CN111220429A - An array high-throughput protein sample pretreatment device - Google Patents
An array high-throughput protein sample pretreatment device Download PDFInfo
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- CN111220429A CN111220429A CN201811406709.6A CN201811406709A CN111220429A CN 111220429 A CN111220429 A CN 111220429A CN 201811406709 A CN201811406709 A CN 201811406709A CN 111220429 A CN111220429 A CN 111220429A
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- 102000004169 proteins and genes Human genes 0.000 title claims description 29
- 108090000623 proteins and genes Proteins 0.000 title claims description 29
- 238000004925 denaturation Methods 0.000 claims description 30
- 230000036425 denaturation Effects 0.000 claims description 30
- 230000009467 reduction Effects 0.000 claims description 29
- 238000010438 heat treatment Methods 0.000 claims description 23
- 239000002904 solvent Substances 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 11
- 238000001802 infusion Methods 0.000 claims description 9
- 238000012545 processing Methods 0.000 claims description 7
- 108010026552 Proteome Proteins 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 4
- 230000005611 electricity Effects 0.000 claims description 2
- 238000005485 electric heating Methods 0.000 claims 4
- 239000012510 hollow fiber Substances 0.000 claims 4
- 239000012528 membrane Substances 0.000 claims 4
- 239000012295 chemical reaction liquid Substances 0.000 claims 1
- 239000003638 chemical reducing agent Substances 0.000 claims 1
- 238000006722 reduction reaction Methods 0.000 description 16
- 239000000243 solution Substances 0.000 description 6
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 5
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 5
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 5
- 239000001099 ammonium carbonate Substances 0.000 description 5
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000003978 infusion fluid Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/44—Sample treatment involving radiation, e.g. heat
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/622—Ion mobility spectrometry
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
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Abstract
本发明涉及一种阵列高通量蛋白质样品预处理装置，是一种可对多个蛋白质样品同时进行在线变性和还原、除盐以及酶解的样品处理系统，包括：蛋白质处理模块，电源模块，加热控制模块，泵及泵控制模块。本发明的特点是在单路蛋白质预处理仪的基础上,增加到八路同时处理,把单路装置的每个操作模块,在电路、控制及液体流路都分别重新做了优化设计，用最优的方式把各个模块集成到一个操作装置中同时控制八路操作。该装置操作简单,控制精确可靠,样品处理量大,可满足大批量样品的检测要求。
The invention relates to an array high-throughput protein sample preprocessing device, which is a sample processing system that can simultaneously perform online denaturation and reduction, desalination and enzymolysis for multiple protein samples, comprising: a protein processing module, a power module, Heating Control Module, Pump and Pump Control Module. The feature of the present invention is that on the basis of the single-channel protein pretreatment instrument, it is increased to eight channels for simultaneous processing, and each operation module of the single-channel device is re-optimized in circuit, control and liquid flow path respectively. The optimal way integrates each module into one operation device and controls eight-way operation at the same time. The device has the advantages of simple operation, accurate and reliable control, and large sample processing capacity, which can meet the detection requirements of large batches of samples.
Description
Technical Field
The invention relates to a high-flux protein sample pretreatment device, which is a sample treatment system capable of simultaneously carrying out online denaturation, reduction, desalination and enzymolysis on a plurality of protein samples.
Background
With the development of protein separation and detection technology, some special parameters and indexes increasingly attract attention in the medical field, and thus, the demand for large-batch and rapid clinical sample detection is generated.
The traditional proteome sample pretreatment comprises the steps of denaturation, reduction, alkylation, desalination, enzymolysis and the like, is usually carried out off-line manually, not only takes long time, but also has the possibility of sample pollution and loss, and seriously influences the result of large-scale protein analysis. Therefore, the development of an on-line protein sample pretreatment method and device has very important significance. However, the existing on-line proteome sample processing system can only integrate a few steps (such as on-line enzymolysis, analytical Chimica acta,2018,1000, 172-179; Talanta,2015,141,235-238.) or can only process one sample at a time, and is difficult to meet the requirement of the field of clinic and the like for large-array sample analysis, so in order to solve the above problems, an array type on-line proteome sample processing system (patent number: 201210517241.4) is further developed on the basis of a high-throughput proteome sample pre-processing device developed in the previous period
The device is protein sample pretreatment equipment with online protein denaturation, reduction, desalting and online enzymolysis functions. The device can realize high-flux and low-loss pretreatment of protein samples, and has good application prospect in proteomics research and clinical examination.
Disclosure of Invention
The invention aims to provide a sample pretreatment device integrating protein denaturation, reduction, desalting and online enzymolysis, and the realization of the function needs to control the transportation of exchange liquid, control high-temperature denaturation and reduction and enzyme reaction at a certain temperature, so the invention adopts the following technical scheme.
An array high-throughput protein sample pretreatment device, comprising: the system comprises a protein pretreatment module, a power supply module, a heating control module, a pump and a pump control module. The method specifically comprises the following steps: a high temperature denaturation and reduction unit component, a solvent displacer component, an enzyme reactor component, a heater power supply, a pump driving power supply, a heating controller, a pump flow controller and an infusion pump.
The high-temperature denaturation and reduction device assembly and the exchange liquid in the solvent displacer assembly are respectively completed by the infusion pump in a serial conveying mode, and the flowing mode can ensure that the flow rate of each assembly of the high-temperature denaturation and reduction device is the same.
The power module is respectively a high-temperature denaturation and reduction device component and an enzyme reactor component heating power supply and a pump power supply, and the high-temperature denaturation and reduction device component and the enzyme reactor component adopt a parallel heating mode respectively to ensure that the temperature of each heating element is the same.
The heating controller provides temperature control for the high-temperature denaturation and reduction component and the enzyme reactor component respectively.
The infusion pump is respectively a high-temperature denaturation and reduction device component, a solvent displacer component and an infusion liquid.
And the pump flow controllers respectively provide flow control for the infusion pumps.
The liquid-exchanging pump uses 4 pumps to divide into 2 groups, which are high-temperature denaturation and reduction components, and the solvent displacer component is used for conveying the exchanging liquid, so that the fluid pressure and the flow rate can be ensured to be stable, and the liquid-exchanging pump adopts the liquid-exchanging mode of connecting flow paths in series.
The high-temperature denaturation and reduction device assembly and the solvent displacer assembly are heated by adopting parallel circuits, so that the heating parts have stable and uniform heat, and the safety voltage of 36V is respectively used to ensure the safety of the electricity environment of the instrument.
The heating controller adopts sensor device parallel connection, can switch the temperature of observing every heating device, avoids every detected parts to detect respectively and control and causes instrument cost to increase, and the volume increases, the scheduling problem to can guarantee that every heating part temperature is unanimous.
The array high-throughput protein pretreatment equipment can be applied to proteome sample treatment of clinical, food safety, environmental exposure crowd and the like.
The invention has the following characteristics:
1, each operation unit module of the whole instrument is optimized, and the modules are optimally matched.
2, the sample processing capacity is large, the power consumption is low, and the method is safe and reliable.
3, the operation and the component replacement are convenient, and the maintenance is simple.
Drawings
FIG. 1 is a diagram of the array device processing of real samples, wherein (a), (b) and (c) are the results of the array device processing Hela samples in three ways;
FIG. 2 is a graph of the array device processing of real samples, wherein six lanes of the array device (a) (b) (c) (d) (e) (f) process plasma sample results separately;
FIG. 3 is a schematic diagram of an array high-throughput protein sample pretreatment apparatus.
Detailed Description
The experiment adopts eight high-throughput protein sample pretreatment devices, eight high-temperature denaturation and reduction devices, eight solvent displacers, eight enzyme reactors and eight samples for simultaneous operation, four pumps are used for dividing the samples into two groups, and exchange liquid is respectively conveyed to the high-temperature denaturation and reduction devices and the solvent displacers,
example 1
As shown in FIG. 1, the pump driving power source (04) was turned on in the connected system, and all of the high temperature denaturing and reducing device modules (06) were filled with a mixed solution of 2M guanidine hydrochloride and 10mM DTT, all of the solvent displacer modules (07) were filled with a 50mM ammonium bicarbonate solution, and a 6cm long enzyme column was connected as an enzyme reactor module (08). Meanwhile, a heater power supply (05) is turned on, and the temperature of the heating controller (03) is set to be 95 ℃.
3 aliquots of 4. mu.L of 8M urea-extracted Hela cellular protein (BCA assay concentration of 3mg/mL) were each diluted ten-fold with 50mM ammonium bicarbonate solution. After the temperature of the component (06) reaches 95 ℃, simultaneously introducing Hela cell protein samples into different pipelines of the whole system at a speed of 1 mu L/min by means of an injector respectively. After the end of the introduction, 20. mu.L of a 50mM ammonium hydrogencarbonate solution was introduced into the system. The solvent displacer component (07) is ensured to be operated all the time in the system operation. Meanwhile, each part was collected to obtain an enzymatic substance, which was 25. mu.L. Formic acid was added to the collected samples to ensure a final mass fraction of 0.1% formic acid in the solution. The latter groups were centrifuged at 16000rpm for 20 min. Each group was analyzed by Q-Exactive mass spectrometry and 5. mu.L each was loaded. The number of the identified proteins is 2100, 2008 and 2156 respectively.
Example 2
The pump driving power supply (04) was turned on in the connected system, and all the high temperature denaturing and reducing device modules (06) were filled with a mixed solution of 6M guanidine hydrochloride and 50mM DTT, all the solvent displacer modules (07) were filled with a 50mM ammonium bicarbonate solution, and a 4cm long enzyme column was connected as the enzyme reactor module (08). Meanwhile, a heater power supply (05) is turned on, and the temperature of the heating controller (03) is set to be 95 ℃.
6 portions of human normal plasma samples diluted to a concentration of 0.1mg/mL with 1 XPBS were taken at 100. mu.L. After the temperature of the component (06) reaches 95 ℃, the plasma samples are simultaneously introduced into different pipelines of the whole system at the speed of 8 mu L/min by means of syringes respectively. After the end of the introduction, 100. mu.L of a 50mM ammonium hydrogencarbonate solution was introduced into the system. The solvent displacer component (07) is ensured to be operated all the time in the system operation. Meanwhile, each part was collected to obtain an enzymatic substance, which was 70. mu.L. After freeze-drying the samples, each group was reconstituted with 8 μ L of 0.1% FA. The latter groups were centrifuged at 16000g for 20 min. Each group was subjected to Q-active mass spectrometry, and 3. mu.L each was loaded. The number of the identified proteins is 220, 218, 225, 206, 236 and 210 respectively.
Claims (7)
1. An array high-throughput protein sample pretreatment device, comprising: more than 2 high-temperature denaturation and reduction devices (06), more than 2 solvent replacers (07), more than 2 enzyme reactors (08), a heater power supply (05), a pump driving power supply (04), a heating controller (03), a pump flow controller (02) and an infusion pump (01);
the method is characterized in that:
the high-temperature denaturation and reduction device (06) is a hollow closed chamber, an exchange solution inlet and an exchange solution outlet are arranged on the chamber, a hollow fiber membrane is arranged in the chamber, and two ends of the hollow fiber membrane penetrate through the side wall of the chamber and are respectively connected with the sample inlet and the sample outlet; the solvent displacer (07) is a hollow closed cavity, an exchange fluid inlet and an exchange fluid outlet are arranged on the cavity, a hollow fiber membrane is arranged in the cavity, and two ends of the hollow fiber membrane penetrate through the side wall of the cavity and are respectively connected with the sample inlet and the sample outlet;
a sample outlet of the high-temperature denaturation and reduction device is connected with a sample inlet of the solvent displacer, the sample outlet of the solvent displacer is connected with a sample inlet of the enzyme reactor (08), and reaction liquid in the enzyme reactor (08) is discharged from the sample outlet to form a continuous protein sample pretreatment structure;
among more than 2 protein sample pretreatment structures: the exchange liquid in more than 2 high-temperature denaturation and reduction devices is connected with a denaturation and reduction exchange liquid storage tank by an infusion pump in a mode of sequential serial delivery; the flowing mode can ensure that the flow of each component of the high-temperature denaturation reducer is the same; the exchange liquid in more than 2 solvent displacers is connected with an exchange liquid storage tank for displacing the solvent by an infusion pump in a mode of sequential serial delivery;
an electric heating element and a temperature sensor are respectively arranged on the high-temperature denaturation and reduction device (06) and the enzyme reactor (08);
the power module includes: a heating power supply (05) and a pump driving power supply (04) which are electric heating elements of a high-temperature denaturation and reduction device (06) and an enzyme reactor (08), wherein the electric heating elements of the high-temperature denaturation and reduction device assembly (06) and the enzyme reactor assembly (08) respectively adopt a parallel heating mode; ensuring that the temperature of each heating element is the same;
the heating controller (03) is used for respectively providing temperature control for the high-temperature denaturation and reduction device assembly (06) and the enzyme reactor assembly (08);
the infusion pump (01) is divided into more than two groups, namely a high-temperature denaturation and reduction device assembly (06) and a solvent displacer assembly (07) which are used for conveying exchange liquid;
and the pump flow controllers (02) respectively provide flow control for the infusion pumps (01).
2. The arrayed high throughput protein pretreatment apparatus according to claim 1, wherein:
the transfer pump (01) of the exchange liquid is divided into 2 groups for 4 pumps, 2 groups are respectively a high-temperature denaturation and reduction device assembly (06), the solvent displacer assembly (07) transfers the exchange liquid, the stability of the fluid pressure and the flow rate can be ensured, and the transfer mode of connecting flow paths in series is adopted.
3. An arrayed high throughput protein pretreatment apparatus according to claim 1, wherein: the high-temperature denaturation and reduction device assembly (06) and the solvent displacer assembly (07) are heated by adopting parallel circuits, so that the heating parts are stable and uniform in heat, and the safety of the electricity environment of the instrument is ensured by respectively using 36V safety voltage.
4. An arrayed high throughput protein pretreatment apparatus according to claim 1, wherein:
the heating controller (03) adopts the parallel connection of the sensing devices, can switch and observe the temperature of each heating device, avoids the problems of instrument cost increase, volume increase and the like caused by the fact that each detected part is respectively detected and controlled, and can ensure the temperature of each heating part to be consistent.
5. The arrayed high-throughput protein pretreatment apparatus according to claim 1 or 4, wherein: the heating controller (03) is a temperature controller and respectively provides temperature control for the high-temperature denaturation and reduction device component (06) and the enzyme reactor component (08); the temperature sensor is in signal connection with the temperature controller, and the electric heating element is connected with the heating power supply (05) through the temperature controller.
6. An arrayed high throughput protein pretreatment apparatus according to claim 1, wherein: the pump flow controller (02) is a pump flow control board.
7. The array high-throughput protein pretreatment apparatus of claim 1, being applicable to proteome sample processing in clinical, food safety or environmental exposure populations.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1263893A (en) * | 1999-02-05 | 2000-08-23 | 上海复旦张江生物医药有限公司 | Renaturation equipment of modified protein |
| CN1331345A (en) * | 2000-07-04 | 2002-01-16 | 清华大学 | Integrated microarray device |
| CN1995319A (en) * | 2007-01-12 | 2007-07-11 | 北京工业大学 | Multiple passage intelligent temperature-control device facing PCR microfluidic chip |
| CN101838609A (en) * | 2010-03-15 | 2010-09-22 | 上海浩源生物科技有限公司 | Sample adding device and application thereof |
| CN102206573A (en) * | 2011-03-22 | 2011-10-05 | 博奥生物有限公司 | Automatic sample introduction device for microarray chip and automatic sample introduction hybridization microarray chip |
| CN103852527A (en) * | 2012-12-05 | 2014-06-11 | 中国科学院大连化学物理研究所 | High-flux protein sample pre-treatment device |
| CN106255542A (en) * | 2014-02-17 | 2016-12-21 | 拜耳公司 | For from buffering or the ultra filtration unit of Medium Exchange continuously of protein solution |
| CN107588997A (en) * | 2016-07-08 | 2018-01-16 | 中国科学院大连化学物理研究所 | A kind of high throughput protein sample pretreatment equipment |
-
2018
- 2018-11-23 CN CN201811406709.6A patent/CN111220429A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1263893A (en) * | 1999-02-05 | 2000-08-23 | 上海复旦张江生物医药有限公司 | Renaturation equipment of modified protein |
| CN1331345A (en) * | 2000-07-04 | 2002-01-16 | 清华大学 | Integrated microarray device |
| CN1995319A (en) * | 2007-01-12 | 2007-07-11 | 北京工业大学 | Multiple passage intelligent temperature-control device facing PCR microfluidic chip |
| CN101838609A (en) * | 2010-03-15 | 2010-09-22 | 上海浩源生物科技有限公司 | Sample adding device and application thereof |
| CN102206573A (en) * | 2011-03-22 | 2011-10-05 | 博奥生物有限公司 | Automatic sample introduction device for microarray chip and automatic sample introduction hybridization microarray chip |
| CN103852527A (en) * | 2012-12-05 | 2014-06-11 | 中国科学院大连化学物理研究所 | High-flux protein sample pre-treatment device |
| CN106255542A (en) * | 2014-02-17 | 2016-12-21 | 拜耳公司 | For from buffering or the ultra filtration unit of Medium Exchange continuously of protein solution |
| CN107588997A (en) * | 2016-07-08 | 2018-01-16 | 中国科学院大连化学物理研究所 | A kind of high throughput protein sample pretreatment equipment |
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