CN106047752A - SILAC (stable isotope labeling by amino acids in cell culture) culture medium capable of labeling bacteria by using arginine as well as preparation method and application of culture medium - Google Patents
SILAC (stable isotope labeling by amino acids in cell culture) culture medium capable of labeling bacteria by using arginine as well as preparation method and application of culture medium Download PDFInfo
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Abstract
The invention discloses a SILAC (stable isotope labeling by amino acids in cell culture) culture medium capable of labeling bacteria by using arginine as well as a preparation method and an application of the culture medium. The culture medium comprises inorganic salt, a carbon source, a nitrogen source, proline and heavy and stable isotope labeled arginine and further comprises glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, threonine, aspartic acid, glutamic acid, lysine, histidine and other amino acids as well as asparagine and glutamine. Proline is used, and heavy and stable isotope labeled arginine is prevented from being transformed into proline, so that the identification number and quantitative accuracy of protein are improved; another 19 common amino acids are added, the culture medium is complete in nutritional ingredient, thus, the culture medium is applicable to multiple bacteria, the bacteria grow rapidly in the culture medium, the labeling speed is high, and labeling can be completed in 1-3 generations; besides, the SILAC technology is applied to bacterial proteome and is simple to operate, good in repeatability and the like.
Description
Technical field
The present invention relates to a kind of culture medium, particularly to a kind of SILAC culture medium utilizing arginine labelling antibacterial and
Preparation method and application.
Background technology
Antibacterial is kind and large number of colony in nature.It is closely bound up with mankind's activity, has many antibacterials to be
Cause a disease, even can be fatal, it is useful for the most also having many antibacterials, and particularly many engineering bacterias serve the mankind, exist simultaneously
The digestive tract of human body also there are many probiotic bacterias contribute to human health.After the Human Genome Project completes, many antibacterials are such as
Escherichia coli etc., its genome is sequenced deciphering the most one after another, this most just development for proteomics provide opportunity.Protein
Group can understand biosis with an intracellular all protein, and this is for finding drug target, biomarker and understanding
Microorganism provides effective means.
In proteomics research method as above, early stage is two-dimensional electrophoresis for study bacterioprotein group
In conjunction with Mass Spectrometric Identification.But, two-dimensional electrophoresis has many limitations, as few in identification of proteins quantity, and experimental arrangement is loaded down with trivial details, has slipped point
Son measures relatively big, molecular weight and extreme isoelectric point, IP protein, many hydrophobic proteins of also having slipped.And in antibacterial,
Hydrophobic memebrane protein occupies the biggest proportion, and therefore bidirectional electrophoresis technique have lost the information of a lot of protein.With iTRAQ it is
The chemical labeling and the label-free that represent can be used for the quantitative of bacterioprotein.Wherein iTRAQ efficient labelling sample
Peptide fragment in product, and not by the physicochemical property of restriction albumen itself, but iTRAQ test kit is expensive, and before labelling respectively
Process sample, particularly downstream is done further the experiment that peptide fragment is enriched with, is readily incorporated random error.But with metabolism mark
Note is compared with label-free technology, and the peptide fragment of chemical labeling identifies negligible amounts.Label-free is low to the requirement of sample, but
It is higher to mass spectrum requirement, and various dimensions pre-separation result in worse repeatability, therefore poor due to the repeatability of experiment
Various reasons limits the extensive application at proteomics field of label-free technology.
Therefore, comparatively speaking, metabolic marker is the method for more satisfactory Quantifying Bacteria proteomics.Generation on antibacterial
Thank in labelling,15Although N labelling is the most well applied in antibacterial, but this requires that antibacterial must be able to be grown in only the most simultaneously
With inorganic15N is in the microorganism of the various nitrogen-containing products such as energy self synthesizing amino acid in nitrogen source, but the microorganism of many is ammonia
Base acid or vitamin deficiency, also have led to antibacterial and cannot be grown in these cultivations being nitrogen source with inorganic ammonium salt
In base.Meanwhile,15N labelling proposes powerful challenge to search engine.Another metabolic labeling approaches-SILAC (Stable
Isotope labeling by amino acid in cell culture, cytotostatic isotope labelling techniques) there is albumen
Matter identifies that quantity is many, the most accurately, easily operates, and the advantage such as applied range.But SILAC is in bacterioprotein group
Application very the fewest, and usually require that antibacterial is auxotrophy strain.Soufi, B. et al. (Stable isotope
labeling by amino acids in cell culture(SILAC)applied to quantitative
Proteomics of Bacillus subtilis.Journal of proteome research 9,3638-3646) provide
A kind of method utilizing lysine tag bacillus subtilis, has carried out the protein of bacillus subtilis by the method simultaneously
Group research, in the method, in order to ensure the labeling effciency of weight cold labeling lysine, the experiment of said method
Person is by the lysine synthetase gene knockout in bacillus subtilis.(one utilizes SILAC labelling escherichia coli egg to Xu Ping et al.
The method of white matter group and special culture media (CN201210276080.4) thereof and paper: Quantitative proteomics
reveals significant changes in cell shape and an energy shift after IPTG
induction via an optimized SILAC approach for Escherichia coli.Journal of
Proteome research 12,5978-5988) provide a kind of utilization weight cold labeling lysine tag large intestine bar
The SILAC culture medium of bacterium.Frohlich F et al. (Native SILAC:metabolic labeling of proteins in
prototroph microorganisms based on lysine synthesis regulation.Molecular&
Cellular proteomics:MCP 12,1995-2005) provide a kind of utilization weight cold labeling lysine tag
Saccharomycetic method.
By the patent delivered above and paper, it can be seen that SILAC technology is applied in the microorganisms such as antibacterial at present
In prior art, all can only realize single lysine tag, even need to knock out the gene of lysine synthetase.But, at egg
During white matter group research, for the usually pancreatin of aminosal, it specifically identifies and cuts off essence in peptide fragment
Propylhomoserin and the peptide bond of lysine C-terminal.Therefore the SILAC technology of single lysine tag antibacterial is used, to C-terminal with arginine
The polypeptide of ending cannot be carried out quantitatively, therefore losing the Protein quantitative analysis of about half.Further, low containing lysine for some
Or do not contain lysine protein cannot, be finally easily caused quantitative result inaccurate.
When SILAC is applied in mammalian cell, all achieve lysine and arginic double labelling.Due to following thin
The reason of bacterium self, causes arginine labelling cannot realize in antibacterial always.One is owing to many antibacterials are aminoacid autotrophy
Type is biological, it is possible to synthesize self required aminoacid.If this antibacterial can synthesize arginine and not utilize offer in environment
The arginine of weight cold labeling, then the arginic labeling effciency causing weight cold labeling is low, and cannot be real
Existing bis-amino acid labelling.Two is due to many antibacterials, such as bacillus subtilis, Bacillus licheniformis, beer yeast aspergillus nidulans
With neurospora crassa, staphylococcus aureus, arginine proline biosynthesis can be utilized.If weight cold labeling essence
Propylhomoserin can labelling bacterioprotein, then when antibacterial utilizes weight cold labeling arginine proline biosynthesis, albumen
Proline in matter will contain weight stable isotope at the same time or separately13C and15N: if arginine is L-Arginine-13C6 15N4
Time, then it is converted into L-Proline13C5 15N1；If arginine is L-Arginine-13C6, then L-Proline it is converted into13C5.Contain
The proline of weight stable isotope will change the quality of polypeptide, changes the mass-to-charge ratio of polypeptide, causes this polypeptide identified to arrive,
The error of the minimizing and quantification of protein that ultimately result in identification of proteins quantity increases.Even, tricarboxylic acids is passed through due to aminoacid
Circulation can be used in synthesizing the material required for its metabolism, if weight cold labeling arginine or lysine pass through tricarboxylic
Acid circulates and synthesizes other materials, and by causing, the protein amounts identified is fewer, the most inaccurate.
Based on the above reason, result in SILAC in antibacterial time can only use single amino acids, i.e. lysine is made
For labeled amino acid.Therefore, cold labeling arginine to be realized is in the proteomics research of antibacterial, it is necessary to
Solve antibacterial self synthesizing amino acid and weight other amino acid whose problems of the amino acid converting one-tenth of cold labeling.
Summary of the invention
What the present invention was primary aims to overcome that the shortcoming of prior art is with not enough, it is provided that one utilizes arginine labelling thin
The SILAC culture medium of bacterium.By with the addition of proline to this culture medium, it is suppressed that heavy chain isotope labelling conversion of Arginine becomes dried meat
Propylhomoserin, promotes arginine can also be applied in the SILAC labelling of antibacterial as labeled amino acid.
A kind of SILAC culture medium utilizing arginine labelling antibacterial, including inorganic salt, carbon source, nitrogen source, also includes proline
With weight cold labeling arginine.
Preferably, described heavy cold labeling arginine is L-argrinine-13C6 14N4Or L-argrinine
-13C6 15N4。
Preferably, the described SILAC culture medium utilizing arginine labelling antibacterial also include glycine, alanine, valine,
Leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, agedoite, glutamy
Amine, threonine, aspartic acid, glutamic acid, lysine, histidine.Described culture medium is restricted culture medium, nutritional labeling phase
To limited, when bacterial growth wherein time, due in culture medium containing sufficient heavy cold labeling arginine, antibacterial appearance
Easily weight cold labeling arginine is carried out deamination, and then makes the weight arginic carbon skeleton of cold labeling
Enter tricarboxylic acid cycle, for synthesizing the material needed for other life processes, as synthesized other aminoacid.Weight stable isotope mark
Remember that arginic carbon skeleton contains isotope13C, can change amino acid whose molecule when this skeleton is used for synthesizing other aminoacid time
Amount, cause the karyoplasmic ratio after spectrometer analysis cannot with database matching and cause identification of proteins amount reduce or quantitative
Mistake.Add other aminoacid, can effectively suppress corresponding amino acid whose biosynthesis, improve the qualification quantity of protein with
And the accuracy of quantification of protein is provided.Next to that many antibacterials are amino acid-deficient, or for needs, need to knock out antibacterial
Some gene, cause antibacterial in lacking certain amino acid whose culture medium to grow, add various common aminoacid and exist
Culture medium can make culture medium have the widest suitability.
Preferably, in the described SILAC culture medium utilizing arginine labelling antibacterial, the content of described glycine is
100～400mg/L；The content of described alanine is 100～400mg/L；The content of described valine is 100～400mg/
L；Described leucic content is 100～400mg/L；The content of described isoleucine is 100～400mg/L；Described
The content of phenylalanine is 100～400mg/L；The content of described tryptophan is 100～400mg/L；Described serine
Content is 100～400mg/L；The content of described tyrosine is 100～400mg/L；The content of described cysteine is 100
～400mg/L；The content of described methionine is 100～400mg/L；The content of described agedoite is 100～400mg/
L；The content of described glutamine is 100～400mg/L；The content of described threonine is 100～400mg/L；Described
The content of aspartic acid is 100～400mg/L；The content of described glutamic acid is 100～400mg/L；Described histidine
Content is 100～400mg/L；The content of described lysine is 100～400mg/L；Inventor is through experiment repeatedly and touches
Rope, finally determines and can either effectively suppress corresponding amino acid whose biological conjunction in this concentration range at above-mentioned various aminoacid
Become, do not result in again antibacterial and synthesize arginine by own metabolism approach, and cause the weight arginic mark of cold labeling
Note efficiency is defective.Meanwhile, antibacterial quickly can also grow in this culture medium.
As preferably, in the described SILAC culture medium utilizing arginine labelling antibacterial, also include citric acid, thiamine
Element, nicotinic acid, pantothenic acid, biotin, Tris alkali.Adding above-mentioned substance can make antibacterial preferably grow.In nature or be
Auxotroph or the auxotroph of some predetermined substance that some needs can cause many antibacterials to be vitamin, increases lemon
Lemon acid, thiamine, nicotinic acid, pantothenic acid, biotin can increase the suitability of described SILAC culture medium.Tris aqueous slkali is good
Good buffer, coordinates HCl to use and can configure required pH, make culture medium be more suitable for the growth of multiple different bacterium.
Preferably, in the described SILAC culture medium utilizing arginine labelling antibacterial, described inorganic salt is calcium chloride, chlorine
Change potassium, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate, ferrous sulfate；Described carbon source is glucose；Described nitrogen source is inorganic
Nitrogen source, more preferably ammonium sulfate.Inventor, through repeatedly testing, on the premise of taking into account various bacteria growth, picks as far as possible
Above-mentioned various inorganic salt, beneficially antibacterial growth conditions in SILAC culture medium provided by the present invention is more preferable.
Preferably, in the described SILAC culture medium utilizing arginine labelling antibacterial, the concentration of described proline is 200
～400mg/L.Inventor finds through experiment, when proline is less than 200mg/L, can not suppress heavily to stablize coordination completely
Element labelling conversion of Arginine becomes proline, and the proline of too high concentration can cause waste.
Preferably, in the described SILAC culture medium utilizing arginine labelling antibacterial, described heavy cold labeling essence
The concentration of propylhomoserin is 30～400mg/L；It is preferably 100～400mg/L；More preferably 200～400mg/L.When heavily stablizing coordination
When the element arginic concentration of labelling is less than 30mg/L, many antibacterials meeting poor growths, such as staphylococcus aureus, result even in
Labeling effciency is too low and is not used to quantitative protein.Secondly prices are rather stiff for weight cold labeling arginine, too high
Concentration can cause greatly waste.
Preferably, in the described SILAC culture medium utilizing arginine labelling antibacterial, the concentration of described calcium chloride is
16.58mg/L, the concentration of described potassium chloride is 3000mg/L, and the concentration of described sodium chloride is 9500mg/L, described magnesium sulfate
Concentration is 633mg/L, and the concentration of described potassium dihydrogen phosphate is 140mg/L, described manganese sulfate H2Concentration 10mg/L of O, described
The concentration of citric acid is 6mg/L, and the concentration of described thiamine is 2mg/L, and the concentration of described nicotinic acid is 2mg/L, described pantothenic acid
Concentration be 2mg/L, the concentration of described biotin is 2mg/L, described ferrous sulfate 7H2The concentration of O is 6mg/L, described sulfur
The concentration of acid ammonium is 4000mg/L, and the concentration of described glucose is 5000mg/L, and the concentration of described Tris-base (Tris alkali) is
12100mg/L；Described glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, cheese
Propylhomoserin, cysteine, methionine, agedoite, glutamine, threonine, aspartic acid, glutamic acid, lysine propylhomoserin, group ammonia
Acid, proline, the weight arginic concentration of cold labeling are 200mg/L.Find through experiment, taking into account bacterial growth
Speed, labeling effciency, on the premise of the problem of amino acid whose mutual conversion each side, it is determined that each group in SILAC culture medium
The optimal concentration divided, under these conditions, antibacterial can quickly grow；The growth of various bacteria can be taken into account simultaneously, can have
Effect prevent amino acid whose mutual conversion, and obtain qualified, i.e. the heavy cold labeling essence ammonia more than or equal to 95%
The labeling effciency of acid.
Preferably, in the described SILAC culture medium utilizing arginine labelling antibacterial, described antibacterial is escherichia coli, gold
At least one in Staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and Acinetobacter bauamnnii.In above-mentioned antibacterial
Contain gram-positive bacterium and gram negative bacteria, and above-mentioned various bacteria be common model organism, above-mentioned carefully
Bacterium can be labeled and show that described SILAC culture medium has the widest suitability.
The SILAC culture medium utilizing arginine labelling antibacterial of the present invention, answering in labelling bacterioprotein group
With falling within protection scope of the present invention.It is marked antibacterial by SILAC culture medium of the present invention and then obtains antibacterial
Quantitative protein group data, have simple to operate, and required time is short, and quantitative accuracy rate is high, identify that protein amounts is many, repeatability
Good advantage.
The present invention has such advantages as relative to prior art and effect:
In the process of research, inventor finds, adds the amino acid whose mark of heavy cold labeling in culture medium to
Note efficiency can improve along with the rising of the amino acid concentration added；Inventor guesses that antibacterial can preferentially utilize the ammonia in culture medium
Base is sour and suppresses himself corresponding amino acid whose synthesis, so that weight cold labeling arginine occupation rate in peptide fragment
Sufficiently high, i.e. ensure sufficiently high labeling effciency.And antibacterial with the addition of growth speed in abundant amino acid whose culture medium this
Degree is fast, and labeling effciency is the highest simultaneously.
The present invention is by adding proline in culture medium, and preventing weight cold labeling conversion of Arginine is dried meat ammonia
Acid, promotes weight cold labeling arginine to be applied to labelling bacterioprotein group, therefore improves the qualification number of protein
Amount and dosing accuracy；With the addition of other 19 kinds of common aminoacid in the medium makes the nutritional labeling of this culture medium neat simultaneously
Entirely so that SILAC culture medium provided by the present invention is applicable to various bacteria, and antibacterial is fast growth in this culture medium, mark
Note speed is fast, and 1～3 generations can complete labelling；And make SILAC technology simple to operate in bacterioprotein group, repeated
OK etc..
Accompanying drawing explanation
Fig. 1 is the quantification of protein correlation analysis figure of embodiment 1.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.
Embodiment one
1, the SILAC culture medium of different ratio is prepared
(1) culture medium A, concrete composition and content are as shown in table 1: solvent is water.
Table 1
Described heavy cold labeling arginine is L-argrinine-13C6 14N4。
(2) culture medium B: with culture medium A differ only in each component that culture medium B also includes in table 2；And described heavy surely
Determining isotope labelling arginine is L-argrinine-13C6 15N4, concentration is 30mg/L；
Table 2
(3) culture medium C: with culture medium B differ only in weight the arginic concentration of cold labeling be 100mg/
L。
(4) culture medium D: with culture medium B differ only in weight the arginic concentration of cold labeling be 200mg/
L。
(5) culture medium E: with culture medium B differ only in weight the arginic concentration of cold labeling be 300mg/
L。
(6) culture medium F: with culture medium B differ only in weight the arginic concentration of cold labeling be 400mg/
L。
(7) culture medium G: culture medium D differ only in described glycine, alanine, valine, leucine, different bright ammonia
Acid, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, agedoite, glutamine, threonine, sky
Winter propylhomoserin, glutamic acid, lysine, the concentration of histidine are 100mg/L.
(8) culture medium H: culture medium D differ only in described glycine, alanine, valine, leucine, different bright ammonia
Acid, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, agedoite, glutamine, threonine, sky
Winter propylhomoserin, glutamic acid, lysine, the concentration of histidine are 400mg/L.
(9) culture medium I: do not contain proline with differing only in of culture medium D.
(10) culture medium J: be 100mg/L with the concentration of proline that differs only in of culture medium D.
(11) culture medium K: be 400mg/L with the concentration of proline that differs only in of culture medium D.
2, the effect detection of the labelling antibacterial of culture medium A～H.
It is respectively used to every kind of culture medium cultivate escherichia coli (BW 25113, Chinese microorganism strain preservation center), gold
Staphylococcus aureus (ATCC 29213), bacillus subtilis (U.S. 168), pseudomonas aeruginosa (ATCC 9027), Bao Man
Acinetobacter calcoaceticus (ATCC 19606), in 37 DEG C, 220rpm shaking table cultivate obtain antibacterial.By document " Soufi, B., Kumar, C.,
Gnad,F.,Mann,M.,Mijakovic,I.,and Macek,B.(2010)Stable isotope labeling by
amino acids in cell culture(SILAC)applied toquantitative proteomics of
Bacillus subtilis.Journal of proteome research 9,3638-3646 " operation, extract and use pancreas
Enzyme hydrolysis bacterioprotein obtains polypeptide；After polypeptide is analyzed with liquid phase tandem mass spectrometer, scan for MaxQuant, searching
In " peptide.txt " file in hitch fruit, according to Search Results contains the weight arginic polypeptide of cold labeling
With the ratio of the ratio containing the non-arginic polypeptide of heavy cold labeling, i.e. heavy chain Yu light chain, calculate containing weight
The occupation rate of the arginic polypeptide of cold labeling, i.e. labeling effciency.Result is as shown in table 3.The present embodiment bacterial strain uses therefor
Buy from American Type Culture Collecti or Chinese microorganism strain preservation center and obtain.
Table 3, the arginic labeling effciency of the heavy cold labeling (%) of various antibacterial
In table 3, "-" represents does not has this data.
3, the effect detection of culture medium D, I, J, K.
Staphylococcus aureus (ATCC 29213) is incubated at culture medium D, I, J, K respectively, it is thus achieved that antibacterial, extracts antibacterial
Protein, then with pancreatin, protein is hydrolyzed；Gained polypeptide liquid phase tandem mass spectrometer is analyzed；The data obtained is used
MaxQuant scans for, and during Selection parameter, selects " Pro6 " in variable modification project.In Search Results, calculate
In " msms.txt " file, Pro6 accounts for the ratio of whole proline；Result is as shown in table 4 below；Described Pro6 is that antibacterial is with the most stable
Isotope labelling arginine (L-argrinine-13C6 15N4) be Material synthesis with weight stable isotope proline (L-
Proline13C5 15N1)。
In table 4, bacterioprotein, Pro6 accounts for the ratio (%) of all proline
| Pro6 occupation rate (%) | |
| Culture medium D | 2.31 |
| Culture medium I | 92.15 |
| Culture medium J | 16.74 |
| Culture medium K | 2.08 |
Owing to the existence of Pro6 can cause B, Y-ion when searching storehouse with database matching, or cannot form mispairing, cause
The minimizing of identification of proteins quantity and cause the most inaccurate.As shown above, the present invention with the addition of dried meat ammonia in the medium
After acid, staphylococcus aureus can be effectively suppressed to utilize arginine proline biosynthesis.When the concentration of proline in culture medium reaches
During to 200mg/L, i.e. culture medium D, the content of Pro6 can be down to 2.31%, be approximated background level.So, be conducive to improving standard
The protein amounts really identified and the accuracy improving quantification of protein information, also promoted the SILAC can in antibacterial simultaneously
Using realization arginine as labeled amino acid, it is applied to antibacterial for double labelling SILAC technology and provides guarantee.
4, quantitative protein group repeatability detection.
Take prepared SILAC culture medium D in 37 DEG C, 220rpm shaking table cultivate staphylococcus aureus ATCC29213, and
And addition ciprofloxacin (CPFX) is 0.6 μ g/L to concentration.Meanwhile, corresponding light chain culture medium (i.e. use corresponding culture medium,
Change the isotope aminoacid in culture medium into nonisotopic labels aminoacid) cultivate staphylococcus aureus ATCC 29213 make
For comparison.By by cold labeling and cold antibacterial mixed in equal amounts carry out quantitative protein group analysis, test into
Row is repeated twice biology, twice changing value of same protein is carried out correlation analysis, tries to achieve Pearson's correlation coefficient r=
0.9578, concrete grammar sees (Native SILAC:metabolic labeling of proteins in prototroph
microorganisms based on lysine synthesis regulation.Molecular&cellular
Proteomics:MCP 12,1995-2005), experiment reproducible results concordance is good, and result is as shown in Figure 1.This result shows,
SILAC technology, when quantitative staphylococcus aureus differential protein group, has excellent stability and high repeatability.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (10)
1. utilize a SILAC culture medium for arginine labelling antibacterial, including inorganic salt, carbon source, nitrogen source, it is characterised in that: also
Including proline and weight cold labeling arginine.
The SILAC culture medium utilizing arginine labelling antibacterial the most according to claim 1, it is characterised in that: also include sweet
Propylhomoserin, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, egg
Propylhomoserin, agedoite, glutamine, threonine, aspartic acid, glutamic acid, lysine, histidine.
The SILAC culture medium utilizing arginine labelling antibacterial the most according to claim 2, it is characterised in that:
The content of described glycine is 100～400mg/L；
The content of described alanine is 100～400mg/L；
The content of described valine is 100～400mg/L；
Described leucic content is 100～400mg/L；
The content of described isoleucine is 100～400mg/L；
The content of described phenylalanine is 100～400mg/L；
The content of described tryptophan is 100～400mg/L；
The content of described serine is 100～400mg/L；
The content of described tyrosine is 100～400mg/L；
The content of described cysteine is 100～400mg/L；
The content of described methionine is 100～400mg/L；
The content of described agedoite is 100～400mg/L；
The content of described glutamine is 100～400mg/L；
The content of described threonine is 100～400mg/L；
The content of described aspartic acid is 100～400mg/L；
The content of described glutamic acid is 100～400mg/L；
The content of described histidine is 100～400mg/L；
The content of described lysine is 100～400mg/L.
The SILAC culture medium utilizing arginine labelling antibacterial the most according to claim 2, it is characterised in that: also include lemon
Lemon acid, thiamine, nicotinic acid, pantothenic acid, biotin, Tris alkali.
The SILAC culture medium utilizing arginine labelling antibacterial the most according to claim 4, it is characterised in that: described inorganic
Salt is calcium chloride, potassium chloride, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate, ferrous sulfate；Described carbon source is glucose；
Described nitrogen source is ammonium sulfate.
The SILAC culture medium utilizing arginine labelling antibacterial the most according to claim 1, it is characterised in that: described dried meat ammonia
The concentration of acid is 200～400mg/L.
The SILAC culture medium utilizing arginine labelling antibacterial the most according to claim 1, it is characterised in that: described heavy surely
Determining the arginic concentration of isotope labelling is 30～400mg/L.
The SILAC culture medium utilizing arginine labelling antibacterial the most according to claim 5, it is characterised in that: described chlorination
The concentration of calcium is 16.58mg/L, and the concentration of described potassium chloride is 3000mg/L, and the concentration of described sodium chloride is 9500mg/L, institute
The concentration stating magnesium sulfate is 633mg/L, and the concentration of described potassium dihydrogen phosphate is 140mg/L, described manganese sulfate H2The concentration of O
10mg/L, the concentration of described citric acid is 6mg/L, and the concentration of described thiamine is 2mg/L, and the concentration of described nicotinic acid is 2mg/
L, the concentration of described pantothenic acid is 2mg/L, and the concentration of described biotin is 2mg/L, described ferrous sulfate 7H2The concentration of O is
6mg/L, the concentration of described ammonium sulfate is 4000mg/L, and the concentration of described glucose is 5000mg/L, and described Tris-base's is dense
Degree is 12100mg/L；Described glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, silk ammonia
Acid, tyrosine, cysteine, methionine, agedoite, glutamine, threonine, aspartic acid, glutamic acid, lysine ammonia
Acid, histidine, proline, the weight arginic concentration of cold labeling are 200mg/L.
9. according to any one of claim 1～8, utilize the SILAC culture medium of arginine labelling antibacterial, it is characterised in that: institute
The antibacterial stated is in escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and Acinetobacter bauamnnii
At least one.
10. utilize the SILAC culture medium of arginine labelling antibacterial at labelling antibacterial egg described in claim 1～8 any one
Application in white matter group.
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| CN101932698A (en) * | 2007-12-20 | 2010-12-29 | 诺华有限公司 | Fermentation processes for cultivating streptococci and purification processes for obtaining cps therefrom |
| CN102243218A (en) * | 2011-05-13 | 2011-11-16 | 复旦大学 | In vivo termini amino acid labeled quantitative proteomics method |
| CN102796682A (en) * | 2012-08-03 | 2012-11-28 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for labeling escherichia coli proteome by using SILAC (Stable Isotope Labeling with Amino Acids in Cell Cultures) and special culture medium |
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| CN101932698A (en) * | 2007-12-20 | 2010-12-29 | 诺华有限公司 | Fermentation processes for cultivating streptococci and purification processes for obtaining cps therefrom |
| CN102243218A (en) * | 2011-05-13 | 2011-11-16 | 复旦大学 | In vivo termini amino acid labeled quantitative proteomics method |
| CN102796682A (en) * | 2012-08-03 | 2012-11-28 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for labeling escherichia coli proteome by using SILAC (Stable Isotope Labeling with Amino Acids in Cell Cultures) and special culture medium |
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| CN110004074A (en) * | 2019-02-28 | 2019-07-12 | 北京蛋白质组研究中心 | A kind of method and its special culture media using SILAC label mycobacterium smegmatis protein |
| CN110004074B (en) * | 2019-02-28 | 2021-06-18 | 北京蛋白质组研究中心 | Method for marking mycobacterium smegmatis protein by SILAC and special culture medium thereof |
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