CN102805052A - Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium - Google Patents
Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium Download PDFInfo
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Abstract
The invention discloses a method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and a special culture medium. The special culture medium for marking drosophila by using the SILAC is prepared according to the following method: a saccharomycete body marked by using the SILAC is coated on a drosophila culture medium to obtain the special culture medium for marking the drosophila by using the SILAC. An experiment proves that the culture medium for marking the drosophila by using the SILAC can be specially used for marking the drosophila proteomes by using the SILAC, can achieve rapid and high-efficient marking, and creates conditions for preparation of quantified interior labels and high-precision quantified proteome studying.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method and special culture media of the SILAC of utilization overall labeling drosophila protein matter group.
Background technology
Fruit bat (Drosophila melanogaster) is individual small insect.Since Morgan began to utilize its research genetics, fruit bat became the model organism of genetics, physiology, growth and evolution biology research gradually.Since fruit bat have growth fast, be prone to raise and breeding, be prone to keep large group, inexpensive, plurality of advantages such as phenotype abundant and most signal pathways are consistent with higher organism, make fruit bat become the golden worm in the modern biology research.
Protein is the direct executor of biological function.Proteomics is that system identifies and quantitative intracellular protein, and studies the new branch of science of its biological function.Along with the completion of drosophila gene group order-checking and the fast development of proteomics research technology, scientist can realize the target that drosophila protein matter group is identified.The quantitative comparison of protein group is the effective means of research gene mutation or biological processes and effect relation thereof, also is the major fields of current proteomics research.So far have the report of fruit bat quantitative proteomics.Like the protein group of fruit bat of having utilized ICAT technology quantitative comparison such as Brunner.Quantitative comparison such as Xun Parkinsonian fruit bat model.Utilizations such as Krijgsveld
15The yeast of the N mark fruit bat of feeding has obtained
15The fruit bat of N mark has begun the research of internal labeling of fruit bat body and quantitative proteomics.But because
15The protein group Isotopic Distribution of N mark is wide; The overlapping quantitative accuracy that causes is low easily between the weight mark; And the weight mark is difficult to distinguish; Be prone to produce technical barriers such as false positive, make quantitative proteomics turn to whole protein group echo technology (stable isotope labeling by amino acid in cell culture, SILAC) goldstandard of this quantitative proteomics research gradually based on the heavy stable isotope labeled amino acid in the cell cultivation process.
Mann research group utilizes the SILAC technology that the SL2 cell-line of fruit bat is studied, and identifies and quantitative more than 6000 protein, and has compared the incidence relation of mRNA and protein abundance.Although SILAC has just begun just to be used for cultured cells, like yeast, mammalian cell etc., the beginning of the heavy stable isotope mark of whole animal has been started in the success of the whole mouse of SILAC mark recently.Sury etc. have utilized SILAC technology mark whole fruit bat, but because of technical limitations, its labeling effciency can only reach about 96%, has influenced the quantitative precision and the sensitivity of detection.
Summary of the invention
An object of the present invention is to provide a kind of SILAC mark fruit bat special culture media.
The present invention provides SILAC mark fruit bat special culture media, prepares according to following method: the yeast thalline of SILAC mark is coated on the fruit bat medium, obtains SILAC mark fruit bat special culture media.
Above-mentioned fruit bat medium is made up of low melting-point agarose, glucose, methyl p-hydroxybenzoate, ethyl acetate and water; The mass ratio of said low melting-point agarose, said glucose, said methyl p-hydroxybenzoate, said ethyl acetate is 0.8:10:0.002:0.8.
The concrete concentration of above-mentioned fruit bat nutrient media components is following: the concentration of said low melting-point agarose in said fruit bat medium is 0.8% (quality percentage composition); The concentration of said glucose in said fruit bat medium is 10% (quality percentage composition); The concentration of said methyl p-hydroxybenzoate in said fruit bat medium is 0.002% (quality percentage composition); The concentration of the said agar of said ethyl acetate in said fruit bat medium is 0.8% (quality percentage composition).
Wherein methyl p-hydroxybenzoate adds in the fruit bat medium with the form of methyl p-hydroxybenzoate ethanolic solution in the fruit bat nutrient media components; The methyl p-hydroxybenzoate ethanolic solution prepares according to following method: methyl p-hydroxybenzoate is dissolved in the mixed liquor that obtains in 95% ethanol water, and the concentration of methyl p-hydroxybenzoate in mixed liquor is 10% (quality percentage composition).
In the above-mentioned special culture media; The yeast thalline of said SILAC mark prepares according to following method: lysine auxotroph yeast strain is cultivated in the SILAC mark yeast special culture media that contains heavy stable isotope mark lysine; Collect thalline, obtain the yeast thalline of SILAC mark.
Lysine auxotroph yeast strain in the embodiments of the invention is knock-out bacterial strain BY4741 △ YBR115C, available from Saccharomyces Genome Deletion Project.
The above-mentioned SILAC mark yeast special culture media that contains heavy stable isotope mark lysine prepares according to following method: lysine, adenosine, uracil, tryptophan, histidine, arginine, methionine, tyrosine, leucine, isoleucine, phenyl alanine, glutamic acid, aspartic acid, valine, threonine, serine and water by yeast nitrogen basis, glucose, heavy stable isotope mark are formed; The concentration of said yeast nitrogen basis in said SILAC mark yeast special culture media is 7g/L; The concentration of said glucose in said SILAC mark yeast special culture media is 20g/L; The lysine L-lysine-of said heavy stable isotope mark
13C
6Concentration in said SILAC mark yeast special culture media is 164.3 μ M; Said adenosine, said uracil, said tryptophan, said histidine, said arginine, the concentration of said methionine in said SILAC mark yeast special culture media are 20mg/L; Said tyrosine, said leucine, the concentration of said isoleucine in said SILAC mark yeast special culture media are 30mg/L; The concentration of said phenyl alanine in said SILAC mark yeast special culture media is 50mg/L; Said glutamic acid and the said aspartic acid concentration in said SILAC mark yeast special culture media is 100mg/L; The concentration of said valine in said SILAC mark yeast special culture media is 150mg/L; The concentration of said threonine in said SILAC mark yeast special culture media is 200mg/L; The concentration of said serine in said SILAC mark yeast special culture media is 400mg/L.The lysine of the heavy stable isotope mark in the above-mentioned SILAC mark yeast special culture media is L-lysine-
13C
6Also can be for the different heavy stable isotope mark lysine of other mass number, like L-lysine-
13C
6 15N
2Deng.
In the saccharomycete preparation of above-mentioned SILAC mark, said best cultivation is 30 ℃ and cultivated for 9 generations, cultivates 18h altogether.
The application of above-mentioned special culture media in SILAC mark drosophila protein matter group also is the scope that the present invention protects.
Another object of the present invention provides a kind of method of SILAC fruit bat medium mark drosophila protein matter group.
Method provided by the invention comprises the steps: that ovum with above-mentioned SILAC mark fruit bat special culture media fruit flies to developing into adult, obtains the fruit bat of SILAC overall labeling drosophila protein matter group, realizes SILAC overall labeling drosophila protein matter group.
In the said method, the ovum of said fruit bat for paired male and female fruit bat on above-mentioned special culture media, cultivate, laying eggs obtains.
Experiment of the present invention proves; A kind of medium that is used for fruit bat SILAC mark of the present invention's exploitation; Can be specifically designed to SILAC mark drosophila protein matter group, and can realize mark fast and efficiently, for condition has been created in target preparation in quantitatively and quantitative protein group research accurately.
Description of drawings
The attach most importance to saccharomycete of the complete mark of stable isotope labeled amino acid of Fig. 1
Fig. 2 is the fruit bat culture apparatus that contains SILAC mark medium
Fig. 3 is the fruit bat of SILAC mark medium breed and the comparison of common fruit bat
Fig. 4 is that SILAC mark medium is cultured the test of fruit bat labeling effciency
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The present invention can more easily understand through following instance, but the present invention is not limited in these instances.
The preparation of embodiment 1, SILAC mark yeast thalline
One, the preparation of SILAC mark yeast special culture media
The component of the SILAC mark yeast special culture media of every 1L (the amino acid whose SC medium that contains heavy stable isotope mark) is following: 7g Difco yeast nitrogen basis (U.S. company BD; 239210), 20g glucose (Chemical Reagent Co., Ltd., Sinopharm Group, article No.: 10010592), the lysine L-lysine-of the heavy stable isotope mark of 164.3 μ mol article No.:
13C
6(U.S. CIL Corp.; Article No.: CNLM-291-0.25), the adenosine of 20mg (U.S. Amresco company, article No.: 0325-50G0325-50G), 20mg uracil (U.S. Amresco company, article No.: 0847-250G0847-250G), 20mg tryptophan (U.S. Amresco company; Article No.: E800-25G), 20mg histidine (U.S. Amresco company; Article No.: E806-100G), 20mg arginine (U.S. Amresco company, article No.: 0877-100G), 20mg methionine (U.S. Amresco company, article No.: E801-100G), 30mg tyrosine (U.S. Amresco company; Article No.: E821-25G), 30mg leucine (U.S. Amresco company; Article No.: E811-100G), 30mg isoleucine (U.S. Amresco company, article No.: E803-25G), 50mg phenyl alanine (U.S. Amresco company, article No.: 0991-100G), 100mg glutamic acid (U.S. Amresco company; Article No.: 0421-1KG), 100mg aspartic acid (U.S. Amresco company; Article No.: 0192-500G), 150mg valine (U.S. Amresco company, article No.: 1B1102-25G), 200mg threonine (U.S. Amresco company, article No.: E808-25G), 400mg serine (U.S. Amresco company; Article No.: 1B1103-25G), water polishing volume.
Two, the acquisition and the checking of SILAC mark yeast thalline
1, SILAC mark yeast
Picking fission yeast lysine auxotrophic strain (knock-out bacterial strain BY4741 △ YBR115C from the YPD solid culture flat board; Available from Saccharomyces Genome Deletion Project) fresh bacterium colony; Be transferred in the YPD liquid nutrient medium (containing 10g bacterium yeast extract, 20g bacto peptone, 20g glucose in every liter); Grow overnight obtains seed culture fluid; Centrifugal seed culture fluid is collected yeast cells under aseptic condition, with aseptic washing twice, and gets a certain amount of above-mentioned SILAC mark yeast special culture media that is inoculated into sterile water after resuspended, makes that the final concentration of initial thalline is 0.0023OD (A
600), cultivated for 9 generations after totally 18 hours for 30 ℃, the final concentration that reaches thalline is 1.2OD (A
600), collected thalline in centrifugal 5 minutes through 10000g again, obtain SILAC mark yeast thalline ,-80 ℃ are frozen subsequent use.
2, the checking of SILAC mark yeast thalline
1) extraction of total protein
With the Tris of 10mM pH8.0, the NaH of 0.1M
2PO
4, 8M urea, 0.02%SDS and 10mM beta-mercaptoethanol be as protein extract, with bead (Biospec Products Inc., Bartlesville) concussion broken wall method cracking SILAC mark yeast thalline.Thalline-bead mixed liquor shook 1 minute the most at a high speed on vortex mixer, cooled on ice 1 minute, obtained pyrolysis product totally for 14 times, with pyrolysis product under the condition of 17000g centrifugal 5 minutes, obtained total protein again.With being used for protease digestion behind the Bradford standard measure commonly used, the result is about 10 μ g/ μ l for the concentration of total protein.
2) digestion
Getting 5 μ g total proteins is that the DTT of 5mM handled 30 minutes through final concentration, and is that the iodoacetamide lucifuge of 20mM was handled 30 minutes down for 25 ℃ with the final concentration, and adding urea to final concentration again is 8M sex change 30 minutes, with 50MmNH
4HCO
3Dilution urea is to 4M concentration, and (article No.: 125-05061) 37 ℃ of digestion is 14 hours, obtains the peptide section for Lys-C, Japanese Wako company with the lysine protease C of 10ng/ μ l.
3) chromatograph-mass spectrometer coupling analysis
With the peptide section of above-mentioned gained with the desalination of C18 post (3M, St.Paul MN), dissolve with 5% acetonitrile of 10 μ l and the chromatograph-mass spectrometer coupling sample-loading buffer of 1% formic acid, get 2 μ l and carry out the chromatograph-mass spectrometer coupling analysis, specifically with reference to Xu, P.; Duong, D.; Peng, J., Systematical optimization of reverse-phase chromatography for shotgun proteomics.J Proteome Res 2009,8 (8), the method for putting down in writing among the 3944-50.Preparing albumen according to the method described above, detect with yeast thalline before the SILAC mark is contrast.
Picked at random ATP combines Protein S SA2 peptide section NQAAMNPANTVFDAK; The result is as shown in Figure 1, can find out, SILAC mark yeast thalline is complete mark yeast thalline; Complete mark after 9 generations, and detect peptide section less than the lysine that contains natural light isotope.
Three, the acquisition of light mark yeast thalline of contrast and contrast yeast thalline
Contrast light mark medium and SILAC mark yeast special culture media component is basic identical, that different is the lysine L-lysine-that will weigh the stable isotope mark
13C
6Replace with common light isotope label lysine (U.S. Amresco company, article No.: 0437-100G)
YPD medium: 1% bacterium yeast extract Bacto-yeast extract (U.S. Amresco company; Article No.: J850-1KG), 2% bacto peptone bacto-peptone (U.S. Amresco company, article No.: J636-500G); 2% glucose (Chemical Reagent Co., Ltd., Sinopharm Group; Article No.: 10010592), 2% bacteria culture media is with agar powder (U.S. Amresco company, article No.: J637-500G).
Adopt above-mentioned two 1 method, yeast BY4741 △ YBR115C is cultivated in the light mark medium of contrast and YPD medium respectively, obtain contrasting light mark yeast thalline and contrast YPD yeast thalline.
The preparation of embodiment 2, SILAC mark fruit bat special culture media
1, SILAC mark fruit bat special culture media preparation
The component of the fruit bat medium of every 1L is following: the quality percentage composition is 0.8% Bio-Rad low melting-point agarose (Bio-Rad Hercules; CA), the quality percentage composition is that 10% glucose, quality percentage composition are that 0.002% methyl p-hydroxybenzoate, final concentration are that 0.8% ethyl acetate and water mix, water constant volume to 1 liter.
SILAC mark fruit bat special culture media prepares according to following method: coating obtains the SILAC mark medium of mark fruit bat by two the 1 SILAC mark yeast thalline yeast thalline of mark (heavy stable isotope) that obtains of embodiment 1 on above-mentioned fruit bat medium.
2, the light mark medium of contrast fruit bat SILAC
The light mark medium of contrast fruit bat SILAC: basic identical with the preparation method of SILAC mark fruit bat special culture media, different is the light mark yeast of the contrast thalline that coating is obtained by three of embodiment 1;
Contrast YPD fruit bat medium: basic identical with the preparation method of SILAC mark fruit bat special culture media, different is the contrast YPD yeast thalline that coating is obtained by three of embodiment 1.
One, SILAC mark
1, material
Select for use wild type fruit bat w118 strain (hereinafter to be referred as fruit bat W118D; Melanogaster w1118; Fruit bat preservation center (Bloomington Drosophila Stock Center at Indiana University) available from U.S. seal ground Anna university makes an experiment, in the fruit bat medium of standard, 25 ℃ and the cultivation down of 70% damp condition.
2, mark
SILAC mark: 100 female fruit bat w118 and 100 male fruit bat w118 are raised together with on above-mentioned SILAC mark fruit bat special culture media lay eggs; Again with ovum subsequently culture transferring to inserting in folding 3 times fruit bat culture tube 3M filter paper and that contain above-mentioned SILAC mark fruit bat special culture media (Fig. 2); About 100 ovum of every pipe; Under 70% damp condition, cultivate in 25 ℃ of incubators, hatch into larva, via the stage of pupa; Develop into ripe fruit bat, obtain SILAC mark fruit bat.
Contrast SILAC light mark fruit bat: adopting uses the same method lays eggs paired fruit bat, cultivates and obtain contrasting the light mark fruit bat of SILAC at the light mark medium of contrast fruit bat SILAC;
Contrast YPD fruit bat: adopting uses the same method lays eggs paired fruit bat, cultivates and obtain the contrasting marking fruit bat at contrast YPD fruit bat medium.
Two, detect
1, profile is observed
SILAC mark fruit bat (heavily marking the SD medium), the contrast light mark fruit bat of SILAC (gently marking the SD medium) and contrasting marking fruit bat (YPD medium) are observed, and the result is as shown in Figure 3, and A is with SILAC mark fruit bat flow chart; B for respectively with the YPD medium, gently mark the SD medium, heavily mark the SD medium culture yeast raising fruit bat pupa shape ratio; C for respectively with the YPD medium, gently mark the SD medium, heavily mark the adult eyes of fruit bat of the yeast raising of SD medium culture; The shape ratio of health and wing; Can find out that with fruit bat behind the mark of above-mentioned medium and method cultivation, the fruit bat that shape and ordinary culture medium are cultivated is in full accord.
2, Mass Spectrometer Method
1) extraction of total protein: with the above-mentioned one SILAC mark fruit bat that obtains according to the second portion 2 of embodiment 1 1) method extract;
2) digestion: with the second portion 2 of embodiment 1 2) identical;
3) chromatograph-mass spectrometer coupling analysis: with the second portion 2 of embodiment 1 3) identical;
The peptide hydrolysis of picked at random protein disulfide bond isomerase (DNEIAIIGFFK), the result is shown in Fig. 4 A, and SILAC mark fruit bat is complete mark fruit bat, after 1 generation, with regard to the complete mark of ability, detects the peptide section less than the lysine that contains natural light isotope.The fruit bat of the complete mark of this heavy stable isotope is suitable for the quantitative proteomics research of high accuracy.
3, the mensuration of the complete mark fruit bat of SILAC labeling effciency
Take a sample zero day (ovum) and 1 phase of larva (L1), 2 phases (L2), 3 phase (L3) and adult stage respectively in the preparation process of above-mentioned SILAC mark fruit bat; Extract total protein, digestion and chromatograph-mass spectrometer coupling analysis, calculating labeling effciency (the amino acid whose ratio of the light mark of mark amino acid replacement of attaching most importance to) according to above-mentioned 2.Quantitative result to each sample carries out gently, the distributional analysis of the logarithm of recalibration value.The experiment triplicate, results averaged.
The result is shown in Fig. 4 B and table 1, and the labeling effciency that is presented at L1, L2, L3 is respectively: 80%, 95% and 96%, and basic detection the in the adult sample less than light target peptide section, system detects and finds that its labeling effciency is more than 98%.Show through a generation and culture, can realize the complete mark of fruit bat.The fruit bat of the complete mark of this heavy stable isotope is suitable for the quantitative proteomics research of high accuracy.
The mark rate of table 1 drosophila protein matter group
#Heavily mark signal/resultant signal behind labeling effciency=mark.
Claims (8)
1. a SILAC mark fruit bat special culture media prepares according to following method: the yeast thalline of SILAC mark is coated on the fruit bat medium, obtains SILAC mark fruit bat special culture media.
2. special culture media according to claim 1 is characterized in that:
Said fruit bat medium is made up of low melting-point agarose, glucose, methyl p-hydroxybenzoate, ethyl acetate and water; The mass ratio of said low melting-point agarose, said glucose, said methyl p-hydroxybenzoate, said ethyl acetate is 0.8:10:0.002:0.8.
3. special culture media according to claim 2 is characterized in that:;
In the said fruit bat medium, the concentration of said low melting-point agarose in said fruit bat medium is 0.8% (quality percentage composition); The concentration of said glucose in said fruit bat medium is 10% (quality percentage composition); The concentration of said methyl p-hydroxybenzoate in said fruit bat medium is 0.002% (quality percentage composition); The concentration of the said agar of said ethyl acetate in said fruit bat medium is 0.8% (quality percentage composition).
4. according to arbitrary described special culture media among the claim 1-3, it is characterized in that:
The yeast thalline of said SILAC mark prepares according to following method: lysine auxotroph yeast strain is cultivated in the SILAC mark yeast special culture media that contains heavy stable isotope mark lysine; Collect thalline, obtain the yeast thalline of SILAC mark.
5. special culture media according to claim 4 is characterized in that:
Said heavy stable isotope mark lysine is L-lysine-
13C
6Said best cultivation is 30 ℃ and cultivated for 9 generations, cultivates 18h altogether.
6. the application of arbitrary described special culture media in SILAC mark drosophila protein matter group among the claim 1-5.
7. the method for a SILAC mark drosophila protein matter group; Comprise the steps: that in claim 1-5 the ovum of fruit flies is to developing into adult on arbitrary described special culture media; Obtain the fruit bat of SILAC overall labeling drosophila protein matter group, realize SILAC overall labeling drosophila protein matter group.
8. method according to claim 7 is characterized in that: the ovum of said fruit bat for will paired male and female fruit bat cultivation on arbitrary described special culture media in claim 1-5, laying eggs obtains.
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