technical problems that paraffin section samples are difficult to extract, the extraction efficiency is low and the experimental period is long in the prior art.
In order to achieve the above object, according to one aspect of the present invention, an automated processing method for proteomics analysis of FFPE samples is provided. The automated processing method comprises the following steps: s1, a first moving module places a sample tube with FFPE samples placed at a first preset position; s2, the identification unit identifies the sample tube to obtain 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, based on the target production mode handle the sample, include: 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 conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into 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 perform 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 to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample; sequentially adding hydration reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample; adding acetone into the crude protein sample through an automatic workstation to perform protein precipitation, centrifuging to remove supernatant, washing twice with acetone, re-dissolving 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 places the protein sample plate into a protein library position.
Further, the automated processing method further comprises: after obtaining a protein sample, generating a detection sheet; the detection sheet is sent to an intelligent production line so as to quantitatively determine the protein of the protein sample by using the intelligent production line.
Further, sending the detection sheet to an intelligent production line to perform protein detection on the protein sample by using the intelligent production line, including: performing enzyme-labeled instrument quantification on the protein sample to obtain protein concentration; performing SDS-PAGE gel running analysis on the protein sample to judge whether the protein sample meets the requirement of subsequent polypeptide treatment or not, so as to obtain a judging result; executing a polypeptide processing flow when the judging result shows that the protein sample meets the condition; and re-extracting the protein sample when the judging result shows that the protein sample does not meet the polypeptide treatment condition.
Further, performing the polypeptide processing procedure includes: and (3) carrying out enzymolysis, positive pressure desalination, quantification and plate rotation on the protein sample meeting the polypeptide treatment conditions.
Further, the automated processing method further comprises: when receiving a customer order, automatically assembling and typesetting polypeptides of different projects under the condition that the polypeptides are determined to be processed, scanning to confirm the number of a new sample plate, and then adding an enzymatic hydrolysis reagent for enzymolysis; then adding an acidification reagent, shaking and uniformly mixing, and centrifuging at a high speed; then taking a sample supernatant to carry out positive pressure desalination, and purifying a polypeptide sample; lyophilizing the desalted polypeptide solution; re-dissolving the polypeptide after freeze-drying, and quantifying a small amount of polypeptide solution; diluting the polypeptide based on the quantitative result; and then, high-speed centrifugation is carried out, the polypeptide sample is transferred into a 96-well plate of the machine, the plate number is recorded, the final polypeptide sample to be machine is obtained, and the system is arranged on a machine list.
According to another aspect of the present invention, an automated processing apparatus for proteomic analysis of FFPE samples is provided. The automated processing apparatus includes: a first movement module arranged to place a sample tube with FFPE sample placed in a first predetermined position; the identifying unit is used for identifying the sample tube to obtain the identification information of the sample tube; a selection unit configured to select a target production method based on the identification information, wherein the target production method is a method 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: an automatic opening and closing cover machine module which is used for opening and closing the cover of the sample tube; a second movement module arranged to carry the sample tube between the modules within the first processing unit by means of a robotic arm; an automated workstation module configured to add various reagents to the sample tube, take a supernatant, or remove a supernatant treatment; the automatic oscillator module is arranged for carrying out oscillation treatment on 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 cover opening and closing machine through the mechanical arm to execute cover opening operation; the second moving module conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into 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 perform 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 to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample; sequentially adding hydration reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample; adding acetone into the crude protein sample through an automatic workstation to perform protein precipitation, centrifuging to remove supernatant, washing twice with acetone, re-dissolving 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 a protein library position.
Further, the automated processing apparatus further includes: the generation module is used for generating a detection list after the protein sample is obtained; the detection module is used for sending the detection list to the intelligent production line so as to detect the protein by utilizing the intelligent production line.
Further, the detection module includes: the processing submodule is used for carrying out sampling processing on the protein sample to obtain a target sample; the code scanning module is used for performing centrifugal code scanning operation on the target sample to obtain a code scanned target sample; the judging submodule is used for setting a result to judge whether the target sample meets the polypeptide processing conditions or not to obtain a judging result; the first determining submodule is used for executing a polypeptide processing flow when the judging result shows that the sample meets the polypeptide processing condition; and the second determination submodule is used for re-acquiring the sample when the judgment result shows that the sample does not meet the polypeptide processing condition.
Further, the first determining submodule includes: the sequencing submodule is used for sequencing samples meeting the polypeptide processing conditions and plywood the re-sequenced samples; and the fragmentation submodule is used for carrying out whole-plate sampling and recording of plate numbers on the samples processed by the folding plates, and carrying out automatic polypeptide processing operation on the samples recorded with the plate numbers to obtain polypeptide samples to be put on the machine.
Further, 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 added with the enzymolysis reagent, add an acidification reagent to the sample after enzymolysis, and obtain an acidified polypeptide sample after high-speed centrifugation; the automated processing unit further comprises a purification module configured to desalt the acidified polypeptide sample and freeze-dry 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 order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal processing on a sample; and the rotating plate unit is used for carrying out rotating plate treatment on the sample after vibration centrifugation.
According to yet another aspect of the present invention, a computer-readable storage medium is provided. The computer readable storage medium includes a stored program that when executed performs an automated processing method for proteomic analysis of any of the FFPE samples described above.
According to yet another aspect of the present invention, an automated sample processing system is provided. The automated processing system comprises a memory having a computer program stored therein and a processor configured to perform an automated processing method of proteomic analysis of any of the FFPE samples described above by the computer program.
By applying the technical scheme of the invention, the purpose of automatically processing the FFPE sample can be realized by the automatic processing method, the technical effect of improving the sample processing efficiency is achieved, and the technical problem that the pretreatment of the FFPE sample proteomics needs to be completed manually in the related technology and has lower efficiency is solved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows a flow chart of a method for automated processing of a sample according to an embodiment of the invention;
FIG. 2 shows a flow chart of automated extraction of FFPE sample proteins in accordance with an embodiment of the present invention;
FIG. 3 shows a flow chart of protein control according to an embodiment of the 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 apparatus for samples according to an embodiment of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The application will be described in detail below with reference to the drawings in connection with embodiments.
According to an exemplary embodiment of the present invention, an automated processing method for proteomic analysis of FFPE samples is provided. The automated processing method comprises the following steps: s1, a first moving module places a sample tube with FFPE samples placed at a first preset position; s2, the identification unit identifies the sample tube to obtain 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, based on the target production mode handle the sample, include: 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 conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into 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 perform 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 to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample; sequentially adding hydration reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample; adding acetone into the crude protein sample through an automatic workstation to perform protein precipitation, centrifuging to remove supernatant, washing twice with acetone, re-dissolving 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 places the protein sample plate into a protein library position.
By applying the technical scheme of the invention, the purpose of automatically processing the FFPE sample can be realized by the automatic processing method, the technical effect of improving the sample processing efficiency is achieved, and the technical problem that the pretreatment of the FFPE sample proteomics needs to be completed manually in the related technology and has lower efficiency is solved.
Wherein, "shake hyperthermia incubation" refers to the general meaning in the art, such as: incubation was performed at 95℃with shaking at 1000 rpm. Wherein, the method is characterized in that the method is carried out by decrosslinking and reductive alkylation at 95 ℃, and the proteins and nucleic acid and proteins are crosslinked after formaldehyde treatment, so that the subsequent treatment is not facilitated, and the crosslinking is removed.
To increase the automation level, the automated processing method further comprises: after obtaining a protein sample, generating a detection sheet; the detection sheet is sent to an intelligent production line so as to quantitatively determine the protein of the protein sample by using the intelligent production line.
In an exemplary embodiment of the present invention, a test sheet is sent to a smart manufacturing line for protein testing of protein samples using the smart manufacturing line, comprising: performing enzyme-labeled instrument quantification on the protein sample to obtain protein concentration; performing SDS-PAGE gel running analysis on the protein sample to judge whether the protein sample meets the requirement of subsequent polypeptide treatment or not, so as to obtain a judging result; executing a polypeptide processing flow when the judging result shows that the protein sample meets the condition; and re-extracting the protein sample when the judging result shows that the protein sample does not meet the polypeptide treatment condition.
To further increase the level of automation, the execution of the polypeptide processing procedure includes: and (3) 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: when receiving a customer order, automatically assembling and typesetting polypeptides of different projects under the condition that the polypeptides are determined to be processed, scanning to confirm the number of a new sample plate, and then adding an enzymatic hydrolysis reagent for enzymolysis; then adding an acidification reagent, shaking and uniformly mixing, and centrifuging at a high speed; then taking a sample supernatant to carry out positive pressure desalination, and purifying a polypeptide sample; lyophilizing the desalted polypeptide solution; re-dissolving the polypeptide after freeze-drying, and quantifying a small amount of polypeptide solution; diluting the polypeptide based on the quantitative result; and then, high-speed centrifugation is carried out, the polypeptide sample is transferred into a 96-well plate of the machine, the plate number is recorded, the final polypeptide sample to be machine is obtained, and the system is arranged on a machine list.
According to an exemplary embodiment of the present invention, an automated processing apparatus for proteomic analysis of FFPE samples is provided. The device comprises: a first movement module arranged to place a sample tube with FFPE sample placed in a first predetermined position; the identifying unit is used for identifying the sample tube to obtain the identification information of the sample tube; a selection unit configured to select a target production method based on the identification information, wherein the target production method is a method 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: an automatic opening and closing cover machine module which is used for opening and closing the cover of the sample tube; a second movement module arranged to carry the sample tube between the modules within the first processing unit by means of a robotic arm; an automated workstation module configured to add various reagents to the sample tube, take a supernatant, or remove a supernatant treatment; the automatic oscillator module is arranged for carrying out oscillation treatment on 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 cover opening and closing machine through the mechanical arm to execute cover opening operation; the second moving module conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into 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 perform 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 to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample; sequentially adding hydration reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample; adding acetone into the crude protein sample through an automatic workstation to perform protein precipitation, centrifuging to remove supernatant, washing twice with acetone, re-dissolving 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 a protein library position.
Preferably, the automated processing apparatus further comprises: the generation module is used for generating a detection list after the protein sample is obtained; the detection module is used for sending the detection list to the intelligent production line so as to detect the protein by utilizing the intelligent production line.
In an exemplary embodiment of the present invention, a detection module includes: the processing submodule is used for carrying out sampling processing on the protein sample to obtain a target sample; the code scanning module is used for performing centrifugal code scanning operation on the target sample to obtain a code scanned target sample; the judging submodule is used for setting a result to judge whether the target sample meets the polypeptide processing conditions or not to obtain a judging result; the first determining submodule is used for executing a polypeptide processing flow when the judging result shows that the sample meets the polypeptide processing condition; and the second determination submodule is used for re-acquiring the sample when the judgment result shows that the sample does not meet the polypeptide processing condition.
Typically, the first determining submodule includes: the sequencing submodule is used for sequencing samples meeting the polypeptide processing conditions and plywood the re-sequenced samples; and the fragmentation submodule is used for carrying out whole-plate sampling and recording of plate numbers on the samples processed by the folding plates, and carrying out automatic polypeptide processing operation on the samples recorded with the plate numbers to obtain polypeptide samples to be put on the machine.
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 added with the enzymolysis reagent, add an acidification reagent to the sample after enzymolysis, and obtain an acidified polypeptide sample after high-speed centrifugation; the automated processing unit further comprises a purification module configured to desalt the acidified polypeptide sample and freeze-dry 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 order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal processing on a sample; and the rotating plate unit is used for carrying out rotating plate treatment on the sample after vibration centrifugation.
According to an exemplary embodiment of the present invention, a computer readable storage medium is provided, the computer readable storage medium comprising a stored program, which when executed performs an automated processing method for performing proteomic analysis of any one of the FFPE samples described above.
According to an exemplary embodiment of the present invention, an automated processing system for samples is provided, comprising a memory having a computer program stored therein and a processor configured to perform an automated processing method for proteomic analysis of any of the FFPE samples described above by the computer program.
The beneficial effects of the present invention will be further illustrated with reference to the following examples, wherein the reagents used in the following examples are conventional in the art.
Example 1
Sample: paraffin section of human tissue
Reagent:
Dewaxing reagent: xylene (P)
Hydrating 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 (acetone)
The DB complex solution comprises: 8M urea and 100mM TEAB.
Setting relevant experimental parameters: adding a dewaxing reagent, shaking and uniformly mixing for 30s, incubating at room temperature for 10min, centrifuging at 6000g for 2min, removing the supernatant, and repeating the operation once; sequentially adding hydration reagent: 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 the protein lysate, shaking and mixing for 30s, and shaking and incubating at 95 ℃ and 1000rpm for 60min; cooling to room temperature, adding four times of cold acetone for precipitation for 2h, centrifuging for 10min at 6000g, removing supernatant, adding cold acetone, shaking, mixing for 30s, centrifuging for 10min at 6000g, removing supernatant, and cleaning repeatedly. Adding DB compound solution, shaking and mixing uniformly for 30s of compound protein.
5 Times of enzymolysis buffer (50 mM ammonium bicarbonate) and pancreatin are added, mixed with shaking for 30s and incubated for 4h at 37 ℃. Adding formic acid water to make the final concentration of formic acid be 0.1% -1%; centrifuging 6000g for 10min, collecting supernatant, slowly passing through C18 desalting column, continuously cleaning with cleaning solution (0.1% formic acid, 3% acetonitrile) for 3 times, adding appropriate amount of eluent (0.1% formic acid, 70% acetonitrile), collecting filtrate, and lyophilizing. And (5) re-dissolving and quantifying.
The automation steps and processes used are described in FIGS. 1-4:
s1, a first moving module places a sample tube with FFPE samples placed at a first preset position;
s2, the identification unit identifies the sample tube to obtain 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, based on the target production mode handle the sample, include: 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 conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into 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 perform 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 to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample; sequentially adding hydration reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample; adding acetone into the crude protein sample through an automatic workstation to perform protein precipitation, centrifuging to remove supernatant, washing twice with acetone, re-dissolving 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 places the protein sample plate into a protein library position.
In this embodiment, the automated processing method further includes: after obtaining a protein sample, generating a detection sheet; the detection sheet is sent to an intelligent production line so as to quantitatively determine the protein of the protein sample by using the intelligent production line. The method specifically comprises the following steps: performing enzyme-labeled instrument quantification on the protein sample to obtain protein concentration; performing SDS-PAGE gel running analysis on the protein sample to judge whether the protein sample meets the requirement of subsequent polypeptide treatment or not, so as to obtain a judging result; executing a polypeptide processing flow when the judging result shows that the protein sample meets the condition; and re-extracting the protein sample when the judging result shows that the protein sample does not meet the polypeptide treatment condition.
In this embodiment, the execution of the polypeptide processing procedure includes: and (3) 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: when receiving a customer order, automatically assembling and typesetting polypeptides of different projects under the condition that the polypeptides are determined to be processed, scanning to confirm the number of a new sample plate, and then adding an enzymatic hydrolysis reagent for enzymolysis; then adding an acidification reagent, shaking and uniformly mixing, and centrifuging at a high speed; then taking a sample supernatant to carry out positive pressure desalination, and purifying a polypeptide sample; lyophilizing the desalted polypeptide solution; re-dissolving the polypeptide after freeze-drying, and quantifying a small amount of polypeptide solution; diluting the polypeptide based on the quantitative result; and then, high-speed centrifugation is carried out, the polypeptide sample is transferred into a 96-well plate of the machine, the plate number is recorded, the final polypeptide sample to be machine is obtained, and the system is arranged on a machine list.
Accordingly, the above-mentioned automation method employs the following automation device, see fig. 5, including:
A first movement module arranged to place a sample tube with FFPE sample placed in a first predetermined position;
the identifying unit is used for identifying the sample tube to obtain the identification information of the sample tube;
a selection unit configured to select a target production method based on the identification information, wherein the target production method is a method 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:
An automatic opening and closing cover machine module which is used for opening and closing the cover of the sample tube;
A second movement module arranged to carry the sample tube between the modules within the first processing unit by means of a robotic arm;
An automated workstation module configured to add various reagents to the sample tube, take a supernatant, or remove a supernatant treatment;
the automatic oscillator module is arranged for carrying out oscillation treatment on 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 cover opening and closing machine through the mechanical arm to execute cover opening operation; the second moving module conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into 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 perform 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 to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample; sequentially adding hydration reagents with different proportions through an automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample; adding acetone into the crude protein sample through an automatic workstation to perform protein precipitation, centrifuging to remove supernatant, washing twice with acetone, re-dissolving 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 a protein library position.
The automated processing apparatus further includes: the generation module is used for generating a detection list after the protein sample is obtained; the detection module is used for sending the detection list to the intelligent production line so as to detect the protein by utilizing the intelligent production line.
The detection module comprises: the processing submodule is used for carrying out sampling processing on the protein sample to obtain a target sample; the code scanning module is used for performing centrifugal code scanning operation on the target sample to obtain a code scanned target sample; the judging submodule is used for setting a result to judge whether the target sample meets the polypeptide processing conditions or not to obtain a judging result; the first determining submodule is used for executing a polypeptide processing flow when the judging result shows that the sample meets the polypeptide processing condition; and the second determination submodule is used for re-acquiring the sample when the judgment result shows that the sample does not meet the polypeptide processing condition.
The first determination submodule includes: the sequencing submodule is used for sequencing samples meeting the polypeptide processing conditions and plywood the re-sequenced samples; and the fragmentation submodule is used for carrying out whole-plate sampling and recording of plate numbers on the samples processed by the folding plates, and carrying out automatic polypeptide processing operation on the samples recorded with the plate numbers to obtain polypeptide samples to be put on the machine.
The control module is also arranged to control the automatic workstation module to add an enzymolysis reagent to the protein sample meeting the polypeptide processing conditions, carry out enzymolysis on the sample added with the enzymolysis reagent, add an acidification reagent to the sample after enzymolysis, and obtain an acidified polypeptide sample after high-speed centrifugation; the automated processing unit further comprises a purification module configured to desalt the acidified polypeptide sample and freeze-dry 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 order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal processing on a sample; and the rotating plate unit is used for carrying out rotating plate treatment on the sample after vibration centrifugation.
After the mass spectrum was put on the machine, the number of proteins obtained after searching the database was identified, and the results are shown in table 1.
TABLE 1
| Sample of | Protein count | Sample of | Protein count | Sample of | Protein count | Sample of | Protein count |
| 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 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, treat the sample without being affected by manpower, reduce the contact between people and toxic reagents, and is carried out in a workbench in the whole process.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. 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, a first moving module places a sample tube with FFPE samples placed at a first preset position;
s2, the identification unit identifies the sample tube to obtain 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 method 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 conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into the sample tube through the automatic workstation;
the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator so as to perform oscillation dewaxing treatment and 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 the automatic workstation to remove supernatant, and adds the dewaxing reagent to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample;
Sequentially adding hydration reagents with different proportions through the automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample;
Adding acetone into the crude protein sample through the automatic workstation to carry out protein precipitation, centrifuging to remove supernatant, washing twice by using acetone, and finally re-dissolving 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 places the protein sample plate into a protein library.
2. The automated processing method of claim 1, further comprising: after the protein sample is obtained, generating a detection sheet; and sending the detection list to an intelligent production line so as to quantitatively determine the protein of the protein sample by using the intelligent production line.
3. The automated processing method of claim 2, wherein sending the test sheet to a smart production line for protein testing of the protein sample using the smart production line comprises:
Performing enzyme-labeled instrument quantification on the protein sample to obtain protein concentration;
performing SDS-PAGE gel running analysis on a protein sample to judge whether the protein sample meets the requirement of subsequent polypeptide treatment or not, so as to obtain a judging result;
executing a polypeptide processing flow when the judging result shows that the protein sample meets the condition;
And re-extracting the protein sample when the judging result shows that the protein sample does not meet the polypeptide treatment condition.
4. The automated processing method of claim 3, wherein the performing a polypeptide processing flow comprises: and (3) 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: when receiving a customer order, automatically assembling and typesetting polypeptides of different projects under the condition that the polypeptides are determined to be processed, scanning to confirm the number of a new sample plate, and then adding an enzymatic hydrolysis reagent for enzymolysis; then adding an acidification reagent, shaking and uniformly mixing, and centrifuging at a high speed; then taking a sample supernatant to carry out positive pressure desalination, and purifying a polypeptide sample; lyophilizing the desalted polypeptide solution; re-dissolving the polypeptide after freeze-drying, and quantifying a small amount of polypeptide solution; diluting the polypeptide based on the quantitative result; and then, high-speed centrifugation is carried out, the polypeptide sample is transferred into a 96-well plate of the machine, the plate number is recorded, the final polypeptide sample to be machine is obtained, and the system is arranged on a machine list.
6. An automated processing apparatus for proteomic analysis of FFPE samples, comprising:
A first movement module arranged to place a sample tube with FFPE sample placed in a first predetermined position;
the identifying unit is used for identifying the sample tube so as to obtain identification information of the sample tube;
A selection unit configured to select a target production method based on the identification information, wherein the target production method is a method 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:
an automatic opening and closing cover machine module which is used for opening and closing the cover of the sample tube;
a second movement module arranged to carry the sample tube between the modules within the first processing unit by a robotic arm;
An automated workstation module configured to add various reagents to the sample tube, take a supernatant, or remove a supernatant treatment;
An automatic oscillator module, configured to perform an oscillation process on a sample in the sample tube;
A high-speed centrifuge module configured to centrifuge samples 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 cover opening operation; the second moving module conveys the sample tube to an automatic workstation through a mechanical arm so as to add dewaxing reagent into the sample tube through the automatic workstation; the second moving module moves the sample tube added with the dewaxing reagent to an automatic oscillator so as to perform oscillation dewaxing treatment and 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 the automatic workstation to remove supernatant, and adds the dewaxing reagent to repeat dewaxing treatment once through the automatic workstation to obtain a dewaxed sample; sequentially adding hydration reagents with different proportions through the automatic workstation, vibrating and centrifuging, removing supernatant to obtain a hydrated sample, adding protein lysate through the automatic workstation, vibrating and incubating at high temperature, centrifuging, and taking supernatant to obtain a crude protein sample;
Adding acetone into the crude protein sample through the automatic workstation to carry out protein precipitation, centrifuging to remove supernatant, washing twice by using acetone, and finally re-dissolving 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 in a protein library position.
7. The automated processing apparatus of claim 6, further comprising:
the generation module is used for generating a detection list after the protein sample is obtained;
And the detection module is used for sending the detection list to an intelligent production line so as to detect the protein by utilizing the intelligent production line.
8. The automated processing unit of claim 7, wherein the detection module comprises:
the processing submodule is used for carrying out sampling processing on the protein sample to obtain a target sample;
the code scanning sub-module is arranged for performing centrifugal code scanning operation on the target sample to obtain a code scanned target sample;
the judging submodule is used for setting a result to judge whether the target sample meets the polypeptide processing condition or not to obtain a judging result;
the first determining submodule is used for executing a polypeptide processing flow when the judging result shows that the sample meets the polypeptide processing condition;
And the second determination submodule is used for re-acquiring the sample when the judgment result shows that the sample does not meet the polypeptide processing condition.
9. The automated processing unit of claim 8, wherein the first determination submodule comprises:
A sequencing submodule, configured to sequence samples meeting the polypeptide processing conditions and to sequence the re-sequenced samples;
And the fragmentation submodule is used for carrying out whole-plate sampling and recording of plate numbers on the samples processed by the folding plates, and carrying out automatic polypeptide processing operation on the samples recorded with the plate numbers to obtain polypeptide samples to be put on the machine.
10. The automated processing apparatus of claim 9, wherein the control module is further configured to control the automated workstation module to add an enzymatic reagent to the protein sample that meets the polypeptide processing conditions, to subject the enzymatic reagent-added sample to an enzymatic treatment, to add an acidifying reagent to the enzymatic treated sample, and to obtain an acidified polypeptide sample after high-speed centrifugation;
The automated processing unit further comprises a purification module configured to desalt the acidified polypeptide sample and freeze-dry 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 order instruction; a centrifugal processing unit configured to perform an oscillating centrifugal processing on a sample; and the rotating plate unit is used for carrying out rotating plate treatment on the sample after vibration centrifugation.
11. A computer-readable storage medium comprising a stored program, characterized in that the program when run performs an automated processing method of proteomic analysis of FFPE samples as claimed in 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 perform an automated processing method for proteomic analysis of FFPE samples according to any of claims 1 to 5 by means of the computer program.
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| WO2019211223A1 (en) * | 2018-04-30 | 2019-11-07 | Biotage Ab | Qualitative analysis of proteins |
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| WO2019211223A1 (en) * | 2018-04-30 | 2019-11-07 | Biotage Ab | Qualitative analysis of proteins |
| CN113267394A (en) * | 2021-06-10 | 2021-08-17 | 谱天（天津）生物科技有限公司 | FFPE reference substance for proteome quality control, and preparation method and application thereof |
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