CN105116078A - Method for treating gram bacterium protein for mass spectrum identification and buffer solution of method - Google Patents
Method for treating gram bacterium protein for mass spectrum identification and buffer solution of method Download PDFInfo
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Abstract
The invention discloses a method for treating gram bacterium protein for mass spectrum identification and a buffer solution of the method. The buffer solution comprises 0.01 mol/L-0.1 mol/L of NaCl, 1 mmol/L-10 mmol/L of Tris-HCl, 0.1 mmol/L-2 mmol/L of EDTA and 0.01 mmol/L-0.2 mmol/L of Triton X-100. The buffer solution is used for washing gram bacteria, then the bacterium protein is extracted to serve as a bacterium identification sample, and the distinguishability and accuracy of bacterium mass spectrum identification are improved; furthermore, the buffer solution is suitable for gram negative bacteria and gram positive bacteria.
Description
Technical field
The application relates to microorganism Mass Spectrometric Identification field, particularly relates to a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification and buffer solution thereof.
Background technology
The authenticate technology development of unknown microorganism, by since for a long time, has described unit the most basic in microbial identification conclusion for belonging to and planting usually.Unit bacterial strain minimum in taxonomical unit is the offspring's pure culture from same individual cells.Traditional way is the qualification utilizing chemical method to carry out bacterium, is sometimes also referred to as bacterial chemistry classification.The standard of classification can according to some multiple chemical classification indexs, can according to the presence or absence of some component, or the ratio of some Compound abundance, obtain a finger-print of classifying significantly.
Along with the development of chemical apparatuses, the pioneer CatherineFenselau in this field show ACSSymposiumSeries and the mass-spectrometric technique of all development be used for bacterial chemistry classification.As Matrix assisted laser desorption ionization (Matrix-assistedlaserdesorptionionization, MALDI) Leaf proteins class hour is successfully applied to, start someone and utilize this technical Analysis bacterioprotein, comprising the protein that may be used for division bacteria.
Matrix assisted laser desorption ionization (MALDI) is one of numerous gasification and ioning method, time of flight mass analyzer (TOF) is that ion accelerates to fly over dirft tube under electric field action, according to the flight time arriving detecting device is different, the detected mass-to-charge ratio (M/Z) namely measuring ion was directly proportional to the flight time of ion, thus detected ion.MALDI-TOF indeed achieves a step and completes gasification and ionization.Analyzing thing and first become gaseous ion by solid phase or liquid phase, is gasify under normal circumstances, and some particle is easy to gasification under the environment of high vacuum, but some molecule will decompose when heating, as biomacromolecule albumen etc.MALDI-TOF can realize realizing by the transformation of solid phase to gas phase under the prerequisite of not saboteur's structure.In MALDI-TOF process, analyze thing and first wrapped up by a large amount of matrix, and be coated in sample disc surface.Sample disc is normally made up of stainless steel, and the solution of sample can volatilize very soon in sample disc, normally water, acetonitrile/water or acetone/water.Normally a kind of weak acid of matrix, and can absorbing laser, whether because the laser of matrix to certain wavelength has strong absorption, therefore, analyzing thing has light absorptive matrix or analyzes thing and all very strong reaction can occur in solid-phase layer.After laser bombardment, heated very soon by the sample spot hit and start to separate from sample panel after obtaining kinetic energy, such matrix gasifies together with analysis thing, due to matrix normally weak acid, the analysis thing of some polarity is as protein, proton can be obtained from the hydroxy-acid group of matrix, carry out separation by the difference of the mass-to-charge ratio analyzing thing in the mass analyser and detect.MALDI-TOF is resolution as the method key point of microbial identification.Because bacterial whole cell is a very complicated mixture, and whole process does not have the separation and purification in early stage, so MALDI-TOF acquired results is the mean value for Bacteria Identification, and analyzes relatively limited for specific protein.
Traditional microorganism classification comprises the mode such as phenotypic classification, Physiology and biochemistry classification, and it is all the different expression form of microprotein difference after all.And directly utilize protein to carry out microorganism classification more to reflect the essence distinguished between different microorganisms, be the most effectively, the most essential microorganism classification and authentication method.The microbial identification system developed based on high-throughout MALDI-TOF, based on the application of mass spectrographic proteomic techniques MALDIBioTyper system in microbial identification and classification, the work of three aspects can be realized: 1. for a series of known microorganisms, MALDI-TOFMS database can be obtained, namely set up the standard protein group fingerprint mass spectrometric data storehouse of known microorganisms; 2. for unknown microorganism, then prepare non-Identifying micro-organisms sample, MALDI-TOFMS is utilized to obtain mass spectrometric data, the software package provided is provided again, the standard protein group fingerprint mass spectrometric data storehouse of the mass spectrometric data of acquisition and known microorganisms is compared, there is with qualification the known microorganisms of same or similar mass spectrometric data, then set up the standard protein group fingerprint mass spectrometric data storehouse of unknown microorganism; 3. the software package instrument provided is provided, the known and unknown microbial standard Leaf proteins fingerprint mass spectrometric data storehouse set up can be utilized for the qualification of clinical, environment, industrial unknown sample.The protein fingerprint obtained by Mass Spectrometer Method is applied to microbial identification.This technology only needs sample and the disposal cost of minute quantity, just Rapid identification can go out not clear bacterium, saccharomycete and mould, can substitute classical microbial identification and sorting technique admirably.
But traditional Gram-negative bacteria processing method of protein being applied to Mass Spectrometric Identification, as being applied to the ethanol/formic acid method of Gram-negative bacteria, a certain amount of bacterium colony pure culture (5-10mg) is put into centrifuge tube, add 300 μ l water mixings, add 900 μ l ethanol mixings, with maximal rate (15000rpm) centrifugal 2min, removing supernatant, repeated centrifugation, remove ethanol completely, add the formic acid of 50 μ l70%, the all bacterium colonies of Eddy diffusion, and concuss 1min, add the pure acetonitrile mixing of 50 μ l, maximal rate (15000rpm) centrifugal 2min, transfer supernatant is in a new centrifuge tube, sample preparation is complete can directly be identified.
Traditional gram-positive bacteria method for extracting protein that is applied to is 80%TFA extraction method.Its main process adds 50ul80%TFA (trifluoroacetic acid) for a certain amount of bacterium colony pure culture (5-10mg) is put into centrifuge tube, and suspension concussion makes thalline mix, and leaves standstill several minutes.Add the deionized water of triploid long-pending (150ul), add 100% acetonitrile of equal-volume (200ul), maximal rate (15000rpm) centrifugal 2min, transfer supernatant is in a new centrifuge tube, and sample preparation is complete can directly be identified.
But traditional sample treatment effectively can not get rid of the chitin material produced in bacteria growth process, exocellular polysaccharide and lipopolysaccharides, the nutrient culture media etc. of microorganism, thus affect the separation and purification of protein, thus had influence on qualification accuracy and sensitivity, and when sample uncertain be gram-positive bacteria and Gram-negative bacteria when, can not determine fast to select disposal route, therefore, the existing Gram-negative bacteria for Mass Spectrometric Identification and positive bacteria processing method of protein can not be general.
Summary of the invention
The application provides a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification, solves the accuracy of traditional gram-bacteria proteomic image qualification and the not high problem of sensitivity.
According to the first aspect of the application, the buffer solution that the application provides a kind of gram-bacteria for Mass Spectrometric Identification to wash, its buffer solution formula comprises 0.01mol/L ~ 0.1mol/LNaCl, 1mmol/L ~ 10mmol/LTris-HCl, 0.1mmol/L ~ 2mmol/LEDTA and 0.01mmol/L ~ 0.2mmol/LTritonX-100.
According to the second aspect of the application, the application provides a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification, comprises the following steps:
Buffer solution is utilized to wash thalline: choose thalline and be placed in container, add buffer solution suspension thalline, supernatant is removed after centrifugal, this buffer solution formula comprises 0.01mol/L ~ 0.1mol/LNaCl, 1mmol/L ~ 10mmol/LTris-HCl, 0.1mmol/L ~ 2mmol/LEDTA and 0.01mmol/L ~ 0.2mmol/LTritonX-100;
Bacterioprotein extracts: add cell pyrolysis liquid, Eddy diffusion thalline toward the thalline after buffer solution washing, first time ultrasonic process, removes supernatant after centrifugal; After add protein lysates, second time ultrasonic process, collected after centrifugation upper strata protein solution.
The beneficial effect of the application is: after being used for the gram-bacteria of Mass Spectrometric Identification by buffer solution washing of the present invention, then extract bacterioprotein as qualification bacteria samples, improve resolution and the accuracy of Mass Spectrometric Identification bacterium.
Further, a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification that the application provides, not only increases resolution and the accuracy of Mass Spectrometric Identification bacterium, is also applicable to the sample treatment of Gram-negative bacteria and positive bacteria simultaneously.
Embodiment
The buffer solution formula that the present invention washs for the gram-bacteria of Mass Spectrometric Identification comprises: 0.01mol/L ~ 0.1mol/LNaCl, 1mmol/L ~ 10mmol/LTris-HCl, 0.1mmol/L ~ 2mmol/LEDTA and 0.01mmol/L ~ 0.2mmol/LTritonX-100.Tris-HCl is three (methylol) aminomethane-hydrochloride buffer; EDTA is EDTA complex agent; TritonX-100 is Triton X-100 non-ionics.
This buffer solution can be applicable to of the present invention for washing thalline step in the gram-bacteria protein treatment process of Mass Spectrometric Identification, also can be applicable to the gram-bacteria processing method of protein of traditional Mass Spectrometric Identification.The core group of buffer solution of the present invention is divided into NaCl, Tris-HCl and EDTA, the Main Function of Tris-HCl and EDTA maintains certain pH value, and change the distribution of the metallic ion around thalline, thus contribute to TritonX-100 effectively in conjunction with cell membrane, or the EPS around cell membrane, LPS and Chitin etc.NaCl maintains certain Premeabilisation of cells pressure simultaneously, prevents cell at this step generation cell rupture, release intracellular organic matter.The concentration of NaCl can not be too high, and Na ion concentration is too high, and the salt ionic concentration of protein extraction process really height can be caused to affect the molecular ionization of protein when carrying out spectrometer analysis.
A kind of gram-bacteria protein treatment process for Mass Spectrometric Identification of the present invention comprises the following steps:
Buffer solution of the present invention is utilized to wash thalline: choose thalline and be placed in container, adding buffer solution suspension thalline, after centrifugal, remove supernatant;
Bacterioprotein extracts: add cell pyrolysis liquid, Eddy diffusion thalline, and first time ultrasonic process, removes supernatant after centrifugal; After add protein lysates, second time ultrasonic process, collected after centrifugation upper strata protein solution.
The present invention's cell pyrolysis liquid used is the aqueous solution of 65% ~ 75% ethanol, classic method must be added 3 parts of water and 9 parts of absolute ethyl alcohols, change the ethanol water directly adding 65% ~ 75% into, operates more simple.
The acetonitrile of the present invention's protein lysates used to be mol ratio be 10:7:1 ~ 10:7:3: formic acid: water.
The centrifugal rotating speed all selecting 10000-15000RPM of the present invention, centrifugal effect is best.
The present invention utilizes buffer solution to wash thalline step and repeats more than 1 time, because the sensitivity of mass spectrum microbial identification technology itself is very high, thalline culture there will be exocellular polysaccharide (ExopolySaccharides in incubation, EPS), lipopolysaccharides (Lipopolysaccharide, and chitin class material (being chitin again, Chitin) LPS).And can not effectively remove in follow-up step, thus likely affect accuracy and the sensitivity of final mass spectrum microbial identification.In addition, some nutrient culture media retains dyestuff (as Congo red nutrient culture media) and other organic substances.Therefore, utilize buffer solution of the present invention to wash and effectively can remove impurity.
Wherein, the inventive method is relatively looser to the condition of culture of thalline, solid culture or liquid culture.
Embodiment 1:
Choose 10mg Escherichia coli (Gram-negative bacteria) solid culture and be placed in centrifuge tube, add 1mlTEST thalline washing buffer solution, complete Eddy diffusion thalline, abandon or adopt supernatant with the centrifugal 1min of 10000RPM rotating speed.TEST washing buffer solution formula: NaCl0.01mol/L, Tris-HCl (pH8.0) 1mmol/L, EDTA (pH8.0) 0.1mmol/L, TritonX-1000.01mmol/L, the concentration of Tris-HCl and the concentration ratio of EDTA are 10:1, and washing once.
Add the ethanol of 65% as cell pyrolysis liquid, Eddy diffusion thalline, and be placed in ultrasonic process 5min on ultrasonic cell disruption instrument, to remove supernatant after the centrifugal 2min of the rotating speed of 10000RPM.
Add acetonitrile: formic acid: water is 10:7:3 lysate 100uL, carry out ultrasound wave process 2min, promote proteolytic.
By the centrifugal 2min of 10000RPM rotating speed, collect upper strata protein liquid, directly carry out next step Analysis and Identification.
Embodiment 2:
Choose 10mg Escherichia coli (Gram-negative bacteria) solid culture and be placed in centrifuge tube, add 1mlTEST thalline washing buffer solution, complete Eddy diffusion thalline, abandon or adopt supernatant with the centrifugal 1min of 10000RPM rotating speed.TEST washing buffer solution formula: NaCl0.05mol/L, Tris-HCl (pH8.0) 5mmol/L, EDTA (pH8.0) 1mmol/L, TritonX-1000.1mmol/L, the concentration of Tris-HCl and the concentration ratio of EDTA are 5:1, wash twice.
Add the ethanol of 70% as cell pyrolysis liquid, Eddy diffusion thalline, and be placed in ultrasonic process 5min on ultrasonic cell disruption instrument, to remove supernatant after the centrifugal 2min of the rotating speed of 15000RPM.
Add acetonitrile: formic acid: water ratio is the lysate 100uL of 10:7:2, carry out ultrasound wave process 3min, promote proteolytic.
By the centrifugal 2min of 15000RPM rotating speed, collect upper strata protein liquid, directly carry out next step Analysis and Identification.
Embodiment 3:
Choose 10mg Escherichia coli (Gram-negative bacteria) solid culture and be placed in centrifuge tube.Add 1mlTEST thalline lavation buffer solution, Eddy diffusion thalline, with the centrifugation 1min of 10000RPM, abandon or adopt supernatant.TEST fills a prescription: NaCl0.1mol/L, Tris-HCl (pH8.0) 10mmol/L, EDTA (pH8.0) 2mmol/L, TritonX-1000.2mmol/L.The concentration ratio of Tris-HCl and EDTA is 5:1, washs three times.
Add the ethanol of 75% as cell pyrolysis liquid, Eddy diffusion thalline, and be placed in ultrasonic process 5min on ultrasonic cell disruption instrument, remove supernatant with the centrifugal 2min of the rotating speed of 10000RPM.
Add acetonitrile: formic acid: the mixed liquor 100ul of water=10:7:1.Ultrasonic process, collects upper strata protein liquid by the centrifugal 2min of centrifugal 10000RPM, directly carries out next step Analysis and Identification.
Embodiment 4:
The difference of embodiment four and embodiment one is rhizobium (Gram-negative bacteria) to instead of Escherichia coli.
Embodiment 5:
The difference of embodiment five and embodiment two is rhizobium (Gram-negative bacteria) to instead of Escherichia coli.
Embodiment 6:
The difference of embodiment six and embodiment three is rhizobium (Gram-negative bacteria) to instead of Escherichia coli.
Embodiment 7:
The difference of embodiment seven and embodiment one is pseudomonad (gram-positive bacteria) to instead of Escherichia coli.
Embodiment 8:
The difference of embodiment eight and embodiment two is pseudomonad (gram-positive bacteria) to instead of Escherichia coli.
Embodiment 9:
The difference of embodiment nine and embodiment three is pseudomonad (gram-positive bacteria) to instead of Escherichia coli.
Embodiment 10:
The difference of embodiment ten and embodiment one is lactobacillus (gram-positive bacteria) to instead of Escherichia coli.
Embodiment 11:
The difference of embodiment 11 and embodiment two is lactobacillus (gram-positive bacteria) to instead of Escherichia coli.
Embodiment 12:
The difference of embodiment 12 and embodiment three is lactobacillus (gram-positive bacteria) to instead of Escherichia coli.
Comparative example 1:
10mg pure culture bacterium colony Escherichia coli are put into centrifuge tube, adds 300 μ l water mixings, add 900 μ l ethanol mixings, with the centrifugal 2min of maximal rate 15000rpm, removing supernatant, repeated centrifugation, remove ethanol completely, add the formic acid of 50 μ l70%, all bacterium colonies of Eddy diffusion, and concuss 1min, add the pure acetonitrile mixing of 50 μ l, with the centrifugal 2min of 15000rpm, transfer supernatant is in a new centrifuge tube, and sample preparation is complete can directly be identified.
Comparative example 2:
10mg pure culture bacterium colony Escherichia coli are put into centrifuge tube, adds 1mlTEST thalline lavation buffer solution, Eddy diffusion thalline, with the centrifugation 1min of 15000RPM, abandon or adopt supernatant.TEST fills a prescription: NaCl0.1mol/L, Tris-HCl (pH8.0) 1mmol/L, EDTA (pH8.0) 0.1mmol/L, TritonX-1000.2mmol/L.The concentration ratio of Tris-HCl and EDTA is 10:1, washs three times.
Add 300 μ l water mixings toward the Escherichia coli after buffer solution washing, add 900 μ l ethanol mixings, with the centrifugal 2min of 15000rpm, removing supernatant, repeated centrifugation, removes ethanol completely, adds the formic acid of 50 μ l70%, the all bacterium colonies of Eddy diffusion, and concuss 1min, adds the pure acetonitrile mixing of 50 μ l, with the centrifugal 2min of 15000rpm, transfer supernatant is in a new centrifuge tube, and sample preparation is complete can directly be identified.
Comparative example 3:
The difference of comparative example three and comparative example one is rhizobium to instead of Escherichia coli.
Comparative example 4:
The difference of comparative example four and comparative example two is rhizobium to instead of Escherichia coli.
Comparative example 5:
The pseudomonad bacterium colony pure culture of 10mg is put into centrifuge tube and adds 50ul80% trifluoroacetic acid (TFA), suspension concussion makes thalline mix, and leaves standstill several minutes.Add the deionized water of 150ul, add 100% acetonitrile of 200ul, with the centrifugal 2min of maximal rate 15000rpm, transfer supernatant is in a new centrifuge tube, and sample preparation is complete can directly be identified.
Comparative example 6:
10mg pseudomonad bacterium colony pure culture is put into centrifuge tube, adds 1mlTEST thalline lavation buffer solution, Eddy diffusion thalline, with the centrifugation 1min of 15000RPM, abandon or adopt supernatant.TEST fills a prescription: NaCl0.1mol/L, Tris-HCl (pH8.0) 1mmol/L, EDTA (pH8.0) 0.1mmol/L, TritonX-1000.2mmol/L.The concentration ratio of Tris-HCl and EDTA is 10:1, washs three times.
Pseudomonad bacterium colony pure culture after being washed by buffer solution puts into centrifuge tube, and add 50ul80% trifluoroacetic acid (TFA), suspension concussion makes thalline mix, and leaves standstill several minutes.Add the deionized water of 150ul, add 100% acetonitrile of 200ul, with the centrifugal 2min of maximal rate 15000rpm, transfer supernatant is in a new centrifuge tube, and sample preparation is complete can directly be identified.
Comparative example 7:
The difference of comparative example seven and comparative example five is lactobacillus to instead of pseudomonad.
Comparative example 8:
The difference of comparative example eight and comparative example six is lactobacillus to instead of pseudomonad.
The method of operating of bacterioprotein Mass Spectrometric Identification and interpretation of result:
Before starting to carry out Mass Spectrometric Identification, first carry out the preparation of matrix solution: carry out under normal room temperature (25 DEG C) condition, be conducive to it and reach capacity.HCCA (alpha-cyano-4-hydroxycinnamic acid) saturated solution, 50% acetonitrile (AN), 2.5% trifluoroacetic acid (TFA).HCCA crystal is put into centrifuge tube and add 300 μ l50%AN/2.5%TFA solution, room temperature concuss number minute, until completely saturated.
Anticipating of sample disc: use cleansing tissue wiping gently, dip suitable organic solution wipe samples dish with cleansing tissue.After be immersed in ultrasound wave 5-10min in methanol/water 1:1 (v/v) solution, with methyl alcohol and ultrapure water drip washing successively, can use after drying.
Get comparative example 1-8 respectively, the direct point sample of sample 1 μ l prepared by embodiment 1-12, to sample disc, dries at nitrogen environment.
Cover on the sample after drying with the matrix solution of 2 μ l, dry in nitrogen environment, HCCAmatrix solution will be covered rapidly after sample dries.
Adopt MALDI-TOF/TOF (UltraFlexTOF/TOFIII, BrukerDaltonikGmbH, the Germany) instrumental analysis of Brooker company.
Data analysis flow process: generate database engine-generation preprocessed data storehouse, the main mass spectrometric data storehouse of mass spectra peak list-build-open-generate main mass spectrometric data storehouse-importing PDB file-to the definition of mass spectrometric data, analysis and classification-MSP qualification process-principal component analysis or cluster analysis.
The MALDI-TOF analysis operation method that the present invention carries out in gram-bacteria qualification process is the software spectrum data Fitting Analysis of prior art field routine, wherein in fit procedure, multiple correlation index (CCI) and Log value two parameters are the index judging microorganism whether same bacterial classification, directly can embody accuracy and the sensitivity of mass spectrometry method qualification bacterioprotein.
The matching index of correlation of the mass spectrogram of known sample in the mass spectrogram that multiple correlation index (CCI value) is testing sample and database, mxm. is 1, namely CCI value is higher, show testing sample bacterioprotein qualification sensitivity and accuracy higher.
Score is a kind of marking situation, its represents the similarity degree of the mass spectrogram of known sample in the mass spectrogram of unknown sample and database, Log value represents takes the logarithm to Score, it has been generally acknowledged that Log value is greater than the 1.7 protein profiling data consistents can thinking testing sample and known sample.Log value is greater than 1.2, thinks that test strains and reference strains are sames.Namely Log value is higher, show testing sample bacterioprotein qualification sensitivity and accuracy higher.
Therefore, by comparative example 1-4, the method for embodiment 1-6 is prepared into sample and carries out MALDI-TOF Analysis and Identification result as following table 1:
Table 1
By comparative example 5-8, the method for embodiment 7-12 is prepared into sample and carries out MALDI-TOF Analysis and Identification result as following table 2:
Table 2
From table 1, table 2 can be found out, relative to traditional gram-bacteria protein extracting method characterization sample, by the CCI value raising 4%-6% of sample mass spectrogram Analysis and Identification prepared with traditional gram-bacteria protein extracting method again after buffer solution of the present invention washing, Log value improve 18-23%, illustrate its sensitivity and accuracy higher; Improve 11%-16%, Log value raising 60%-68% by the CCI value of the sample mass spectrogram Analysis and Identification prepared by gram-bacteria protein extracting method of the present invention again after buffer solution of the present invention washing, illustrate its sensitivity and accuracy the highest.
And the gram-bacteria protein treatment process for Mass Spectrometric Identification of the present invention, be suitable for gram-positive bacteria and Gram-negative bacteria simultaneously, operate more convenient in bacterium blind sample identification and analysis.
Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made.
Claims (8)
1. the buffer solution that washs of the gram-bacteria for Mass Spectrometric Identification, it is characterized in that: described buffer solution formula comprises 0.01mol/L ~ 0.1mol/LNaCl, 1mmol/L ~ 10mmol/LTris-HCl, 0.1mmol/L ~ 2mmol/LEDTA and 0.01mmol/L ~ 0.2mmol/LTritonX-100.
2. the buffer solution that washs of a kind of gram-bacteria for Mass Spectrometric Identification according to claim 2, is characterized in that: the molar concentration rate of described Tris-HCl and EDTA is 5:1 ~ 10:1.
3., for a gram-bacteria protein treatment process for Mass Spectrometric Identification, it is characterized in that: comprise the following steps:
The buffer solution described in claim 1 or 2 is utilized to wash thalline: choose thalline and be placed in container, adding the buffer solution suspension thalline described in claim 1 or 2, after centrifugal, remove supernatant;
Bacterioprotein extracts: add cell pyrolysis liquid, Eddy diffusion thalline toward the thalline after the buffer solution washing described in claim 1 or 2, first time ultrasonic process, removes supernatant after centrifugal; After add protein lysates, second time ultrasonic process, collected after centrifugation upper strata protein solution.
4. a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification according to claim 3, is characterized in that: described cell pyrolysis liquid comprises the aqueous solution containing 65%-75% ethanol.
5. a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification according to claim 3, is characterized in that: described protein lysates comprises the acetonitrile that mol ratio is 10:7:1 ~ 10:7:3: formic acid: water.
6. a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification according to claim 3, is characterized in that: the described centrifugal rotating speed all selecting 10000-15000RPM.
7. a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification according to claim 3, is characterized in that: utilize the buffer solution described in claim 1 or 2 to wash thalline step and repeat more than 2 times or 2 times.
8. a kind of gram-bacteria protein treatment process for Mass Spectrometric Identification according to claim 3, is characterized in that: described first time, ultrasonic process was at least 2min, and second time sonication treatment time is at least 2min.
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| CN107917950A (en) * | 2017-10-31 | 2018-04-17 | 中国疾病预防控制中心传染病预防控制所 | Tiretube process microorganism mass spectrum detection kit with biological safety |
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