CN214310345U - Separation device and analysis system for separating high-abundance and low-abundance proteins of tobacco through liquid chromatography - Google Patents
Separation device and analysis system for separating high-abundance and low-abundance proteins of tobacco through liquid chromatography Download PDFInfo
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- CN214310345U CN214310345U CN202022113321.6U CN202022113321U CN214310345U CN 214310345 U CN214310345 U CN 214310345U CN 202022113321 U CN202022113321 U CN 202022113321U CN 214310345 U CN214310345 U CN 214310345U
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Abstract
The utility model provides a separation device and an analysis system for separating high-low abundance proteins of tobacco by liquid chromatography. The apparatus comprises a first dimension chromatography unit, a second dimension chromatography unit and a fraction collector; the first dimension chromatography unit comprises a first pump, a first multi-way valve with at least 6 interfaces, a first chromatographic column, a first detector and a second multi-way valve with at least 4 interfaces; the second dimension chromatographic unit comprises a second pump, a third multi-way valve with at least three interfaces, a flow divider with at least 2 channels, a trapping column array with at least 2 trapping columns, a flow dividing valve array with at least 2 flow dividing valves and a second chromatographic column array with at least 2 second chromatographic columns. The analysis system comprises the separation device, a protein content determination unit, a high-abundance protein MS analysis unit and a low-abundance protein LC-MS analysis unit. The separation device and the analysis system can rapidly and efficiently separate, analyze and identify the high-abundance and low-abundance proteins of the tobacco.
Description
Technical Field
The utility model relates to a tobacco protein analysis technical field, concretely relates to separator and analytic system of liquid chromatography separation tobacco height abundance albumen.
Background
In the field of plant biochemical research, tobacco is widely studied as an important model plant. In 2011, the first whole genome of tobacco is successfully drawn, the tobacco enters proteomics research, and plant genes are often tetraploid or even octaloid, so that the transcription and translation of plant proteins are more complicated. The tobacco proteins are various in types and large in content difference, and when low-abundance proteins are analyzed by the traditional protein analysis and identification method, the traditional protein analysis and identification method is greatly influenced by the high-abundance proteins, so that the detection result is adversely affected. At present, two-dimensional electrophoresis (2-DE), two-dimensional fluorescence differential gel electrophoresis (DIGE), isotope affinity tag (ICAT), and Isobaric Tags for Relative and Absolute Quantification (iTRAQ) are the main techniques for detecting plant proteome. The 2-DE process is gradually replaced by the DIGE process due to poor reproducibility. The DIGE method still has the problem of contaminating protein spots. The ICAT technology introduces active reaction groups, improves the sensitivity of protein identification, but has low marking efficiency, and restricts the accuracy and the repeatability of protein identification. iTRAQ can only analyze the polypeptide on the label and the corresponding protein, limiting the range of detection of the target protein.
SUMMERY OF THE UTILITY MODEL
In view of the above-mentioned shortcomings of the prior art, the present invention provides a separation device and an analysis system for separating high and low abundance proteins of tobacco by liquid chromatography. The separation device is used for separating through the array type two-dimensional chromatographic device, and the multiple chromatographic columns are high in separation and detection efficiency at the same time, and have the characteristics of high flux and good separation effect. The analysis system adopts array type multidimensional liquid chromatography tandem mass spectrometry to analyze and identify the high-abundance protein and the low-abundance protein respectively, and combines detection results to obtain complete biological information of the tobacco protein. The separation device and the analysis system can rapidly and efficiently separate, analyze and identify the high-abundance and low-abundance proteins of the tobacco, the identified low-abundance proteins are rich in information, and the high-abundance proteins can realize position positioning in the array two-dimensional chromatogram.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a separation device for separating high and low abundance proteins from tobacco by liquid chromatography, comprising a first dimension chromatography unit, a second dimension chromatography unit and a fraction collector;
the first dimension chromatography unit comprises a first pump, a first multi-way valve with at least 6 interfaces, a first chromatographic column, a first detector and a second multi-way valve with at least 4 interfaces;
the second dimension chromatographic unit comprises a second pump, a third multi-way valve with at least three interfaces, a flow divider with at least 2 channels, a trapping column array with at least 2 trapping columns, a flow dividing valve array with at least 2 flow dividing valves and a second chromatographic column array with at least 2 second chromatographic columns;
the first multi-way valve is respectively communicated with the first pump and the sample inlet end of the first chromatographic column, and the sample outlet end of the first chromatographic column, the first detector and the second multi-way valve are sequentially communicated along the sample flowing direction;
the flow dividing valve array is respectively communicated with the sample injection ends of the second multi-way valve, the trapping column array and the second chromatographic column array, the third multi-way valve is respectively communicated with the second pump and the flow divider, and the flow divider is communicated with the trapping column array;
and the sample outlet end of the second chromatographic column array is communicated with the fraction collector.
The utility model discloses the second aspect provides an array multidimensional liquid chromatography tandem mass spectrometry tobacco high-low abundance protein's analytic system, include:
the separation device is used for separating the high-abundance protein and the low-abundance protein of the tobacco from the tobacco sample through the array type multidimensional liquid chromatography and collecting fractions;
the protein content measuring unit is used for measuring the protein content of the collected fractions obtained by the separation device and determining fractions containing high-abundance proteins and fractions containing low-abundance proteins;
the high-abundance protein MS or LC-MS analysis unit is used for detecting the proteomics information of each high-abundance protein fraction or the proteomics information of the full high-abundance protein fraction;
and the low-abundance protein LC-MS analysis unit is used for detecting the proteomics information of the all-low-abundance protein fraction.
The utility model discloses at least one in following beneficial effect has:
1) the utility model has the advantages that the multiple chromatographic columns are separated and analyzed simultaneously, and the detection efficiency is high;
2) the utility model effectively removes the influence of the abundant protein through the array type fraction transfer mode, and the identified low abundant protein has rich information;
3) the utility model realizes the position positioning of the abundant protein in the array two-dimensional chromatogram;
4) the utility model discloses the system founds simply, degree of automation is high, and adjustable parameter is many.
Drawings
FIG. 1 is a schematic diagram of the separation device for separating high-low abundance proteins from tobacco by liquid chromatography.
FIG. 2 is a diagram of an analysis system for analyzing high-low abundance proteins of tobacco by liquid chromatography-tandem mass spectrometry of the present invention.
FIG. 3 shows the result of the separation and identification of part of the high-abundance protein in the separation device and analysis system for separating the high-abundance and low-abundance proteins of tobacco by liquid chromatography.
Reference numerals
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The present invention can also be implemented or applied through other different specific embodiments, and various details in the present specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.
As shown in figure 1, a separation device for separating high-abundance and low-abundance proteins of tobacco by liquid chromatography comprises a first dimension chromatographic unit 1, a second dimension chromatographic unit 2 and a fraction collector 3;
the first dimension chromatographic unit 1 comprises a first pump 11, a first multi-way valve 12 with at least 6 interfaces, a first chromatographic column 13, a first detector 14 and a second multi-way valve 15 with at least 4 interfaces;
the second dimension chromatographic unit 2 comprises a second pump 21, at least three interface third multi-way valves 22, at least 2 channels of flow dividers 23, at least 2 trapping column arrays 24, at least 2 flow divider valve arrays 25 and at least 2 second chromatographic column arrays 26;
the first multi-way valve 12 is respectively communicated with the first pump 11 and the sample injection end of the first chromatographic column 13, and the sample outlet end of the first chromatographic column 13, the first detector 14 and the second multi-way valve 15 are sequentially communicated along the sample injection direction;
the flow dividing valve array 25 is respectively communicated with the sample injection ends of the second multi-way valve 15, the trapping column array 24 and the second chromatographic column array 26, the third multi-way valve 22 is respectively communicated with the second pump 21 and the flow divider 23, and the flow divider 23 is communicated with the trapping column array 24;
the sample outlet end of the second chromatographic column array 26 is communicated with the fraction collector 3.
The utility model discloses separator uses two-dimensional chromatogram as the basis, builds ion exchange-reversed phase liquid phase (IEC-RP-RP) device. Separating the tobacco protein extraction product in an array two-dimensional liquid chromatography, firstly, respectively collecting fractions into a trapping column array 24 by adopting a first dimension chromatography unit 1 such as an ion exchange chromatography; second, the fraction is switched by the third multi-way valve 22 to be transferred to a second chromatography column array 26 such as a reverse phase liquid chromatography column array; finally, the fraction separated by the second chromatography column array 26 is collected by the fraction collector 3 to be analyzed. The tobacco protein extract can be extracted by phenol method.
In a preferred embodiment, the first multi-way valve 12 is a two-position multi-way valve, the first multi-way valve 12 is at least provided with a first multi-way valve first interface 121, a first multi-way valve second interface 122, a first multi-way valve third interface 123, a first multi-way valve fourth interface 124, a first multi-way valve fifth interface 125 and a first multi-way valve sixth interface 126, the first multi-way valve first interface 121 is a sample inlet, the first multi-way valve second interface 122 is communicated with the first multi-way valve fifth interface 125, the first multi-way valve third interface 123 is communicated with the first pump 11, the first multi-way valve fourth interface 124 is communicated with the first chromatography column 13, and the first multi-way valve sixth interface 126 is a waste discharge port.
The first multi-way valve 12 has at least 6 ports, such as a six-way valve, an eight-way valve, etc., and if there are more than 6 ports, other ports are closed by plugs.
In a preferred embodiment, the first multi-way valve 12 is a two-position multi-way valve;
the first position relationship is: the first multi-way valve first port 121 is communicated with the first multi-way valve sixth port 126; the first multi-way valve second port 122 is communicated with the first multi-way valve third port 123; the first multi-way valve fourth port 124 is communicated with the first multi-way valve fifth port 125; the position is an injection (Inject) position, and the mobile phase conveyed by the first pump pushes the sample into the first chromatographic column 13 and the first detector 14 for analysis;
the second position relation is as follows: the first multi-way valve first port 121 is communicated with the first multi-way valve second port 122; the third port 123 of the first multi-way valve is communicated with the fourth port 124 of the first multi-way valve; the fifth port 125 of the first multi-way valve is communicated with the sixth port 126 of the first multi-way valve. This position is a sample (Load) position and excess sample is drained from the first multi-way valve sixth port 126.
In a preferred embodiment, the second multi-way valve 15 is provided with 4-18 connectors, and the flow divider 23 is provided with 2-16 channels; the trapping column array 24 is provided with 2-16 trapping columns; the diverter valve array 25 is provided with 2-16 diverter valves; the second chromatographic column array 26 is provided with 2-16 second chromatographic columns. The second multi-way valve 15, the flow divider 23, the trapping column array 24, the flow dividing valve array 25 and the second chromatographic column array 26 are correspondingly arranged to form an array type two-dimensional liquid chromatogram.
In a preferred embodiment, the second multi-way valve 15 is provided with a second multi-way valve first interface 151, a second multi-way valve second interface 152 and two or more second multi-way valve interfaces, the second multi-way valve first interface 151 is communicated with the first chromatographic column 13, and the second multi-way valve second interface 152 is communicated with any one of the two or more second multi-way valve interfaces through switching.
As shown in fig. 1, the second multi-way valve 15 is a ten-way valve, and is provided with a second multi-way valve first interface 151, a second multi-way valve second interface 152, 8 second multi-way valve interfaces, i.e., a second multi-way valve third interface 153, a second multi-way valve fourth interface 154, a second multi-way valve fifth interface 155, a second multi-way valve sixth interface 156, a second multi-way valve seventh interface 157, a second multi-way valve eighth interface 158, a second multi-way valve ninth interface 159, and a second multi-way valve tenth interface 1510, and one interface is sealed by a plug, the second multi-way valve first interface 151 is communicated with the first color spectrum column 13, and the second multi-way valve second interface 152 is switched with the second multi-way valve third interface 153, the second multi-way valve fourth interface 154, the second multi-way valve fifth interface 155, the second multi-way valve sixth interface 156, the second multi-way valve seventh interface 157, and the second multi-way valve seventh interface 157, And any one of the second multi-way valve eighth port 158, the second multi-way valve ninth port 159 and the second multi-way valve tenth port 1510 is selectively communicated.
In a preferred embodiment, the third multi-way valve 22 is at least provided with a third multi-way valve first port 221, a third multi-way valve second port 222 and a third multi-way valve third port 223, the first port 221 is communicated with the second pump 21, the second port 222 is communicated with the flow divider 23, and the third port 223 is a waste discharge port.
In a preferred embodiment, the third multi-way valve 22 is a two-position multi-way valve;
the first position relationship is: the third multi-way valve first port 221 is communicated with the third multi-way valve second port 222;
the second position relation is as follows: the third multi-way valve second port 222 is in communication with the third multi-way valve third port 223.
In a preferred embodiment, the flow divider 23 is provided with a flow divider inlet 231 and 2 or more flow divider channels, the flow divider inlet 231 is communicated with the third multi-way valve 22, and the 2 or more flow divider channels are respectively communicated with the trapping columns of the trapping column array 24.
As shown in fig. 1, the flow divider 23 is provided with a flow divider inlet 231 and 8 flow divider channels, i.e., a flow divider first channel 232, a flow divider second channel 233, a flow divider third channel 234, a flow divider fourth channel 235, a flow divider fifth channel 236, a flow divider sixth channel 237, a flow divider seventh channel 238 and a flow divider eighth channel 239, wherein the flow divider inlet 231 is communicated with the third multi-way valve 22, and the flow divider first channel 232, the flow divider second channel 233, the flow divider third channel 234, the flow divider fourth channel 235, the flow divider fifth channel 236, the flow divider sixth channel 237, the flow divider seventh channel 238 and the flow divider eighth channel 239 are respectively communicated with the trapping columns of the trapping column array 24.
In a preferred embodiment, the first chromatographic column 13 is a WAX ion exchange chromatographic column.
In a preferred embodiment, the first detector 14 is an ultraviolet detector.
In a preferred embodiment, the second dimension chromatography unit 2 is a reverse phase liquid chromatography.
In a preferred embodiment, the fraction collector 3 comprises a collection needle array 31 having at least 2 collection needles, a moving platform 32 and a multi-well collection plate 33 having at least 24 wells, the sample outlet end of the second chromatography column array 26 is connected to the collection needle array 31, the collection needle array 31 is disposed on the moving platform 32, and the collection needle array 31 is disposed above the multi-well collection plate 33.
An analysis system for analyzing high-low abundance proteins of tobacco by array type multidimensional liquid chromatography tandem mass spectrometry, as shown in fig. 2, comprises:
the separation device I-1 is used for separating high-abundance and low-abundance proteins of tobacco from a tobacco sample through array type multidimensional liquid chromatography and collecting fractions;
the protein content measuring unit I-2 is used for measuring the protein content of the collected fraction obtained by the separating device I-1 and determining the fraction containing the high-abundance protein and the fraction containing the low-abundance protein;
the high-abundance protein MS or LC-MS analysis unit I-3 is used for detecting the proteomic information of each high-abundance protein fraction or the proteomic information of the full high-abundance protein fraction;
and the low-abundance protein LC-MS analysis unit I-4 is used for detecting the proteomic information of the all-low-abundance protein fraction.
Fractions separated by the array type multi-dimensional liquid chromatography are collected by a fraction collector 3 in a separation device I-1, the protein content is measured by a protein content measuring unit I-2, and the collected fractions are classified into two types including high-abundance proteins and low-abundance proteins. For fractions containing high-abundance proteins, performing mass spectrum analysis one by a high-abundance protein MS analysis unit to obtain proteomic information in each fraction, and positioning the high-abundance proteins in an array two-dimensional chromatogram, or combining the fractions consisting of the high-abundance proteins, and performing LC-MS analysis by a high-abundance protein LC-MS analysis unit to obtain the proteomic information of the full high-abundance protein fraction; fractions consisting of the low-abundance proteins are combined and analyzed by a low-abundance protein LC-MS analysis unit I-4, so that the overall analysis result of the low-abundance proteins is obtained. And finally, combining the detection results of the two parts to obtain complete biological information of the tobacco extracted protein.
Example 1
1. Materials and reagents
Sucrose, potassium chloride, ethylenediamine tetraacetic acid, Tris, hydrogen chloride, trifluoroacetic acid, saturated phenol in Tris-HCl, acetonitrile (HPLC grade), methanol (HPLC grade), acetone (HPLC grade), ammonium acetate, ammonium bicarbonate, dithiothreitol, iodoacetamide, trypsin were purchased from Sigma-Aldrich.
2. Tobacco pretreatment step
About 5g of tobacco leaves were cut into small pieces, a certain amount of liquid nitrogen was added to the mortar, and the tobacco leaves were ground to powder. Adding sucrose extract (0.7M sucrose; 0.1 MKCl; 50mM EDTA; 0.5M Tris-HCl, pH 7.5) to a total volume of about 25mL, shaking for 10min, adding 25mL of saturated phenol Tris-HCl solution, shaking for 10min, centrifuging at 5000g for 30min, collecting supernatant, adding sucrose extract of equal volume, shaking for 5min, and centrifuging at 5000g for 25 min. The above extraction was repeated once, and 40mL of a methanol solution containing 0.1M ammonium acetate was added to the extracted supernatant, and the solution was cooled to-20 ℃ and stored overnight. The sample was centrifuged at 5000g for 30min, the precipitate was washed twice with 15mL of ice methanol and twice with 15mL of ice acetone and evaporated to dryness at room temperature. The resulting product powder was redissolved in 10mL of 7M urea and the solution was filtered using a 0.22 μ M needle filter. And (3) carrying out ultrafiltration concentration on the solution by 10 times by using a 3kDa ultrafiltration centrifugal tube, diluting the solution to the original volume, repeating the concentration for five times, and finally diluting the solution to 2mL, wherein the protein content of the obtained solution is determined by using a BCA method.
3. Construction of separation device for separating high-abundance and low-abundance proteins of tobacco by liquid chromatography
A separation device for separating high-low abundance proteins of tobacco by liquid chromatography is shown in FIG. 2. The first column 13 was a WAX ion exchange column (TSKgel DEAE-3SW, 7.5 × 75mM, 10 μ M), mobile phase a was a Tris-HCl solution (20mM, pH 6.8), mobile phase B was a Tris-HCl solution (20mM, pH 6.8) to which 1.5M NaCl was added; the sample injection amount is 1.5 mL; the flow rate is 0.2 muL/min; the column temperature is 35 ℃; the first detector 14 is an ultraviolet detector with the detection wavelength of 215 nm; the mobile phase gradient was: 0min, 100% A; 15min, 100% A; 20min, 96% A; 74min, 78% A; 116min, 45% A; 120min, 100% A; 130min, 100% A;
during the separation of the first chromatographic column 13, one fraction is collected every 10 minutes from 30 to 110 minutes for a total of 8 fractions, and the separation is performed while the separation result of the sample is detected on line by the first detector 14. In this stage, the portion (between 25 and 26) of the splitter valve array 25 connected to the second chromatography column array 26 was in a closed state, and 8 fractions were transferred to the trap column array 24 through the second multi-way valve 15 for trapping, respectively, and then the trap column was further washed with the first-dimensional mobile phase for 3 minutes.
The second column array 26 consisted of 8 reverse phase columns (WelchXtimate C8, 2.1 x 250mm, 5 μm) and the mobile phase delivered by the second pump 21 was divided equally into eight parts by the splitter 23. At this time, the portion of the diverter valve array 25 connected to the second column array 26 is opened, and the mobile phase elutes the components from the trap column and transfers the components to the second column array 26 for subsequent separation. In the second dimension chromatography unit 2, mobile phase a was 95% water + 5% acetonitrile + 0.1% TFA, and mobile phase B was 95% acetonitrile + 5% water + 0.1% TFA; the sample introduction amount is 2 mL; the flow rate is 0.2 mL/min; the column temperature is 35 ℃; the detector is an ultraviolet detector, and the detection wavelength is 215 nm; the mobile phase gradient was: 0min, 100% A; 5min, 100% A; 15min, 75% A; 25min, 67% A; 60min, 60% A; 80min, 46% A; 85min, 33% A; 85.1min, 0% A; 95min, 0% A; 95.1min, 100% A; 110min, 100% A;
the fractions separated by reverse phase chromatography are collected by a fraction collector 3, one fraction is collected from 0 to 96 minutes per minute, 96 fractions are collected in total, the protein content in each fraction is determined by a protein content determination unit I-2, and a method for determining the protein content by using BCA can be used.
LC-MS/MS analysis and data analysis
Separating tobacco sample by a separation device for separating high-abundance and low-abundance proteins of tobacco through liquid chromatography, collecting fractions, respectively freeze-drying the fractions, fully dissolving the fractions by 20 mu L of 50mM ammonium bicarbonate, heating the fractions at 95 ℃ for 10 minutes to denature the proteins, adding 10 mu L of 30Mm DTT, and carrying out oscillation reaction at 50 ℃ for half an hour; adding 10 mu L of 55mM IAA, and reacting for half an hour at room temperature in the dark; finally, 10. mu.L of 25mM DTT was added and the reaction was allowed to proceed for 5 minutes at 37 ℃ to quench excess IAA. 50uL of 20 ng/. mu.L trypsin was added, the reaction was carried out at 37 ℃ for 15 hours, and after completion of the enzymatic hydrolysis, 1% TFA was added to terminate the enzymatic hydrolysis.
After combining fractions of low abundance proteins, subsequent LC-MS/MS analysis was performed by Q-ExactiveOrbitrap Mass Spectrometry (Thermo Fisher Scientific, San Jose, Calif., USA). The separation column was Acclaim Pep Map C18 (Thermo Fisher Scientific, San Jose, Calif., USA). Mobile phase a3 was water (plus 0.1% formic acid) and B3 was acetonitrile (plus 0.1% formic acid). The flow rate of the separation column is 300nL/min, and the mobile phase gradient for peptide fragment separation is as follows: 0-90 minutes: 5-30% (B3), 90-100 min: 30-80%, 100-: 80-5%, 101-: 5 percent. The mass spectrum parameters were as follows: full scan, mass to charge ratio scan range: 350-; resolution ratio: 70000; MS/MS scanning: resolution 17500.
Mass spectrometry results were analyzed by Proteome Discover (PD version 1.4.0.288, thermo Fisher Scientific) using a MASCOT engine (Matrix Science, London, UK, version 2.6.0) matched to the tobacco database _ UP 000084051. The parameters of the mass spectrum were set as follows: scanning range of parent ion mass: 350-; the minimum peak number of an MS/MS spectrogram is 10; the minimum signal-to-noise ratio is 1.5. The database matching parameters are set as follows: maximum number of missed cut sites allowed: 2, the number of the cells is 2; FDR: 0.05; urea methylation is set as a fixed modification, and methionine is oxidized into a variable modification; the enzyme digestion type is as follows: (ii) trypsin.
5. Results and discussion
In top-down analysis of a protein layer, components are complex and difficult to separate, single sample analysis time is long, fraction collection is complicated, and the defects are effectively overcome by an array two-dimensional chromatographic system. The first chromatographic column 13 adopts a WAX ion exchange chromatographic column to separate a sample, a fraction is collected every 10 minutes within 30-110 minutes, 8 collected fractions are conveyed to the trapping column array 24 for temporary storage, then the fractions are eluted from the trapping column in the trapping column array 24, and second-dimensional separation is carried out in 8 second chromatographic columns (reversed-phase chromatographic columns), the separation result is shown in FIG. 3, 8 second-dimensional chromatograms have better discrimination with each other, and the separation effect of the first-dimensional chromatogram is better; meanwhile, the components are further separated through a second-dimensional reversed-phase chromatography, so that the subsequent mass spectrometry is facilitated. The separation principles of two-dimensional chromatography are different, the two-dimensional chromatography is separated from two different properties of the charge state and the hydrophilicity/hydrophobicity of the protein respectively, and the orthogonality is good, so that the peak capacity of the constructed two-dimensional chromatography system is high, and the separation effect is better compared with that of the traditional one-dimensional chromatography. Furthermore, since the single sample analysis time of the two-dimensional chromatography is long (1D: 130 min; 2D: 110 min), if the fractions of the first dimension are subjected to subsequent reversed-phase chromatographic separation one by one, the time is too long and the fraction collection process is cumbersome. The two-dimensional chromatographic array system is used for simultaneously carrying out second-dimensional chromatographic separation on 8 fractions, so that the flux is greatly improved, and the analysis time is saved; fractions separated by the reverse phase chromatography are directly stored in a 96-well plate through a fraction collector, so that a large amount of labor cost is saved.
The above embodiments are merely illustrative of the principles and effects of the present invention, and are not to be construed as limiting the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A separation device for separating high and low abundance proteins of tobacco by liquid chromatography is characterized by comprising a first dimension chromatography unit (1), a second dimension chromatography unit (2) and a fraction collector (3);
the first dimension chromatographic unit (1) comprises a first pump (11), a first multi-way valve (12) with at least 6 interfaces, a first chromatographic column (13), a first detector (14) and a second multi-way valve (15) with at least 4 interfaces;
the second dimension chromatographic unit (2) comprises a second pump (21), a third multi-way valve (22) with at least three interfaces, a flow divider (23) with at least 2 channels, a trapping column array (24) with at least 2 trapping columns, a flow divider valve array (25) with at least 2 flow divider valves and a second chromatographic column array (26) with at least 2 second chromatographic columns;
the first multi-way valve (12) is respectively communicated with the first pump (11) and the sample inlet end of the first chromatographic column (13), and the sample outlet end of the first chromatographic column (13), the first detector (14) and the second multi-way valve (15) are sequentially communicated along the sample flowing direction;
the flow dividing valve array (25) is respectively communicated with the sample injection ends of the second multi-way valve (15), the trapping column array (24) and the second chromatographic column array (26), the third multi-way valve (22) is respectively communicated with the second pump (21) and the flow divider (23), and the flow divider (23) is communicated with the trapping column array (24);
the sample outlet end of the second chromatographic column array (26) is communicated with the fraction collector (3).
2. The separation device for separating high and low abundance proteins of tobacco by liquid chromatography according to claim 1, it is characterized in that the first multi-way valve (12) is at least provided with a first multi-way valve first interface (121), a first multi-way valve second interface (122), a first multi-way valve third interface (123), a first multi-way valve fourth interface (124), a first multi-way valve fifth interface (125) and a first multi-way valve sixth interface (126), the first multi-way valve first interface (121) is a sample inlet, the first multi-way valve second interface (122) is communicated with the first multi-way valve fifth interface (125), the third interface (123) of the first multi-way valve is communicated with the first pump (11), the fourth port (124) of the first multi-way valve is communicated with the first chromatographic column (13), and the sixth port (126) of the first multi-way valve is a waste discharge port.
3. The separation device for separating high and low abundance proteins of tobacco by liquid chromatography as claimed in claim 2, wherein the first multi-way valve (12) is a two-position multi-way valve;
the first position relationship is: the first multi-way valve first interface (121) is communicated with the first multi-way valve sixth interface (126); the first multi-way valve second connector (122) is communicated with the first multi-way valve third connector (123); the fourth port (124) of the first multi-way valve is communicated with the fifth port (125) of the first multi-way valve;
the second position relation is as follows: the first multi-way valve first interface (121) is communicated with the first multi-way valve second interface (122); the third interface (123) of the first multi-way valve is communicated with the fourth interface (124) of the first multi-way valve; the fifth interface (125) of the first multi-way valve is communicated with the sixth interface (126) of the first multi-way valve.
4. The separation device for separating the high and low abundance proteins in the tobacco by liquid chromatography as claimed in claim 1, wherein the second multi-way valve (15) is provided with 4-18 interfaces, and the flow divider (23) is provided with 2-16 channels; the trapping column array (24) is provided with 2-16 trapping columns; the shunt valve array (25) is provided with 2-16 shunt valves; the second chromatographic column array (26) is provided with 2-16 second chromatographic columns.
5. The separation device for separating the high and low abundance proteins in tobacco by liquid chromatography as claimed in claim 1, wherein the second multi-way valve (15) is provided with a first multi-way valve interface (151), a second multi-way valve second interface (152) and more than two second multi-way valve interfaces, the first multi-way valve interface (151) is communicated with the first chromatographic column (13), and the second multi-way valve second interface (152) is selectively communicated with any one of the more than two second multi-way valve interfaces by switching.
6. The separation device for separating the high and low abundance proteins in the tobacco by liquid chromatography as claimed in claim 1, wherein the third multi-way valve (22) is at least provided with a first interface (221), a second interface (222) and a third interface (223) of the third multi-way valve, the first interface (221) is communicated with the second pump (21), the second interface (222) is communicated with the flow divider (23), and the third interface (223) is a waste discharge port.
7. The separation device for separating high and low abundance proteins of tobacco by liquid chromatography as claimed in claim 6, wherein the third multi-way valve (22) is a two-position multi-way valve;
the first position relationship is: the third multi-way valve first interface (221) is communicated with the third multi-way valve second interface (222);
the second position relation is as follows: the third multi-way valve second port (222) is communicated with the third multi-way valve third port (223).
8. The separation device for separating high and low abundance proteins of tobacco by liquid chromatography according to claim 1, wherein the splitter (23) is provided with a splitter inlet (231) and more than 2 splitter channels, the splitter inlet (231) is communicated with the third multi-way valve (22), and the more than 2 splitter channels are respectively communicated with the trapping columns of the trapping column array (24).
9. The separation device for separating the high and low abundance proteins of tobacco by liquid chromatography according to claim 1, further comprising at least one of the following technical features:
1) the first chromatographic column (13) is a WAX ion exchange chromatographic column;
2) the first detector (14) is an ultraviolet detector;
3) the second dimension chromatography unit (2) is a reverse phase liquid chromatography;
4) fraction collector (3) include collection needle array (31) of 2 at least collection needles, moving platform (32) and porous collection board (33) of 24 at least holes, the play appearance end of second chromatographic column array (26) with collect needle array (31) intercommunication, collection needle array (31) are located on moving platform (32), collection needle array (31) are located the top of porous collection board (33).
10. An analysis system for analyzing high-low abundance proteins of tobacco by liquid chromatography-tandem mass spectrometry is characterized by comprising:
the separation device (I-1) according to any one of claims 1 to 9, for separating tobacco high and low abundance proteins from a tobacco sample by liquid chromatography and collecting fractions;
a protein content measuring unit (I-2) for measuring the protein content of the collected fraction obtained by the separation device (I-1) and determining a fraction in which the high-abundance protein is present and a fraction in which the low-abundance protein is present;
a high-abundance protein MS or LC-MS analysis unit (I-3) for detecting proteomics information of each high-abundance protein fraction or proteomics information of all high-abundance protein fractions;
and the low-abundance protein LC-MS analysis unit (I-4) is used for detecting the proteomic information of the full low-abundance protein fraction.
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