CN107064338A - A kind of method based on iTRAQ marker determination nicotine inducing cell differential expression proteins - Google Patents
A kind of method based on iTRAQ marker determination nicotine inducing cell differential expression proteins Download PDFInfo
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- G—PHYSICS
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- 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
Abstract
There is provided a kind of analysis method of nicotine exposure to nerves Cell differentials protein group for a kind of method based on iTRAQ marker determination nicotine inducing cell differential expression proteins by the present invention.Specifically, nerve cell is after the medium culture containing nicotine, the extracting through protein, the measure of protein concentration, sample quality and collimation, enzymolysis, after iTRAQ marks are analyzed and identified with nano LC Q TOF, and storehouse analysis is searched in progress.The method quantitative analysis 1mmol/L nicotine of the present invention exposes the differential expression of 1h SHSY 5Y protein, wherein participate in the proteasome protein of MAPK cascade bioprocess and proteasome signal path, it may be played a significant role in nicotine addiction, so as to reach the purpose for the bioprocess that the differential expression protein and differential protein that understand nicotine inducing nerve cell are participated in.
Description
Technical field
The present invention relates to technical field of biological, more particularly it relates to poor in nicotine exposure to nerves cell
The assay method of different marking protein.
Background technology
Nicotine is the habitual main component smoked of reward, is to cause the main cause of smoking addiction.During smoking, nicotine with
Flue gas direct oral cavity is reached after lung, by the capillary absorbance in alveolar, is moved by pulmonary venous circulatory through heart into body circulation
Vein system is united, and enters brain within about 10-20 seconds after smoking, is combined with nAChR (nAChR), leads to the ion of acceptor
Road is opened, and calcium ion and potassium ion etc. enter neuron, is induced a series of change of bioprocess and is caused habituation.
Protein is the agent of vital movement, and research shows that contacting dependence producing drug repeatedly includes nicotine, morphine, hemp etc.
The change of brain specific region protein expression level can be caused, the change of this expression is not only involved in single neuron and phase
Close the regulation of neural circuit, it is also possible to which influence is produced on the related abnormal behaviour of habituation.Therefore, identification participates in forming and maintaining medicine
The albumen that thing is relied on is the key for understanding the potential molecular mechanism of drug habit.
Assay method report at present on protein expression is a lot.Proteomic techniques being capable of dynamic monitoring protein
Change, be the main method for identifying the expression of nicotine inducible protein.
Relative and absolute quantitation isotope marks (iTRAQ) are a kind of external same positions of the same race researched and developed by AB SCIEX companies
The relative and absolute quantitation technology of element mark.The technology utilizes a variety of isotope reagent labelled protein polypeptide N-terminals or lysine
Side-chain radical, through high-precision mass spectrograph Tandem analysis, the expressing quantity of several samples can be compared simultaneously, be to quantify egg in recent years
The conventional High Throughput Screening Assay of white matter group.But, there is not yet the technology to be used for the differential protein of nicotine function cells
The report of matter group research.
The content of the invention
For above-mentioned technical problem, the purpose of the present invention is to overcome the deficiencies in the prior art there is provided a kind of flux height, repeat
The Study on Different Proteomics method of the good nicotine function cells of property.
The purpose of the present invention is achieved through the following technical solutions.
The present invention provides a kind of method based on iTRAQ marker determination nicotine inducing cell differential expression proteins, described
Method comprises the following steps：
1) sample pre-treatments：Cell is divided into experimental group and control group, respectively in the cell culture medium with or without nicotine
Middle culture, then extracts cell holoprotein；
2) protein digestion：Respectively to step 1) the cell holoprotein of obtained experimental group and control group reduces successively
With alkylation processing, trypsase is then added under conditions of enzymolysis is adapted to, the peptide fragment that enzymolysis is obtained is collected；
3) iTRAQ is marked：Using the iTRAQ reagents markers step 2 of different molecular weight) the obtained peptide fragment of experimental group and right
According to the peptide fragment of group, two groups of labeled peptide fragments are mixed, desalination, freezed；
4) nano-LC-Q-TOF is determined：By step 3) obtained peptide fragment mixture is loaded in nano-LC-Q-TOF, enters
Row LC-MS analysis；
5) differential protein is screened：By step 4) obtained data import mascot softwares to Uniprot-human databases
Storehouse is searched in progress, screens differential expression protein.
In the method that the present invention is provided, in the step 1) in, the cell is preferably nerve cell, more preferably god
Through female oncocyte；
Preferably, concentration of the nicotine in cell culture medium is 1mol/L；
Preferably, the step 1) comprise the following steps：
Cell is randomly divided into two groups of experimental group and control group, in 37 DEG C and 5%CO2Under, experimental group is containing 1mmol/L cigarettes
In the culture medium of alkali, control group cultivated 1 hour in without nicotine identical culture medium, then collect cell, extract cell complete
Albumen, wherein the culture medium is to contain the dual anti-DMEM culture mediums of 10% serum and 1%.
In the method that the present invention is provided, it is preferable that in the step 2) in, cell holoprotein is carried out using reducing agent
Reduction treatment, the reducing agent is 10mMDTT, 8M urea, 0.1MTris-HCL, pH8.5；
Preferably, using alkylating agent carry out cell holoprotein alkylation handle, the alkylating agent be 50mM IAA,
8M urea, 0.1MTris-HCL, pH8.5；
Preferably, reduction and the alkylation processing includes：Add described in 4 times of volumes and reduce into cell holoprotein solution
Agent, is incubated 1 hour at 37 DEG C, the alkylating reagent with reducing agent same volume is then added under the conditions of lucifuge,
Place 20 minutes at room temperature.
In the method that the present invention is provided, it is preferable that in the step 2) in, the trypsin digestion includes：By thin
The mass ratio of born of the same parents' holoprotein and enzyme is 20:1 ratio adds pancreas egg to by reducing and being alkylated in the cell holoprotein of processing
White enzyme；
Preferably, the trypsin digestion includes：The mass ratio for pressing cell holoprotein and enzyme is 20:1 ratio is to warp
Trypsase is added in the cell holoprotein for crossing reduction and alkylation processing, is incubated 16 hours at 37 DEG C, molecular weight is collected small
In 10kd peptide fragment.
Preferably, the step 2) comprise the following steps：
2-1) take 100 μ g cells holoproteins to be configured to the cell holoprotein solution of 30 μ L volumes with cell pyrolysis liquid, add
Reducing agent now with 4 times of volumes, is incubated 1 hour at 37 DEG C, is then added under the conditions of lucifuge and reducing agent same volume
Alkylating reagent, is placed 20 minutes at room temperature；
It will 2-2) be moved into through reducing and being alkylated the protein solution of processing in 10kd super filter tube, 12000 × g at 4 DEG C
Centrifugation 40 minutes, then adds 150 μ L 0.3M TEAB into super filter tube, and 12000 × g is centrifuged 40 minutes at 4 DEG C, is repeated
TEAB is centrifuged 3 times, to remove unreacted liquid；
The mass ratio for 2-3) pressing cell holoprotein and enzyme is 20:1 ratio adds trypsase into super filter tube, at 37 DEG C
Lower to be incubated 16 hours, then 12000 × g is centrifuged 10 minutes at 4 DEG C, then adds 100 μ L0.2M TEAB into super filter tube, 4
12000 × g is centrifuged 20 minutes at DEG C, is repeated TEAB and is centrifuged 2 times；
The peptide fragment of super filter tube ttom of pipe 2-4) is collected, after freeze-drying, adding 100 μ L 0.2M TEAB redissolves it, fully
Mix.
In the method that the present invention is provided, it is preferable that in the step 3) in, the iTRAQ reagents are 114,115,116
With 117 4 mark reagents.
Preferably, the step 3) include：
Different iTRAQ reagents are dissolved in 100 μ L ethanol, then with step 2) the obtained peptide fragment of experimental group and compare
The peptide fragment of group reacts 2 hours at room temperature, then adds 200 μ L water terminating reaction 30 minutes, two groups of labeled peptide fragments are mixed
Close, then through sep-park C18 post desalinations, freeze.
In the method that the present invention is provided, it is preferable that in the step 4) in, in the liquid chromatogram, Trap posts are
Acclamin pepmap (100 μm of * 2cm, C18,5 μm), analytical column be acclamin pepmap RPLC (75 μm of * 50cm,
C18,2 μm)；
Preferably, the liquid chromatogram includes following condition：
55 DEG C of column temperature；
Mobile phase A：0.1% aqueous formic acid, B：0.1% formic acid acetonitrile solution, eluent gradient is as follows, flow velocity
0.4μl/min：
Eluent gradient
Preferably, the liquid chromatogram includes：
By step 3) obtained peptide fragment mixture is dissolved in 100 μ l 0.1% aqueous formic acid, with 2 μ l sample size
Carry out chromatogram 150 minutes.
In the method that the present invention is provided, it is preferable that in the step 4) in, the mass spectrum includes following condition：
CaptiveSpray ion guns, sweep cation scan pattern, capillary voltages 1600V entirely；Mass spectrum full scan matter
Measure 50~2200m/z of scope, second order mses full scan 50~2200m/z of mass range, circulation time 3s；MS/MS High：16,
Energy range：85%-100%.
In the method that the present invention is provided, it is preferable that in the step 5) in, searching the condition in storehouse includes：
Parameter：Maximum leakage enzyme site number 2；Cysteine urea to methylate fixes modification, the variable oxidative modification of methionine, N-terminal
The variable modification of acetylation；
It is quantitative：ITRAQ 4 is marked；
Peptide end allowable error：20ppm；
MS/MS allowable errors：0.1Da；
Remarkable threshold：P ＜ 0.05.
In the method that the present invention is provided, it is preferable that in the step 5) in, screen the process bag of differential expression protein
Include：First according at least one unique peptide fragment, and score >=25 filter out credible albumen, then quantitative according at least two
Peptide fragment, | ratio | >=1.5 times and single-sample t-test P<0.05 filters out differential expression protein.
Specifically, the content of the invention of technical solution of the present invention is as follows.
A kind of assay method of nicotine inducible protein differential expression, including herein below：
A, solution preparation
(1) preparation of bovine serum albumin(BSA) standard liquid：2g/ml bovine serum albumin solutions, PBS is diluted to 1.5 successively,
1.0、0.75、0.5、0.25、0.125、0.025、0g/ml；
(2) buffer solution 1 (8M urea, 0.1M Tris-HCl (pH8.5))：Accurately weigh urea 24.024g and Tris
0.6057g, distilled water constant volume to 50ml；
(3) (the 8M urea of buffer solution 2；0.1M Tris-HCl(pH8.0))：Accurately weigh urea 24.024g and Tris
0.6057g, distilled water constant volume to 50ml；
(4) reducing agent (buffer solution 1+10mM DTT)：Accurately weigh urea 24.024g, Tris 0.6057g and DTT
0.0771g, distilled water constant volume to 50ml, pH is adjusted to 8.5；
(5) alkylating reagent (buffer solution 1+50mM IAA)：Accurately weigh urea 24.024g, Tris 0.6057g and IAA
0.4624g, distilled water constant volume to 50ml, pH is adjusted to 8.5 (prepare and preservation is both needed to lucifuge)；
(6)TEAB1(0.3mol/L)：It is accurate to measure 15mL 1M TEAB, add chromatogram water and be settled to 50mL；
(7)TEAB2(0.2mol/L)：It is accurate to measure 10mL 1M TEAB, add chromatogram water and be settled to 50mL；Enzymolysis liquid：
Take μ g, the TEAB1 dissolvings of trypsase 20.
(8) prepared by gel：30% acrylamide storing liquid：29.2g acrylamides and 0.8g bisacrylamides accurately are weighed,
Distilled water constant volume is to 100ml, and 0.45 μm of membrane filtration is preserved；10% ammonium persulfate：Accurately 0.5g ammonium persulfates are weighed to be settled to
5ml；Separation gel and concentration glue prepare such as table 1.By the separation gel of mixing pour into offset plate at top edge about 1cm, take advantage of do not coagulate when
Cover one layer of water, it is ensured that gel is uniform and gel upper surface is straight, after gelling to be separated is solid, water is blotted into addition concentration glue, it is fast
Speed plugs comb, after gelling to be concentrated is solid.
The separation concentration glue of table 1
B, sample pre-treatments
The extracting of protein example：Cell inoculation after, using containing the dual anti-DMEM culture mediums of 10% serum and 1% 37
DEG C, 5%CO2Cultivated in incubator, when degrees of fusion reaches 80%, be randomly divided into experimental group and each 3 bottles of two groups of control group, respectively
After being cultivated in the culture medium with and without nicotine, enzymolysis, digestion are collected, and protein extract and protease are added after PBS
Inhibitor, fully shaking (operation is carried out on ice above), 4 DEG C of placement 20min centrifuge (12000 × g, 4 DEG C) 20min, taken
Clearly.
The concentration of BCA method determination sample protein：The albumen of 25 μ L various concentrations is separately added into every hole of 96 orifice plates
Solution standard and 25 μ L testing sample protein, add 200 μ L BCA dyeing liquors, mix, and are incubated 30min and are in wavelength
The absorbance of detection protein is carried out under 565nm.Standard curve is drawn using the absorbance for measuring known protein, and calculates sample
Product concentration.
The detection of protein quality and sample collimation：Protein sample presses volume 3 with sample-loading buffer (4*):1 ratio is mixed
Close (use of 4* buffer solutions), boiling water bath heating 7min makes albumen be denatured completely, takes a certain amount of protein sample to be loaded to gel
On electrophoresis, when the collimation between protein quality and each sample is good, the enzymolysis of protein is carried out.
Protein digestion：100 μ g cell holoproteins are taken, the reducing agent now with 4 times of volumes, 37 DEG C of incubation 1h is added；Lucifuge
Under the conditions of add alkylating reagent, room temperature places 20min；In the super filter tube that protein solution is moved into 10kd, 40min is centrifuged
(12000 × g, 4 DEG C), unreacted liquid is all centrifuged；150 μ L0.3M TEAB are added into each super filter tube, are centrifuged
40min (12000 × g, 4 DEG C), by unreacted liquid, all centrifugation is gone down, in triplicate；Then 20 are pressed:1 ratio adds pancreas
7 DEG C of incubation 16h of protease 3；The good sample of digestion is centrifuged into 10min (12000 × g, 4 DEG C), plus 100 μ L 0.2M TEAB, from
Heart 20min (12000 × g, 4 DEG C), repeats the above steps 2 times；Peptide fragment solution centrifugal after enzymolysis, digestion is in collecting pipe bottom；Freezing
After drying, 100 μ L 0.2M TEAB are added thereto redissolves it, fully mixes.
ITRAQ is marked：100 μ L second are added into 4 pipe iTRAQ reagents (molecular weight is respectively 114,115,116 and 117)
Alcohol, experimental group and control group proteolysis peptide fragment are marked respectively；React at room temperature after 2h, plus 200 μ L chromatograms water are terminated instead
Answer 30min；Through sep-park C18 post desalinations after mixing, freeze.
C, nano-LC-Q-TOF are determined
(1) nano-LC-Q-TOF conditions
Chromatographic condition：Trap post acclamin pepmap (100 μm of * 2cm, C18,5 μm), analytical column acclamin
Pepmap RPLC (75 μm of * 50cm, C18,2 μm), 55 DEG C of column temperature；Mobile phase A：0.1% aqueous formic acid, B：0.1% formic acid second
Nitrile solution, eluent gradient is as shown in table 2, flow velocity：0.4 μ l/min, the μ l of sample size 2.
The eluent gradient of table 2
| Time (min) | 0 | 5 | 120 | 125 | 135 | 135.1 | 150 |
| Mobile phase B (%) | 5 | 5 | 30 | 80 | 80 | 5 | 5 |
Mass Spectrometry Conditions：Mass spectrum is furnished with captiveSpray ion guns, and cation scan pattern, capillary voltages are swept entirely
1600V；Mass spectrum full scan 50~2200m/z of mass range, second order mses full scan 50~2200m/z of mass range, during circulation
Between 3s；MS/MS High：16, energy range：85%-100%.
(2) measure of differential protein
The peptide fragment sample for digesting mark is redissolved in the aqueous formic acids of 100 μ L 0.1%, sample size is that 2 μ L are loaded to
Nano-LC-Q-TOF is measured.
D, differential protein carry out screening analysis
Differential protein is analyzed data measured importing mascot data, uses Mascot softwares (Version
2.4.1) search engine searches storehouse to Uniprot-human databases.Search storehouse parameter：Maximum leakage enzyme site number 2；Cysteine urea first
Baseization fixes modification, the variable oxidative modification of methionine, the variable modification of N-terminal acetylation；It is quantitative：ITRAQ 4 is marked；Peptide end allows to miss
Difference：20ppm；MS/MS allowable errors：0.1Da, remarkable threshold：P ＜ 0.05.
Due to the presence of system deviation, to reduce the result of false positive, first according at least one unique peptide fragment, and
Points >=25 filter out credible albumen, then according at least two quantitative peptide fragments, | ratio | >=1.5 times and single-sample t-test P<
0.05 filters out differential protein.
The present inventor is based on iTRAQ technologies and combines the triple level Four bar flight time mass spectrums of nanoliter liquid chromatogram
(nano-LC-Q-TOF) method for, developing the differential expression for determining nicotine inducing nerve cell protein, this method passes through excellent
Change pre-treatment and testing conditions, establish extraction and enzymolysis, iTRAQ marks, the detection nicotine induction god of nerve cell protein
Differential expression through cell protein is determined.
It is demonstrated experimentally that the note efficiency of this method is 96.7%, labeling effciency is good.Repeating label quantifies egg to control group twice
The confidential interval of white ratio 95% is distributed in (1.091,1.147)；Experimental group repeating label Quantitative Western ratio 95% twice
Confidential interval is distributed in (1.099,1.142), shows reproducible, experimental result is reliable.Using the method for the present invention, ginseng is found
Important work may be played in nicotine addiction with the MAPK proteasome proteins for cascading bioprocess and proteasome signal path
With, reached understand nicotine inducing nerve cell differential expression protein and differential protein participate in bioprocess mesh
's.
Brief description of the drawings
Hereinafter, embodiment of the present invention is described in detail with reference to accompanying drawing, wherein：
Fig. 1 shows the analysis process for the protein difference expression that SHSY-5Y cells are induced according to the nicotine of embodiment 1.
Fig. 2 is shown according to the different disengaging time chromatograms of the liquid chromatogram of embodiment 1.
Fig. 3 shows the identification according to the different-energy range protein matter of embodiment 1.
Embodiment
Illustrate the present invention referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, it does not limit the scope of the present invention in any way.
Experimental method in following embodiments, is conventional method unless otherwise specified.Medicine used in following embodiments
Material raw material, reagent material etc., unless otherwise specified, are commercially available products.
Embodiment 1
To investigate the differential expression of nicotine exposed cell protein, the culture 1mol/mol nicotine exposures 1h female knurl of nerve is thin
Born of the same parents, albumen is extracted through neuroprotein lysate, and the collimation to the concentration, quality and sample of the albumen of extraction is measured, and is passed through
Ultrafiltration is digested, and is marked and mixed with iTRAQ, by sep-park C18 post desalinations, is loaded to nano-LC-Q-TOF, analyzed
Identification.Flow chart is shown in Fig. 1.
1. instrument, reagent and material
SHSY-5Y cells：American ACT T cell storehouse；
DMEM culture mediums and mycillin：Hyclone companies of the U.S.；
Top grade hyclone：Gibco companies of the U.S.；
BCA kits, PBS, dithiothreitol (DTT) (DTT), iodoacetamide (IAA), acrylamide, N-N- intersects acryloyl
Amine, urea and teabrom (TEAB)：Sigma Co., USA；
Acetonitrile (CAN), urea, methanol, formic acid (FA), mass spectrum level pancreatin and N-PER neuroprotein extract solutions：Promega
Company；
ITRAQ kits：American AB Sciex companies；
Three (methylol) aminomethanes (Tris HCl), ammonium persulfate, lauryl sodium sulfate (SDS), sample-loading buffer,
Glycine, quick blue transfection reagent WB, SDS, and the RIPA lysate of gel：Green skies company of China；
Nano-LC(Thermo)-Q-TOF：German Bruker；
Multi-function microplate reader：U.S. Molecular Devices；
Vacuum freeze drier：German CHRIST；
ImageScanner scanners：GE Healthcare.
2. sample treatment
After cell inoculation, in 37 DEG C, 5%CO2Under the conditions of cultivate, trained using the dual anti-DMEM of 10% serum and 1% is contained
Support base, be randomly divided into two groups of experimental groups and each 3 bottles of two groups of control group when the degrees of fusion of cell reaches 80%, experimental group containing
In the culture medium of 1mmol/L nicotine, control group is cultivated after 1h in without nicotine identical culture medium, and enzymolysis, digestion is collected thin
Born of the same parents, protein extract, BCA method protein quantifications are added after being cleaned using phosphate buffered saline solution (PBS).
100 every group of μ g cell holoproteins are taken to do 2 repetition experiments, constant volume to 30ul respectively；Plus 4 times of volumes (10mM DTT,
8M urea, 0.1M Tris-HCl (pH8.5)) reducing agent, 37 DEG C of water-bath 1h；The alkylation with reducing agent in equal volume is added to try
Agent (50mM IAA, 8M urea, 0.1M Tris-HCl (pH8.5)) room temperature lucifuge 20min；Protein solution is moved into the super of 10kd
4 DEG C in chimney filter, 12000g, 40min all centrifuge unreacted liquid；150 μ L 0.3M is added into each super filter tube
TEAB, 4 DEG C, 12000g, 40min, by unreacted liquid, all centrifugation is gone down, in triplicate；Then 20 are pressed:1 ratio is added
37 DEG C of enzymolysis 16h of trypsase；The good sample of digestion is centrifuged to 12000g, centrifugation 10min, plus 100ul 0.2M at room temperature
TEAB, room temperature 12000g centrifuge 20min, repeat the above steps 2 times；Peptide fragment solution centrifugal after enzymolysis, digestion is in collecting pipe bottom；
After freeze-drying, 100ul 0.2M TEAB are added thereto redissolves it, fully mixes.
100ul ethanol is added into 4 pipes correspondence iTRAQ (molecular weight is respectively 114,115,116 and 117 label),
115th, 116 mark control group, 114,117 labelling experiment groups；React at room temperature 2h, plus 200ul chromatogram water terminating reactions 30min；It is mixed
Through sep-park C18 post desalinations after conjunction, freeze.
Sample after will be lyophilized is redissolved with 0.1% aqueous formic acid, and identification is separated in upper nano-LC-Q-TOF.
Chromatographic condition：Trap post acclamin pepmap (100 μm of * 2cm, C18,5 μm), analytical column acclamin
Pepmap RPLC (75 μm of * 50cm, C18,2 μm), 55 DEG C of column temperature；Mobile phase A：0.1% aqueous formic acid, B：0.1% formic acid second
Nitrile solution, flow velocity：0.4 μ l/min, the μ l of sample size 2, under different disengaging times and gradient, by separation chromatography figure (such as Fig. 2)
Understand that more preferable separation can be realized under 150min, eluent gradient is as shown in table 3.
The eluent gradient of table 3
| Time (min) | 0 | 5 | 120 | 125 | 135 | 135.1 | 150 |
| Mobile phase B (%) | 5 | 5 | 30 | 80 | 80 | 5 | 5 |
Mass Spectrometry Conditions：Mass spectrum is furnished with captiveSpray ion guns, and cation scan pattern, capillary voltages are swept entirely
1600V；Mass spectrum full scan 50~2200m/z of mass range, second order mses full scan mass range 50~2200m/z, cycle
time 3s；MS/MS High：16.Collision energy is optimized, it is known that (Fig. 3) energy range：85%-100%, signal phase
Should be stronger.
3. differential protein analysis
Storehouse is searched to Uniprot-human databases using Mascot (Version 2.4.1) search engines, Fig. 3 is confrontation
The optimization of spectral condition collision energy range, shows the identification of different-energy range protein matter.
Result identifies 1316 kinds of protein altogether in nicotine exposure group and experimental group, wherein 1273 kinds of albumen have quantitative letter
Breath, iTRAQ labeling effciencies are 96.7%, show that labeling effciency is good.Control group repeating label Quantitative Western ratio 95% twice
Confidential interval be distributed in (1.091,1.147)；The confidential interval point of experimental group repeating label Quantitative Western ratio 95% twice
Cloth shows reproducible, experimental result is reliable in (1.099,1.142).
Due to the presence of system deviation, to reduce the result of false positive, at least selection have two quantitative peptide fragments, | ratio | >=
1.5 times and single-sample t-test P<0.05 albumen is differential protein, finally, 132 species diversity albumen, wherein albumen is filtered out altogether
Mei Ti families, ribosomal protein family, eukaryotic translation initiation factor, aminoacyl-tRNA synthetase and the family A1 of acetaldehyde dehydrogenase 18
(such as table 4) is located at differential protein network interaction center, may be played a significant role in nicotine addiction.
The differential protein network interaction center protein of table 4
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deformed, and without departing from the spirit of the present invention, all should belong to the model of appended claims of the present invention
Enclose.
Claims (10)
1. a kind of method based on iTRAQ marker determination nicotine inducing cell differential expression proteins, methods described includes following
Step：
1) sample pre-treatments：Cell is divided into experimental group and control group, trained respectively in the cell culture medium with or without nicotine
Support, then extract cell holoprotein；
2) protein digestion：Respectively to step 1) the cell holoprotein of obtained experimental group and control group reduced and alkane successively
Baseization processing, then adds trypsase under conditions of enzymolysis is adapted to, and collects the peptide fragment that enzymolysis is obtained；
3) iTRAQ is marked：Using the iTRAQ reagents markers step 2 of different molecular weight) the obtained peptide fragment and control group of experimental group
Peptide fragment, labeled two groups of peptide fragments are mixed, desalination, freezed；
4) nano-LC-Q-TOF is determined：By step 3) obtained peptide fragment mixture is loaded in nano-LC-Q-TOF, carries out liquid
Phase Spectrometry；
5) differential protein is screened：By step 4) obtained data import mascot softwares and Uniprot-human databases are carried out
Storehouse is searched, differential expression protein is screened.
2. according to the method described in claim 1, it is characterised in that the step 1), the cell is nerve cell, preferably
For neural female oncocyte；
Preferably, concentration of the nicotine in cell culture medium is 1mmol/L；
Preferably, the step 1) comprise the following steps：
Cell is randomly divided into two groups of experimental group and control group, in 37 DEG C and 5%CO2Under, experimental group is containing 1mmol/L nicotine
In culture medium, control group cultivated 1 hour in without nicotine identical culture medium, then collect cell, extract cell holoprotein,
Wherein described culture medium is to contain the dual anti-DMEM culture mediums of 10% serum and 1%.
3. method according to claim 1 or 2, it is characterised in that the step 2) in, cell is carried out using reducing agent complete
The reduction treatment of albumen, the reducing agent is 10mM DTT, 8M urea, 0.1M Tris-HCl, pH 8.5；
Preferably, the alkylation for carrying out cell holoprotein using alkylating agent is handled, and the alkylating agent is urinated for 50mM IAA, 8M
Element, 0.1M Tris-HCl, pH 8.5；
Preferably, reduction and the alkylation processing includes：Reducing agent described in 4 times of volumes is added into cell holoprotein solution,
It is incubated at 37 DEG C 1 hour, the alkylating reagent with reducing agent same volume is then added under the conditions of lucifuge, in room temperature
It is lower to place 20 minutes.
4. according to the method in any one of claims 1 to 3, it is characterised in that the step 2), by cell holoprotein
Mass ratio with enzyme is 20:1 ratio adds trypsase to by reducing and being alkylated in the cell holoprotein of processing；
Preferably, the trypsin digestion includes：The mass ratio for pressing cell holoprotein and enzyme is 20:1 ratio is to by also
Trypsase is added in former and alkylation processing cell holoprotein, is incubated 16 hours at 37 DEG C, is collected molecular weight and be less than
10kd peptide fragment.
5. method according to any one of claim 1 to 4, it is characterised in that the step 2) comprise the following steps：
2-1) take 100 μ g cells holoproteins to be configured to the cell holoprotein solution of 30 μ L volumes with cell pyrolysis liquid, add and now match somebody with somebody 4
The reducing agent of times volume, is incubated 1 hour at 37 DEG C, the alkylation with reducing agent same volume is then added under the conditions of lucifuge
Reagent, is placed 20 minutes at room temperature；
It will 2-2) be moved into through reducing and being alkylated the protein solution of processing in 10kd super filter tube, 12000 × g is centrifuged at 4 DEG C
40 minutes, 150 μ L 0.3M TEAB are then added into super filter tube, 12000 × g is centrifuged 40 minutes at 4 DEG C, repeats TEAB
Centrifugation 3 times, to remove unreacted liquid；
The mass ratio for 2-3) pressing cell holoprotein and enzyme is 20:1 ratio adds trypsase into super filter tube, is incubated at 37 DEG C
Educate 16 hours, then 12000 × g is centrifuged 10 minutes at 4 DEG C, then adds 100 μ L 0.2M TEAB into super filter tube, at 4 DEG C
Lower 12000 × g is centrifuged 20 minutes, is repeated TEAB and is centrifuged 2 times；
The peptide fragment of super filter tube ttom of pipe 2-4) is collected, after freeze-drying, adding 100 μ L 0.2M TEAB redissolves it, fully mixes.
6. method according to any one of claim 1 to 5, it is characterised in that the step 3) in, the iTRAQ examinations
Agent is that molecular weight 114,115,116 and 117 4 marks reagent；
Preferably, the step 3) include：
Different iTRAQ reagents are dissolved in 100 μ L ethanol, then with step 2) the obtained peptide fragment of experimental group and control group
Peptide fragment reacts 2 hours at room temperature, then adds 200 μ L water terminating reaction 30 minutes, two groups of labeled peptide fragments is mixed, so
By sep-park C18 post desalinations, freeze.
7. method according to any one of claim 1 to 6, it is characterised in that the step 4) in, the liquid chromatogram
In, Trap posts are acclamin pepmap (100 μm of * 2cm, C18,5 μm), and analytical column is acclamin pepmap RPLC (75
μm * 50cm, C18,2 μm)；
Preferably, the liquid chromatogram includes following condition：
55 DEG C of column temperature；
Mobile phase A：0.1% aqueous formic acid, B：0.1% formic acid acetonitrile solution, eluent gradient is as follows, the μ l/ of flow velocity 0.4
min：
Eluent gradient
Preferably, the liquid chromatogram includes：
By step 3) obtained peptide fragment mixture is dissolved in the aqueous formic acids of 100 μ L 0.1%, and color is carried out with 2 μ l sample size
Spectrum 150 minutes.
8. method according to any one of claim 1 to 7, it is characterised in that the step 4) in, the mass spectrum includes
Following condition：
CaptiveSpray ion guns, sweep cation scan pattern, capillary voltages 1600V entirely；Mass spectrum full scan quality model
Enclose 50~2200m/z, second order mses full scan 50~2200m/z of mass range, circulation time 3s；MS/MS High：16, energy
Scope：85%-100%.
9. method according to any one of claim 1 to 8, it is characterised in that the step 5) in, search the condition bag in storehouse
Include：
Parameter：Maximum leakage enzyme site number 2；Cysteine urea to methylate fixes modification, the variable oxidative modification of methionine, N-terminal acetyl
Change variable modification；
It is quantitative：ITRAQ 4 is marked；
Peptide end allowable error：20ppm；
MS/MS allowable errors：0.1Da；
Remarkable threshold：P ＜ 0.05.
10. method according to any one of claim 1 to 9, it is characterised in that the step 5) in, screen difference table
Process up to protein includes：First according at least one unique peptide fragment, and score >=25 filter out credible albumen, Ran Hougen
According at least two quantitative peptide fragments, | ratio | >=1.5 times and single-sample t-test P<0.05 filters out differential expression protein.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107860852A (en) * | 2017-11-23 | 2018-03-30 | 南宁科城汇信息科技有限公司 | A kind of Differential Proteomic based on iTRAQ joints LC, MALDI |
| CN108627585A (en) * | 2018-05-28 | 2018-10-09 | 天津大学 | The method that significant difference expresses albumen in D-ALPHA-Hydroxypropionic acid fermentation process is identified based on iTRAQ protein sciences |
| CN112858692A (en) * | 2021-01-24 | 2021-05-28 | 南华大学 | BeSO4Method for detecting and analyzing lung tissue differential expression protein of infected rat |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101287991A (en) * | 2005-09-15 | 2008-10-15 | 阿尔克-阿贝洛有限公司 | A method for quantification of allergens |
| CN103163257A (en) * | 2011-12-13 | 2013-06-19 | 上海聚类生物科技有限公司 | Processing method of cow urine iTRAQ test data |
| CN104820103A (en) * | 2015-05-06 | 2015-08-05 | 华南农业大学 | Method of researching change of proteome of rice responding rice blast bacterial infection through iTRAQ technology |
| CN105467022A (en) * | 2014-09-05 | 2016-04-06 | 天士力制药集团股份有限公司 | Analysis method for neuron injury related differentially expressed protein |
-
2017
- 2017-03-01 CN CN201710116236.5A patent/CN107064338B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101287991A (en) * | 2005-09-15 | 2008-10-15 | 阿尔克-阿贝洛有限公司 | A method for quantification of allergens |
| CN103163257A (en) * | 2011-12-13 | 2013-06-19 | 上海聚类生物科技有限公司 | Processing method of cow urine iTRAQ test data |
| CN105467022A (en) * | 2014-09-05 | 2016-04-06 | 天士力制药集团股份有限公司 | Analysis method for neuron injury related differentially expressed protein |
| CN104820103A (en) * | 2015-05-06 | 2015-08-05 | 华南农业大学 | Method of researching change of proteome of rice responding rice blast bacterial infection through iTRAQ technology |
Non-Patent Citations (4)
| Title |
|---|
| INAS A. ABDALLAHA等: "A fully validated LC–MS/MS method for simultaneous determinationof nicotine and its metabolite cotinine in human serum and itsapplication to a pharmacokinetic study after using nicotinetransdermal delivery systems with standard heat application in adult", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| QING XU等: "Nicotine modulates expression of dynamin 1 in rat brain and SH-SY5Y cells", 《NEUROSCIENCE LETTERS》 * |
| 李鹏飞等: "稳定同位素ITRAQ标记／高效液相色谱同时定量分析人体中42种氨基酸及典型病例", 《分析化学研究学报》 * |
| 雷波等: "基于iTRAQ技术分析两个不同生态区烟草叶片蛋白组的表达差异", 《三峡生态环境监测》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107860852A (en) * | 2017-11-23 | 2018-03-30 | 南宁科城汇信息科技有限公司 | A kind of Differential Proteomic based on iTRAQ joints LC, MALDI |
| CN108627585A (en) * | 2018-05-28 | 2018-10-09 | 天津大学 | The method that significant difference expresses albumen in D-ALPHA-Hydroxypropionic acid fermentation process is identified based on iTRAQ protein sciences |
| CN112858692A (en) * | 2021-01-24 | 2021-05-28 | 南华大学 | BeSO4Method for detecting and analyzing lung tissue differential expression protein of infected rat |
| CN112858692B (en) * | 2021-01-24 | 2024-03-12 | 南华大学 | BeSO 4 Method for detecting and analyzing differential expression protein of lung tissue of infected rat |
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