CN104596816B - A kind of Plant nematode pre-treating method detected for MALDI TOF MS - Google Patents
A kind of Plant nematode pre-treating method detected for MALDI TOF MS Download PDFInfo
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- CN104596816B CN104596816B CN201510012165.5A CN201510012165A CN104596816B CN 104596816 B CN104596816 B CN 104596816B CN 201510012165 A CN201510012165 A CN 201510012165A CN 104596816 B CN104596816 B CN 104596816B
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- aqua sterilisa
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Abstract
The invention discloses a kind of Plant nematode pre-treating method detected for MALDI TOF MS, comprise the following steps：(1) extract solution is added dropwise on the slide glass of sterilizing, 58 μ l are added dropwise for every Plant nematode in the dripping quantity of the extract solution；(2) the cleaned Plant nematode is transferred in the extract solution on the slide glass under stereomicroscope, and crushes the Plant nematode, the extract solution is used to extract the protein of the Plant nematode and the protein is hydrolyzed into polypeptide；(3) under stereomicroscope, it is added dropwise together in sample panel with the extract solution in liquid-transfering gun aspiration step (2) and the related Plant nematode crushed, and dry；(4) add matrix solution in the sample panel after drying, the polypeptide is dispersed in the matrix solution, and dry the sample for obtaining detecting for MALDI TOF MS, every Plant nematode adds the 1 1.5 μ l matrix solution.Method of the invention is easily operated, result is reproducible, background signal is low and sensitivity is high.
Description
Technical field
It is more particularly to a kind of to be used for MALDI-TOF MS (Matrix- the invention belongs to analytical technique of mass spectrum field
Assisted laser desorption/ionization time-of-flight mass spectrometry abbreviation,
Chinese full name be Matrix-assisted laser desorption ionization) detection Plant nematode pre-treating method.
Background technology
MALDI-TOF MS instruments are mainly made up of two parts：MALDI (MALDI) and
TOF (TOF).MALDI principle is with laser irradiating sample and substrate formed cocrystallization film, base
Matter absorbs energy transmission to biomolecule from laser, and by proton translocation to biomolecule or from biomolecule in ionization process
Proton is obtained, and makes the process of biomolecule ionization.It is a kind of Soft ionization techniques, it is adaptable to mixture and large biological molecule
Determine.TOF principle is that ion accelerates to fly over dirft tube under electric field action, different according to the flight time for reaching detector
And be detected.The features such as MALDI-TOF-MS has high sensitivity, the high degree of accuracy and high resolution, is that the fields such as life science are carried
A kind of strong analysis means of testing is supplied.In recent years, the technology is increasingly used for vegetative bacteria, fungi and the detection of nematode
It is central.But be difficult operation to the pre-treating method of Plant nematode before detection, so as to get result stability is poor, more than background signal and
Miscellaneous, sensitivity is low so as to influence the detection to Plant nematode.
The content of the invention
In order to make up above-mentioned the deficiencies in the prior art, the present invention proposes a kind of plant line detected for MALDI-TOF MS
Worm pre-treating method.
The technical problem of the present invention is solved by following technical scheme：
A kind of Plant nematode pre-treating method detected for MALDI-TOF MS, comprises the following steps：
(1) extract solution is added dropwise on the slide glass of sterilizing, the dripping quantity of the extract solution is added dropwise for every Plant nematode
5-8μl；
(2) the cleaned Plant nematode is transferred in the extract solution on the slide glass under stereomicroscope, and
The Plant nematode is crushed, the extract solution is for extracting the protein of the Plant nematode and being many by the protein degradation
Peptide；
(3) under stereomicroscope, with the extract solution in liquid-transfering gun aspiration step (2) and the related Plant nematode one crushed
Rise and be added dropwise in sample panel, and dry；
(4) add matrix solution in the sample panel after drying, the polypeptide is dispersed in the matrix solution, and dry
The sample detected for MALDI-TOF MS is obtained, every Plant nematode adds the 1-1.5 μ l matrix solution.
Preferably, the one kind of the extract solution in following group：
The mixed solution of trifluoroacetic acid and aqua sterilisa, in the mixed solution, the volumetric concentration of trifluoroacetic acid is 0.1-
0.5%, surplus is aqua sterilisa；
The mixed solution of ethanol and aqua sterilisa, in the mixed solution, the volumetric concentration of ethanol is 90-95%, and surplus is
Aqua sterilisa；
The mixed solution of acetone and aqua sterilisa, in the mixed solution, the volumetric concentration of acetone is 90-95%, and surplus is
Aqua sterilisa；
The mixed solution of formic acid, acetonitrile and aqua sterilisa, the volumetric concentration of formic acid described in the mixed solution is 33-
37%, the volumetric concentration of the acetonitrile is 48-52%, and surplus is aqua sterilisa；
The mixed solution of formic acid, isopropanol and aqua sterilisa, the volumetric concentration of formic acid described in the mixed solution is 17-
22%, the volumetric concentration of the isopropanol is 33-35%, and surplus is aqua sterilisa.
Preferably, the matrix solution in step (4) is by formed below：It is molten by adding one suitable for the matrix of polypeptide
Until matrix supersaturation in agent.
It is further preferred that the matrix is alpha-cyano -4- hydroxycinnamic acids (α-cyano-4-
Hydroxycinnamicacid, CHCA), sinapic acid (sinapinic acid), forulic acid (Ferulic Acid), 2- (4- hydroxyls
Base imines) benzoic acid (2- (4-hydroxyphenylazo) benzoic acid, HABA), the chloro- 2- sulfydryls phenylpropyl alcohol thiazole (5- of 5-
chloro-2-mercaptobenzothiazo1e,CMBT)。
It is further preferred that the one kind of the solvent in following group：
The mixed solvent of acetonitrile, trifluoroacetic acid and aqua sterilisa, is 33- in the volumetric concentration of the in the mixed solvent acetonitrile
50%, the volumetric concentration of trifluoroacetic acid is 2.5-3.3%, and surplus is aqua sterilisa；
The mixed solvent of ethanol and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent ethanol, surplus is to go out
Bacterium water；
The mixed solvent of methanol and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent methanol, surplus is to go out
Bacterium water；
The mixed solvent of acetonitrile and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent acetonitrile, surplus is to go out
Bacterium water；
The mixed solvent of tetrahydrofuran and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent tetrahydrofuran,
Surplus is aqua sterilisa；
The mixed solvent of acetone and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent acetone, surplus is to go out
Bacterium water.
Sample and matrix formation cocrystallization film, matrix absorbs energy transmission to sample molecule from laser, and makes sample
Molecular ionization, the formation of cocrystallization film is the key of detection.The solvent for the dissolved matrix selected above can be protected and promote base
Matter and sample formation cocrystallization film, beneficial to detection.
Preferably, cleaning step is also included before the step (1)：The Plant nematode is transferred in centrifuge tube,
1-1.2mL aqua sterilisas are added, vortex 5-8min, 4000-5000rpm centrifugation 5-10min removes supernatant；Then 1-1.2mL is added
Aqua sterilisa, vortex 5-8min, 4000-5000rpm centrifugation 5-10min, removes supernatant；Add 1-1.2mL aqua sterilisas, vortex 5-
8min, 4000-5000rpm centrifuge 5-10min, remove supernatant；Finally plus 1-1.2mL aqua sterilisas, it is standby.
The beneficial effect that the present invention is compared with the prior art is：The pre-treating method of the Plant nematode of the present invention is for single
In the processing that bar Plant nematode is carried out, the extract solution that wall scroll Plant nematode is chosen to slide glass (such as slide), in stereomicroscope
Under crush nematode, make protein release into extract solution, Plant nematode be transferred to sample with liquid-transfering gun under stereomicroscope
On plate, then add matrix solution to carry out subsequent detection, this method is easily operated, the result repeatability obtained after determining is repeated several times
It is good, as a result stablize；Plant nematode after treatment detects the background signal of obtained polypeptide mass spectrogram with MALDI-TOF MS
Low, the ratio for being extracted and being hydrolyzed due to the protein in Plant nematode is more thoroughly so that sensitivity is higher, and method of the invention is fitted
With MALDI-TOFMS is to the quarantine of Plant nematode, detection and identifies.
Brief description of the drawings
Fig. 1 is the polypeptide mass-spectrogram of the wall scroll root-knot nematode in the embodiment of the present invention 1.
Embodiment
Below against accompanying drawing and with reference to preferred embodiment the invention will be further described.
The present invention provides a kind of Plant nematode pre-treating method detected for MALDI-TOF MS, in a kind of embodiment
In, comprise the following steps：
(1) extract solution is added dropwise on the slide glass of sterilizing, the dripping quantity of the extract solution is added dropwise for every Plant nematode
5-8μl；
(2) the cleaned Plant nematode is transferred in the extract solution on the slide glass under stereomicroscope, and
The Plant nematode is crushed, the extract solution is used to extract the protein of the Plant nematode and is hydrolyzed to the protein many
Peptide；
(3) under stereomicroscope, with the extract solution in liquid-transfering gun aspiration step (2) and the related Plant nematode one crushed
Rise and be added dropwise in sample panel, and dry；
(4) add matrix solution in the sample panel after drying, the polypeptide is dispersed in the matrix solution, and dry
The sample detected for MALDI-TOF MS is obtained, every Plant nematode adds the 1-1.5 μ l matrix solution.
Wherein：
Extract solution has broken wall function, the protein release in Plant nematode can be promoted to go forward side by side an one-step hydrolysis for polypeptide, often
The amount for the extract solution that bar Plant nematode needs is 5-8 μ l, can be 5 μ l, 6 μ l, 7 μ l, 8 μ l etc. in certain embodiments.
The matrix in matrix solution in step (4) is the matrix detected for MALDI-TOF MS, in present embodiment
In, selection is applied to the matrix of scattered polypeptide, and every Plant nematode adds 1-1.5 μ l matrix solution, in certain embodiments may be used
To add 1 μ l, 1.2 μ l, 1.5 μ l etc..
The present invention will be described in detail by taking plant root-knot nematode as an example below.
Embodiment 1
1. the cleaning of root-knot nematode
The root-knot nematode of the wall scroll obtained from plant is transferred in 1.5mL centrifuge tubes (Eppendorf), is vortexed
5min, 4000rpm centrifuge 10min, remove supernatant；1mL aqua sterilisas are added, vortex 5min, 4000rpm centrifugation 10min removes supernatant
Liquid；1mL aqua sterilisas are added, vortex 5min, 4000rpm centrifugation 10min removes supernatant, plus 1mL aqua sterilisas, standby.
2. the pre-treatment of wall scroll root-knot nematode
(1) the 5 μ l extract solutions (mixed solutions of formic acid, acetonitrile and aqua sterilisa, in the mixing are added dropwise on the slide of sterilizing
The volumetric concentration of formic acid is 35% in solution, and the volumetric concentration of acetonitrile is 50%, and surplus is aqua sterilisa).
(2) turned under stereomicroscope with pin (it can be insect needle to choose pin) one cleaned root-knot nematode of picking is chosen
Move on in the extract solution on slide, nematode is crushed with pin is chosen.
(3) 1.5 μ l extract solutions (related root-knot nematode is together) are drawn with liquid-transfering gun under stereomicroscope, is added drop-wise to sample
On plate, and dry.
(4) in the sample panel after drying plus 1 μ l CHCA matrix solution (matrix solution is：By CHCA add acetonitrile,
The in the mixed solvent of trifluoroacetic acid and aqua sterilisa is until CHCA supersaturation, and the mixing of wherein acetonitrile, trifluoroacetic acid and aqua sterilisa is molten
In agent, the volumetric concentration of acetonitrile is 33%, and the volumetric concentration of trifluoroacetic acid is 3.3%, and surplus is aqua sterilisa), and dry and obtain
The sample detected for MALDI-TOF MS.
3.MALDI-TOF MS are detected
After the above method is handled wall scroll root-knot nematode, the sample panel dried is put into MALDI-TOFMS sample
Product are detected in storehouse, detect that the polypeptide mass spectrogram of obtained root-knot nematode is as shown in Figure 1.Test, obtain by multiple repetition
Very well, background signal is relatively low for the repeatability of the polypeptide mass spectrogram arrived, and sensitivity is high.
In certain embodiments, the difference with embodiment 1 is：Extract solution in (1) of step 2 is trifluoroacetic acid and gone out
The mixed solution of bacterium water, in the mixed solution, the volumetric concentration of trifluoroacetic acid is 0.1%, and surplus is aqua sterilisa；Or extract
Liquid is the mixed solution of ethanol and aqua sterilisa, in the mixed solution, and the volumetric concentration of ethanol is 90%, and surplus is aqua sterilisa；
Or the mixed solution that extract solution is acetone and aqua sterilisa, in the mixed solution, the volumetric concentration of acetone is 90%, and surplus is
Aqua sterilisa；Or the mixed solution that extract solution is formic acid, isopropanol and aqua sterilisa, the volume of formic acid described in the mixed solution
Concentration is 17%, and the volumetric concentration of the isopropanol is 33%, and surplus is aqua sterilisa.
In further embodiments, the difference with embodiment 1 is：Matrix in (4) of step 2 can for sinapic acid,
Forulic acid, HABA or CMBT.
In some other embodiments, the difference with embodiment 1 is：The solvent in matrix solution in (4) of step 2 can
Think：The mixed solvent of ethanol and aqua sterilisa, is 45% in the volumetric concentration of the in the mixed solvent ethanol, surplus is aqua sterilisa；
Or the mixed solvent that this is methanol and aqua sterilisa, it is 50% in the volumetric concentration of the in the mixed solvent methanol, surplus is aqua sterilisa；
Or be acetonitrile and the mixed solvent of aqua sterilisa, it is 45% in the volumetric concentration of the in the mixed solvent acetonitrile, surplus is aqua sterilisa；
Or be tetrahydrofuran and the mixed solvent of aqua sterilisa, it is 55%, surplus in the volumetric concentration of the in the mixed solvent tetrahydrofuran
For aqua sterilisa；Or be acetone and the mixed solvent of aqua sterilisa, it is 45%, surplus in the volumetric concentration of the in the mixed solvent acetone
For aqua sterilisa.
The condition in embodiment more than is tested by multiple repetition, obtained polypeptide mass spectrogram and embodiment 1
In Fig. 1 repeatability it is all fine, background signal is relatively low, and sensitivity is high.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For those skilled in the art, do not taking off
On the premise of from present inventive concept, some equivalent substitutes or obvious modification can also be made, and performance or purposes are identical, all should
When being considered as belonging to protection scope of the present invention.
Claims (6)
1. a kind of Plant nematode pre-treating method detected for MALDI-TOF MS, it is characterised in that before the Plant nematode
Processing method is the processing carried out for wall scroll Plant nematode, is comprised the following steps：
(1) 5-8 μ l extract solution is added dropwise on the slide of sterilizing；
(2) the cleaned wall scroll Plant nematode is transferred in the extract solution on the slide under stereomicroscope,
And crushing the wall scroll Plant nematode, the extract solution is used for the protein for extracting the wall scroll Plant nematode and by the albumen
Matter is hydrolyzed to polypeptide；
(3) under stereomicroscope, dripped together with the extract solution in liquid-transfering gun aspiration step (2) and the related Plant nematode crushed
It is added in sample panel, and dries；
(4) add 1-1.5 μ l matrix solution in the sample panel after drying, the polypeptide is dispersed in the matrix solution,
And dry the sample for obtaining detecting for MALDI-TOF MS.
2. the Plant nematode pre-treating method detected as claimed in claim 1 for MALDI-TOF MS, it is characterised in that：Institute
State the one kind of extract solution in following group：
The mixed solution of trifluoroacetic acid and aqua sterilisa, in the mixed solution, the volumetric concentration of trifluoroacetic acid is 0.1-0.5%,
Surplus is aqua sterilisa；
The mixed solution of ethanol and aqua sterilisa, in the mixed solution, the volumetric concentration of ethanol is 90-95%, and surplus is sterilizing
Water；
The mixed solution of acetone and aqua sterilisa, in the mixed solution, the volumetric concentration of acetone is 90-95%, and surplus is sterilizing
Water；The mixed solution of formic acid, acetonitrile and aqua sterilisa, the volumetric concentration of formic acid described in the mixed solution is 33-37%, described
The volumetric concentration of acetonitrile is 48-52%, and surplus is aqua sterilisa；
The mixed solution of formic acid, isopropanol and aqua sterilisa, the volumetric concentration of formic acid described in the mixed solution is 17-22%,
The volumetric concentration of the isopropanol is 33-35%, and surplus is aqua sterilisa.
3. the Plant nematode pre-treating method detected as claimed in claim 1 for MALDI-TOF MS, it is characterised in that：Step
Suddenly the matrix solution in (4) is by formed below：It will be added suitable for the matrix of polypeptide a solvent until the matrix
Supersaturation.
4. the Plant nematode pre-treating method detected as claimed in claim 3 for MALDI-TOF MS, it is characterised in that：Institute
Matrix is stated for alpha-cyano -4- hydroxycinnamic acids, sinapic acid, forulic acid, 2- (4- hydroxyl imides) benzoic acid and the chloro- 2- sulfydryls benzene of 5-
One kind in third thiazole.
5. the Plant nematode pre-treating method detected as claimed in claim 3 for MALDI-TOF MS, it is characterised in that：Institute
State the one kind of solvent in following group：
The mixed solvent of acetonitrile, trifluoroacetic acid and aqua sterilisa, is 33-50%, three in the volumetric concentration of the in the mixed solvent acetonitrile
The volumetric concentration of fluoroacetic acid is 2.5-3.3%, and surplus is aqua sterilisa；
The mixed solvent of ethanol and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent ethanol, surplus is sterilizing
Water；
The mixed solvent of methanol and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent methanol, surplus is sterilizing
Water；
The mixed solvent of acetonitrile and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent acetonitrile, surplus is sterilizing
Water；
The mixed solvent of tetrahydrofuran and aqua sterilisa, is 45-55%, surplus in the volumetric concentration of the in the mixed solvent tetrahydrofuran
For aqua sterilisa；
The mixed solvent of acetone and aqua sterilisa, is 45-55% in the volumetric concentration of the in the mixed solvent acetone, surplus is sterilizing
Water.
6. the Plant nematode pre-treating method detected as claimed in claim 1 for MALDI-TOF MS, it is characterised in that：
Also include cleaning step before the step (1)：The Plant nematode is transferred in centrifuge tube, 1-1.2mL aqua sterilisas are added,
Vortex 5-8min, 4000-5000rpm centrifugation 5-10min, removes supernatant；Then add 1-1.2mL aqua sterilisas, vortex 5-8min,
4000-5000rpm centrifuges 5-10min, removes supernatant；Add 1-1.2mL aqua sterilisas, vortex 5-8min, 4000-5000rpm
5-10min is centrifuged, supernatant is removed；Finally plus 1-1.2mL aqua sterilisas, it is standby.
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| CN106404474B (en) * | 2015-12-25 | 2018-03-06 | 首都医科大学附属北京同仁医院 | Filamentous fungi Mass Spectrometric Identification protein extracting process |
| CN107926766B (en) * | 2017-10-17 | 2023-07-14 | 杭州晶雁电子科技有限公司 | Automatic moving method |
| CN107991491A (en) * | 2017-10-31 | 2018-05-04 | 北京毅新博创生物科技有限公司 | Correct the method and product of the accuracy rate of Mass Spectrometer Method protein sample |
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| CN102426187A (en) * | 2011-11-21 | 2012-04-25 | 程金生 | Graphene matrix and application of graphene matrix in matrix-assisted laser desorption/ionization-time of flight-mass spectrometry detection |
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