CN106047752B - A kind of SILAC culture medium and the preparation method and application thereof using arginine label bacterium - Google Patents
A kind of SILAC culture medium and the preparation method and application thereof using arginine label bacterium Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
- G01N2333/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella or Psychrobacter
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
Abstract
The present invention discloses a kind of SILAC culture medium and the preparation method and application thereof using arginine label bacterium.The culture medium includes inorganic salts, carbon source, nitrogen source, proline and weight stable isotope labeling arginine.The culture medium further include it is sweet, third, figured silk fabrics, bright, different bright, phenylpropyl alcohol, dried meat, color, silk, junket, half Guang, egg, Soviet Union, asparagus fern, paddy, rely, group etc. propylhomoserins and asparagine, glutamine.The present invention uses proline, and preventing weight stable isotope labeling conversion of Arginine is proline, to improve the identification quantity and dosing accuracy of protein；And be added to other 19 kinds of common amino acid and make the nutritional ingredient of the culture medium complete, so that the culture medium is suitable for various bacteria, bacterium speed of growth in the culture medium is fast, and signature velocity is fast, and 1~3 generation can complete to mark；And make SILAC technology for easy to operate, reproducible etc. in bacterio protein group.
Description
Technical field
The present invention relates to a kind of culture medium, in particular to it is a kind of using arginine label bacterium SILAC culture medium and its
Preparation method and application.
Background technique
Bacterium is the group that type and quantity are numerous in nature.It is closely bound up with mankind's activity, is there are many bacterium
It is pathogenic, or even can be fatal, on the contrary also there are many bacterium be it is beneficial, especially many engineering bacterias serve the mankind, at the same
Also facilitate human health there are many probiotics in the alimentary canal of human body.From after the completion of the Human Genome Project, many bacteriums are such as
Escherichia coli etc., genome are also sequenced interpretation one after another, this also just provides opportunity for the development of proteomics.Protein
Group, which is learned, to interpret biological phenomena with intracellular all protein, this is searching drug target, biomarker and understanding
Microorganism provides effective means.
It is in early days dielectrophoresis for study bacterio protein group in proteomics research method as described above
In conjunction with Mass Spectrometric Identification.However, dielectrophoresis has many limitations, if protein identification quantity is few, experimental arrangement is cumbersome, has slipped point
Son amount is larger, molecular weight is smaller and extreme isoelectric point protein, many hydrophobic proteins of also having slipped.And in bacterium,
Hydrophobic memebrane protein occupies very big specific gravity, therefore bidirectional electrophoresis technique has lost the information of many protein.It is with iTRAQ
The chemical labeling and label-free of representative can be used for quantifying for bacterio protein.Wherein iTRAQ efficiently marks sample
Peptide fragment in product, without the physicochemical property by restriction albumen itself, however iTRAQ kit is expensive, and before label respectively
Sample is handled, the experiment that peptide fragment is enriched with especially further is done to downstream, is readily incorporated random error.But it is marked with metabolism
Note is compared with label-free technology, and the peptide fragment of chemical labeling identifies negligible amounts.Requirement of the label-free to sample is low, but
It is more demanding to mass spectrum, and various dimensions pre-separation results in worse reproducibility, therefore since the repeatability of experiment is poor
Various reasons limit the extensive use in proteomics field of label-free technology.
Therefore, in contrast, metabolic marker is the method for more satisfactory Quantifying Bacteria proteomics.In the generation of bacterium
It thanks in label,15Although N label is applied in bacterium well, this also requires bacterium to must be able to be grown in only simultaneously
With inorganic15N is in the microorganism of various nitrogen-containing products such as energy itself synthesizing amino acid of nitrogen source, however many microorganisms are ammonia
Base acid or vitamin deficiency, these can not be grown in using inorganic ammonium salt as the culture of nitrogen source by also having led to bacterium
In base.Meanwhile15N label 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 more, quantitative accurate, easy to operate, and the advantages that have a wide range of application.However SILAC is in bacterioprotein group
Application it is very few, and usually require that bacterium be 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) it provides
A kind of method using lysine tag bacillus subtilis, while with this method having carried out the protein of bacillus subtilis
Group research, in the method, in order to guarantee the labeling effciency of weight stable isotope labeling lysine, the experiment of the above method
Person is by the lysine synthetase gene knockout in bacillus subtilis.Xu Ping et al. is (a kind of to mark Escherichia coli egg using SILAC
The method and its special culture media (CN201210276080.4) and paper of white matter group: 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 stable isotope 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 stable isotope labeling lysine tag
The method of saccharomycete.
By the patent and paper delivered above, it can be seen that SILAC technology is applied in the microorganisms such as bacterium at present
In the prior art, it can only realize single lysine tag, or even need to knock out the gene of lysine synthetase.However, in egg
During white matter group research, what it is for aminosal is usually pancreatin, it specifically identifies and cuts off smart in peptide fragment
The peptide bond of propylhomoserin and lysine C-terminal.Therefore using the SILAC technology of single lysine tag bacterium, to C-terminal with arginine
The polypeptide of ending can not be quantified, therefore lose the Protein quantitative analysis of about half.Also, for certain low containing lysine
Or the protein without containing lysine can not, be finally easy to cause quantitative result inaccurate.
When SILAC is applied in mammalian cell, lysine and arginic double labelling are realized.Due to following thin
The reason of bacterium itself, causes arginine label that can not realize in bacterium always.First is that since many bacteriums are amino acid autotrophy
Type biology, can synthesize amino acid required for itself.If the bacterium can synthesize arginine without using providing in environment
The arginine of weight stable isotope labeling then causes the arginic labeling effciency of weight stable isotope labeling low, and can not be real
Existing bis-amino acid label.Second is that due to many bacteriums, such as bacillus subtilis, bacillus licheniformis, brewer's yeast aspergillus nidulans
With neurospora crassa, staphylococcus aureus, arginine proline biosynthesis can be utilized.If weight stable isotope labeling essence
Propylhomoserin can mark bacterio protein, then when bacterium is using weight stable isotope labeling arginine proline biosynthesis, albumen
Proline in matter will be simultaneously or separately containing weight stable isotope13C and15N: if arginine is L-Arginine-13C6 15N4
When, then it is converted into L-Proline13C5 15N1；If arginine is L-Arginine-13C6, then it is converted into L-Proline13C5.Contain
The proline of weight stable isotope will change the quality of polypeptide, changes the mass-to-charge ratio of polypeptide, causes the polypeptide that can not be accredited,
The error of the reduction and quantification of protein that eventually lead to protein identification quantity increases.Even, since amino acid passes through tricarboxylic acids
Circulation can be used in synthesizing substance required for it is metabolized, if weight stable isotope labeling arginine or lysine pass through tricarboxylic
Acid recycles and synthesizes other substances, and the protein amounts that will lead to identification are fewer, quantitative more inaccurate.
Based on the above reason, single amino acids can only be used when resulting in SILAC in bacterium, i.e. lysine is made
For labeled amino acid.Therefore, Yao Shixian stable isotope labeling arginine is in the proteomics research of bacterium, it is necessary to
Solve the problems, such as that bacterium itself synthesizing amino acid and weight stable isotope labeling are amino acid converting at other amino acid.
Summary of the invention
The present invention it is primary the shortcomings that aiming to overcome that the prior art with it is insufficient, provide it is a kind of marked using arginine it is thin
The SILAC culture medium of bacterium.By being added to proline to the culture medium, it is suppressed that heavy chain isotope labelling conversion of Arginine is at dried meat
Propylhomoserin promotes arginine to can also be used as labeled amino acid applied in the SILAC label of bacterium.
A kind of SILAC culture medium using arginine label bacterium, including inorganic salts, carbon source, nitrogen source, further include proline
With weight stable isotope labeling arginine.
Preferably, the heavy stable isotope labeling arginine is L-argrinine-13C6 14N4Or L-argrinine
-13C6 15N4。
Preferably, it is described using arginine label bacterium SILAC culture medium further include glycine, alanine, valine,
Leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamy
Amine, threonine, aspartic acid, glutamic acid, lysine, histidine.The culture medium is restricted culture medium, nutritional ingredient phase
To limited, when bacterial growth wherein when, since, containing sufficient heavy stable isotope labeling arginine, bacterium is held in culture medium
Weight stable isotope labeling arginine is easily subjected to deamination, and then makes the weight arginic carbon skeleton of stable isotope labeling
Other amino acid are such as synthesized for substance needed for synthesizing other life processes into tricarboxylic acid cycle.Weight stable isotope mark
Remember that arginic carbon skeleton contains isotope13C can change the molecule of amino acid when the skeleton is used for and synthesizes other amino acid
Amount, cause the karyoplasmic ratio after spectrometer analysis can not with database matching and cause the reduction of protein identification amount or quantitative
Mistake.Other amino acid are added, can effectively inhibit the biosynthesis of corresponding amino acid, improve the identification quantity of protein with
And provide the accuracy of quantification of protein.Followed by many bacteriums are amino acid-deficient, or in order to need, need to knock out bacterium
Certain genes, cause the bacterium in the culture medium for lacking certain amino acid that can not grow, various common amino acid be added and exist
It can make culture medium that there is widest applicability in culture medium.
Preferably, in the SILAC culture medium using arginine label bacterium, the content of the glycine is
100~400mg/L；The content of the alanine is 100~400mg/L；The content of the valine is 100~400mg/
L；The content of the leucine is 100~400mg/L；The content of the isoleucine is 100~400mg/L；Described
The content of phenylalanine is 100~400mg/L；The content of the tryptophan is 100~400mg/L；The serine
Content is 100~400mg/L；The content of the tyrosine is 100~400mg/L；The content of the cysteine is 100
~400mg/L；The content of the methionine is 100~400mg/L；The content of the asparagine is 100~400mg/
L；The content of the glutamine is 100~400mg/L；The content of the threonine is 100~400mg/L；Described
The content of aspartic acid is 100~400mg/L；The content of the glutamic acid is 100~400mg/L；The histidine
Content is 100~400mg/L；The content of the lysine is 100~400mg/L；Inventor is by experiment repeatedly and touches
Rope, final determine can either effectively inhibit the biology of corresponding amino acid to close in above-mentioned various amino acid in the concentration range
At, and not will cause bacterium and arginine is synthesized by own metabolism approach, and lead to the weight arginic mark of stable isotope labeling
Remember that efficiency is unqualified.Meanwhile bacterium can also quickly grow in the culture medium.
It preferably, further include citric acid, thiamines in the SILAC culture medium using arginine label bacterium
Element, niacin, pantothenic acid, biotin, Tris alkali.Addition above-mentioned substance can be such that bacterium preferably grows.In nature or it is
Certain auxotrophs for needing will cause auxotroph or certain predetermined substances that many bacteriums are vitamin, increase lemon
Lemon acid, thiamine, niacin, pantothenic acid, biotin can increase the applicability of the SILAC culture medium.Tris aqueous slkali is good
Good buffer, cooperation HCl make culture medium be more suitable for the growth of a variety of different bacteriums using that can configure required pH.
Preferably, in the SILAC culture medium using arginine label bacterium, the inorganic salts are calcium chloride, chlorine
Change potassium, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate, ferrous sulfate；The carbon source is glucose；The nitrogen source is inorganic
Nitrogen source, more preferably ammonium sulfate.Inventor under the premise of taking into account various bacteria growth as far as possible, picks by testing repeatedly
It is more preferable to be conducive to bacterium growth conditions in SILAC culture medium provided by the present invention for above-mentioned various inorganic salts.
Preferably, in the SILAC culture medium using arginine label bacterium, the concentration of the proline is 200
~400mg/L.Inventor can not completely inhibit again stable same position it is discovered by experiment that when proline is lower than 200mg/L
Element label conversion of Arginine is at proline, and the proline of excessively high concentration will cause waste.
Preferably, in the SILAC culture medium using arginine label bacterium, the heavy stable isotope labeling essence
The concentration of propylhomoserin is 30~400mg/L；Preferably 100~400mg/L；More preferably 200~400mg/L.When stable same position again
When element marks arginic concentration to be lower than 30mg/L, many bacteriums meeting slow growths, such as staphylococcus aureus is resulted even in
Labeling effciency is too low and is not used to quantitative protein.Secondly prices are rather stiff for weight stable isotope labeling arginine, excessively high
Concentration will cause great waste.
Preferably, in the SILAC culture medium using arginine label bacterium, the concentration of the calcium chloride is
16.58mg/L, the concentration of the potassium chloride are 3000mg/L, and the concentration of the sodium chloride is 9500mg/L, the magnesium sulfate
Concentration is 633mg/L, and the concentration of the potassium dihydrogen phosphate is 140mg/L, the manganese sulfate H2The concentration 10mg/L of O, it is described
The concentration of citric acid is 6mg/L, and the concentration of the thiamine is 2mg/L, and the concentration of the niacin is 2mg/L, the pantothenic acid
Concentration be 2mg/L, the concentration of the biotin is 2mg/L, the ferrous sulfate 7H2The concentration of O is 6mg/L, the sulphur
The concentration of sour ammonium is 4000mg/L, and the concentration of the glucose is 5000mg/L, and the concentration of the Tris-base (Tris alkali) is
12100mg/L；The glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, junket
Propylhomoserin, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine propylhomoserin, group ammonia
Acid, proline, the weight arginic concentration of stable isotope labeling are 200mg/L.It is discovered by experiment that taking into account bacterial growth
Speed, labeling effciency, amino acid the problem of mutually converting various aspects under the premise of, it is determined that each group in SILAC culture medium
The optimal concentration divided, under the above conditions, bacterium can quickly grow；The growth that various bacteria can be taken into account simultaneously, can have
Effect prevents mutually converting for amino acid, and obtains qualification, that is, is more than or equal to 95% heavy stable isotope labeling essence ammonia
The labeling effciency of acid.
Preferably, in the SILAC culture medium using arginine label bacterium, the bacterium is Escherichia coli, gold
At least one of staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and Acinetobacter bauamnnii.In above-mentioned bacterium
Gram-positive bacterium and gramnegative bacterium are contained, and above-mentioned various bacteria is common model organism, it is above-mentioned thin
Bacterium, which can be labeled, shows that the SILAC culture medium has widest applicability.
SILAC culture medium of the present invention using arginine label bacterium, answering in label bacterio protein group
With also belonging to protection scope of the present invention.Bacterium is marked with SILAC culture medium of the present invention and then obtains bacterium
Quantitative protein group data, have easy to operate, and required time is short, and quantitative accuracy rate is high, and identification protein amounts are more, repeatability
Good advantage.
The present invention has the following advantages and effects with respect to the prior art:
In the process of research, inventors have found that the mark of the amino acid for the heavy stable isotope labeling being added in culture medium
Note efficiency can be improved with the raising of the amino acid concentration of addition；Inventor guesses that bacterium can preferentially utilize the ammonia in culture medium
Base is sour and inhibits the synthesis of its own corresponding amino acid, to make occupation rate of the weight stable isotope labeling arginine in peptide fragment
It is sufficiently high, that is, guarantee sufficiently high labeling effciency.And bacterium grows speed in this culture medium for being added to abundant amino acid
Degree is fast, while labeling effciency is also high.
For the present invention by adding proline into culture medium, preventing weight stable isotope labeling conversion of Arginine is dried meat ammonia
Acid promotes weight stable isotope labeling arginine to be applied to label bacterio protein group, therefore improves the identification number of protein
Amount and dosing accuracy；Being added to other 19 kinds of common amino acid in the medium simultaneously makes the nutritional ingredient of the culture medium neat
Entirely, so that SILAC culture medium provided by the present invention is suitable for various bacteria, bacterium speed of growth in the culture medium is fast, mark
Remember that speed is fast, 1~3 generation can complete to mark；And make SILAC technology for easy to operate, repeated in bacterio protein group
OK etc..
Detailed description of the invention
Fig. 1 is the quantification of protein correlation analysis figure of embodiment 1.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment one
1, the SILAC culture medium of different ratio is prepared
(1) culture medium A, specific ingredient and content are as shown in table 1: solvent is water.
Table 1
The heavy stable isotope labeling arginine is L-argrinine-13C6 14N4。
(2) culture medium B: the difference with culture medium A is only that culture medium B further includes each component in table 2；And it is described heavy steady
Determining isotope labelling arginine is L-argrinine-13C6 15N4, concentration 30mg/L；
Table 2
(3) culture medium C: the difference with culture medium B is only that the weight arginic concentration of stable isotope labeling is 100mg/
L。
(4) culture medium D: the difference with culture medium B is only that the weight arginic concentration of stable isotope labeling is 200mg/
L。
(5) culture medium E: the difference with culture medium B is only that the weight arginic concentration of stable isotope labeling is 300mg/
L。
(6) culture medium F: the difference with culture medium B is only that the weight arginic concentration of stable isotope labeling is 400mg/
L。
(7) culture medium G: the difference of culture medium D is only that the glycine, alanine, valine, leucine, different bright ammonia
Acid, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, day
Aspartic acid, glutamic acid, lysine, histidine concentration be 100mg/L.
(8) culture medium H: the difference of culture medium D is only that the glycine, alanine, valine, leucine, different bright ammonia
Acid, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, day
Aspartic acid, glutamic acid, lysine, histidine concentration be 400mg/L.
(9) culture medium I: the difference with culture medium D is only that without containing proline.
(10) culture medium J: the difference with culture medium D is only that concentration of proline is 100mg/L.
(11) culture medium K: the difference with culture medium D is only that concentration of proline is 400mg/L.
2, culture medium A~H marks the effect detection of bacterium.
Every kind of culture medium is respectively used to culture Escherichia coli (BW 25113, Chinese microorganism strain collection), gold
Staphylococcus aureus (ATCC 29213), bacillus subtilis (U.S. 168), pseudomonas aeruginosa (ATCC 9027), Bao Man
Acinetobacter calcoaceticus (ATCC 19606) obtains bacterium in 37 DEG C, 220rpm shaking table culture.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 bacterio protein obtains polypeptide；After polypeptide is analyzed with liquid phase tandem mass spectrometer, is scanned for, searched with MaxQuant
In " peptide.txt " file in hitch fruit, the arginic polypeptide of stable isotope labeling is weighed according to containing in search result
With the ratio for containing the non-heavy arginic polypeptide of stable isotope labeling, the i.e. ratio of heavy chain and light chain, it is calculated containing weight
The occupation rate of the arginic polypeptide of stable isotope labeling, i.e. labeling effciency.The results are shown in Table 3.The present embodiment bacterial strain uses therefor
It is obtained from American Type Culture Collecti or the purchase of Chinese microorganism strain collection.
Table 3, various bacteriums the arginic labeling effciency of heavy stable isotope labeling (%)
"-" is indicated without this data in table 3.
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, obtains bacterium, extracts bacterium
Protein, then protein is hydrolyzed with pancreatin；Gained polypeptide is analyzed with liquid phase tandem mass spectrometer；The data obtained is used
MaxQuant is scanned for, and when selection parameter, selects " Pro6 " in variable modification project.In search result, calculate
Pro6 accounts for the ratio of whole proline in " msms.txt " file；As a result as shown in table 4 below；The Pro6 is bacterium with again stable
Isotope labelling arginine (L-argrinine-13C6 15N4) it is Material synthesis with the proline (L- for weighing stable isotope
Proline13C5 15N1)。
Pro6 accounts for the ratio (%) of all proline in table 4, bacterio protein
| Pro6 occupation rate (%) | |
| Culture medium D | 2.31 |
| Culture medium I | 92.15 |
| Culture medium J | 16.74 |
| Culture medium K | 2.08 |
It can not cause with database matching, or formation mispairing since the presence of Pro6 will lead to B, Y-ion when searching library
The reduction of protein identification quantity and cause quantitative inaccuracy.As shown above, the present invention is being added to dried meat ammonia in the medium
After acid, staphylococcus aureus can effectively be inhibited to utilize arginine proline biosynthesis.When the concentration of proline in culture medium reaches
When to 200mg/L, i.e. the content of culture medium D, Pro6 can be down to 2.31%, be approximately equal to background level.In this way, being conducive to improve quasi-
The accuracy of the protein amounts and raising quantification of protein information really identified, while also having promoted SILAC to be used for can in bacterium
It uses arginine as labeled amino acid using realization, is applied to bacterium for double labelling SILAC technology and provides guarantee.
4, quantitative protein group repeatability detects.
Take prepared SILAC culture medium D in 37 DEG C, 220rpm shaking table culture staphylococcus aureus ATCC29213, and
And addition Ciprofloxacin (CPFX) to concentration is 0.6 μ g/L.Meanwhile corresponding light chain culture medium (i.e. with corresponding culture medium,
Change the isotope amino acid in culture medium into nonisotopic labels amino acid) the culture work of staphylococcus aureus ATCC 29213
For control.Will by stable isotope labeling and non-marked bacterium mixed in equal amounts carry out quantitative protein group analysis, test into
Row biology is repeated twice, and is carried out correlation analysis to the changing value twice of same protein, is acquired Pearson correlation coefficient r=
0.9578, specific method is referring to (Native SILAC:metabolic labeling of proteins in prototroph
microorganisms based on lysine synthesis regulation.Molecular&cellular
Proteomics:MCP 12,1995-2005), experiment reproducible results consistency is good, as a result as shown in Figure 1.Should the result shows that,
When SILAC technology is used for quantitative staphylococcus aureus differential protein group, there is excellent stability and high repeatability.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (8)
1. a kind of SILAC culture medium using arginine label bacterium, including inorganic salts, carbon source, nitrogen source, it is characterised in that: institute
State nitrogen source include proline, glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine,
Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, histidine
With weight stable isotope labeling arginine；
The arginic concentration of heavy stable isotope labeling is 200~400mg/L.
2. the SILAC culture medium according to claim 1 using arginine label bacterium, it is characterised in that:
The content of the glycine is 100~400mg/L；
The content of the alanine is 100~400mg/L；
The content of the valine is 100~400mg/L；
The content of the leucine is 100~400mg/L；
The content of the isoleucine is 100~400mg/L；
The content of the phenylalanine is 100~400mg/L；
The content of the tryptophan is 100~400mg/L；
The content of the serine is 100~400mg/L；
The content of the tyrosine is 100~400mg/L；
The content of the cysteine is 100~400mg/L；
The content of the methionine is 100~400mg/L；
The content of the asparagine is 100~400mg/L；
The content of the glutamine is 100~400mg/L；
The content of the threonine is 100~400mg/L；
The content of the aspartic acid is 100~400mg/L；
The content of the glutamic acid is 100~400mg/L；
The content of the histidine is 100~400mg/L；
The content of the lysine is 100~400mg/L.
3. the SILAC culture medium according to claim 1 using arginine label bacterium, it is characterised in that: further include lemon
Lemon acid, thiamine, niacin, pantothenic acid, biotin, Tris alkali.
4. the SILAC culture medium according to claim 3 using arginine label bacterium, it is characterised in that: described inorganic
Salt is calcium chloride, potassium chloride, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, manganese sulfate, ferrous sulfate；The carbon source is glucose；
The nitrogen source further includes ammonium sulfate.
5. the SILAC culture medium according to claim 1 using arginine label bacterium, it is characterised in that: the dried meat ammonia
The concentration of acid is 200~400mg/L.
6. the SILAC culture medium according to claim 4 using arginine label bacterium, it is characterised in that: the chlorination
The concentration of calcium is 16.58mg/L, and the concentration of the potassium chloride is 3000mg/L, and the concentration of the sodium chloride is 9500mg/L, institute
The concentration for stating magnesium sulfate is 633mg/L, and the concentration of the potassium dihydrogen phosphate is 140mg/L, the manganese sulfate H2The concentration of O
10mg/L, the concentration of the citric acid are 6mg/L, and the concentration of the thiamine is 2mg/L, and the concentration of the niacin is 2mg/
L, the concentration of the pantothenic acid are 2mg/L, and the concentration of the biotin is 2mg/L, the ferrous sulfate 7H2The concentration of O is
6mg/L, the concentration of the ammonium sulfate are 4000mg/L, and the concentration of the glucose is 5000mg/L, the concentration of the Tris alkali
For 12100mg/L；The glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine,
Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine propylhomoserin, group
Propylhomoserin, proline, the weight arginic concentration of stable isotope labeling are 200mg/L.
7. any one SILAC culture medium using arginine label bacterium according to claim 1~6, it is characterised in that: institute
The bacterium stated is in Escherichia coli, staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and Acinetobacter bauamnnii
At least one.
8. using the SILAC culture medium of arginine label bacterium in label bacterioprotein described in claim 1~7 any one
Application in matter group.
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| 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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| 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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| Christopher Löβner等.Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions.《Analytical Biochemistry》.2011,第412卷123–125. * |
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