CN107860922A - A kind of method of protein group in detection colorectal cancer - Google Patents
A kind of method of protein group in detection colorectal cancer Download PDFInfo
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Abstract
Research in terms of the present invention relates to cancer metastasis, specifically a kind of method for detecting protein group in colorectal cancer.The present invention screens the colon-cancer cell HCT116 I8 of high invasion and attack using Transwell, and carries out the quantitative group of identification of proteins learned with reference to SILAC technologies and find the protein or path related to cell invasion ability.Proteomic techniques can panoramically detect protein expression profile from the Expression level of the intracellular protein of the high invasive ability of overall angle analysis.Using height invasion and attack intestinal cancer model and protein science screening invasion and attack correlation function albumen this technological invention can high flux, high accuracy, the change for detecting to high-resolution the protein group in the cell of different invasive abilities in colorectal cancer, and can be found that the sneak circuit related with attacking transfer.Can be with the mechanism of the diseases such as efficient exploration cancer using this technology, and provide confidence level higher drug target.
Description
Technical field
The invention belongs to medical biotechnology field, and in particular to a kind of method for detecting protein group in colorectal cancer.
Background technology
Cancer metastasis process is related to various kinds of cell biological behaviour, such as adhesion, motion, propagation, extracellular matrix degradation
It is the result of the common coordinative role of different kinds of molecules etc. multiple links, and traditional single factor analysis can not reflect and illustrate that cancer turns
Complicated pathomechanism during shifting, quantitative proteomicses technology are that the protein for participating in cancer metastasis invasion and attack is ground
The powerful mean studied carefully.Proteomic techniques can be from the dynamic table of the intracellular protein of the high invasive ability of overall angle analysis
Up to level, protein expression profile can be panoramically detected, the early molecule event occurred during metastases is excavated, is metastasis of cancer
Complex mechanism research provide new thought.Transwell technologies are to establish the Critical policies of high invasive ability cell model,
It is exactly briefly to be separated the nutrient solution of the nutrient solution of high nutrition and low nutrition with one layer of film for being covered with matrigel, in low nutrition liquid
In cell can oriented high nutrition nutrient solution invasion and attack trend, invasion and attack to the cell dissociation in hyperalimentation fluid are got off to carry out down
The invasion and attack screening of one wheel.Single colorectal cancer cell lines are carried out with repeatedly migration invasion and attack screening, it is high to pick out migration invasive ability
Cancer cell subbreed carry out proteomics research.High invasive ability cell model is established using Transwell technologies also to obtain
More application.Established using Transwell technologies in the application of high invasive ability cell model, majority screens 1 wheel~3
Wheel.Then the invasive ability of cell can be improved.But the characteristic that can not attack the height keeps permanent1,2.(1. Lexus；The residence of a high official
Cyanines；Wang Yunjie；Zhao Xin；The screening of Zhang Chaodong, Transwell cell high aggressive C6 cells MMP-2 and TIMP-2 expression tumours
Prevent and treat magazine 2005, (10), 742-745.2.Li, B.；Xu,W.W.；Lam,A.K.Y.；Wang,Y.；Hu,H.F.；Guan,
X.Y.；Qin,Y.R.；Saremi,N.；Tsao,S.W.；He,Q.Y.；Cheung,A.L.M.,Significance of PI3K/
AKT signaling pathway in metastasis of esophageal squamous cell carcinoma and
its potential as a target for anti-metastasis therapy.Oncotarget 2017,8,(24),
38755-38766.)
Cold labeling technology (Stable isotope labeling with amino under cell culture condition
Acids in cell culture, SILAC) it is to utilize to contain N15And C13Deng heavy chain isotope marks amino acid or contain
N14And C12Cell is cultivated Deng light chain amino acid, converses change in protein ratio and to egg by Mass Spectrometric Identification afterwards
White matter change is quantified.Quantitative mark is carried out to the cell of different invasive abilities using SILAC quantitative proteomicses technology,
The change of the high-throughout identification protein related to cell invasion transfer, is to find the new egg related to cancer metastasis
In vain, the effective means of albumen New function known to exploring.
In the research in terms of application quantitative proteomicses technology carries out cell transfer, there has been no research team's combination
Transwell technologies establish the precedent that the method for high invasive ability colorectal cancer cell model is studied.
In conventional cell invasion transfer research, most researchers are merely capable of the list to participating in cell invasion transfer
Individual protein is explored.It is relatively low that this research method takes longer and precision, it is impossible to large-scale to probe into cell transfer invasion and attack
The change of the species and content of middle protein.
And in terms of tumor correlated albumen is screened, someone has been successfully established people's cancer using a variety of methods such as mice foot-pad inoculations
Cell transfer model.But Tumor Heterogeneity problem and something lost can not be still overcome very well for traditional metastases cell model
Background difference problem is passed, Tumor Heterogeneity refers to the existing tumorigenic cell subgroup of intra-tumor, also there is non-tumorigenic cell subgroup.Its daughter cell
The change in terms of molecular biology or gene is showed, so that the speed of growth of tumour, invasive ability, the sensitivity to medicine
Property, each side such as prognosis produce difference.Increase the treatment difficulty of cancer.The high invasive ability that traditional cell screening obtains it is thin
Its gene of born of the same parents, all can difference in terms of molecule.Cause the reduction of the reliabilities such as ensuing functional study result.
The content of the invention
It is an object of the invention to provide one kind can high flux, high accuracy, detect in colorectal cancer to high-resolution not
With the change of protein group in the cell of invasive ability, and the method for caning be found that the sneak circuit related to invasion and attack transfer.Profit
Can be with the mechanism of the diseases such as efficient exploration cancer with this technology, and provide confidence level higher drug target
Point.
The idiographic flow of technical scheme is shown in Fig. 1, Fig. 2.Comprise the following steps that：
The method of protein group, comprises the following steps in a kind of detection colorectal cancer：
(1) with Transwell methods screening cell；
(2) with SILAC methods mark cell；
(3) super filter tube enzymolysis protein sample solution is used；
(4) mass spectrometric data bioinformatic analysis；
(5) bioinformatic analysis differentially expressed protein.
The detailed step of wherein step (1) includes：
(i) with 1 × PBS rinse cell wild type HCT116 and RKO cell twice, digested and collected with 0.05% pancreatin
Get off, centrifugation removes supernatant, and cell is resuspended with serum free medium 1640；
(ii) cell count, according to formula：Cell density (individual/mL)=(4 big lattice sum/4) × 104× dilution times
Number；
(iii) 20 × 10 are taken4Individual cell adds the upper chamber of Transwell experimental provisions, is filled out with plasma-free DMEM medium
Volume difference between benefit group；
(iv) 600 μ L nutrient solutions are added in the lower room of Transwell experimental provisions；
(v) 37 DEG C, 5%CO are placed in272h is incubated in cell culture incubator；
(vi) when migrating 72h, the cell pancreatin through upper chamber being digested, continues to cultivate, cell concentration reaches 20 ×
104Afterwards, repeat step (i), referred to as 1 wheel；After often taking turns, the time can be gradually shortened, and the concentration for the serum that lower room contains gradually decreases；
(vii) after cell screening is taken turns to the 2nd~8, the phenotype checking of protein is done.
Wherein, step (iv), which adds, contains 20% serum free culture system liquid；The cell screening 8 of step (vii) is taken turns, and obtains I8 cells.
Wherein, the detailed step of step (2) includes：
(i) operate under sterile conditions, every bottle of culture medium in SILAC Kit is poured out into 50mL, adding 50mL dialysis
The hyclone crossed；
(ii) 50mg 13C6L-Lysine-2HCl and 50mg L-Arginine-HCl are thoroughly dissolved in 10mL steps
(i) in the culture medium configured；
(iii) 13C6L-Lysine-2HCl and L- dissolved with 0.22 μm of membrane filtration sterilization step (ii)
Arginine-HCl；
(iv) 13C6L-Lysine-2HCl and L-Arginine-HCl that have filtered are added to the step that 490mL configured
Suddenly in the culture medium of (i), mix, 4 DEG C of preservations, obtain heavy chain culture medium；
(v) again with culture medium dissolving 50mg L-Lysine-2HCl and the 50mg L-Arginine- of 10mL steps (i)
HCl, the membrane filtration with 0.22 μm is degerming, is then added in 490mL culture medium, mixes, and 4 DEG C of preservations, obtains light chain culture
Base；
(vi) HCT116 cells are cultivated respectively with heavy chain culture medium and light chain culture medium.
Wherein, step (vi) cultivates 8 generation HCT116 cells.
Wherein, peptide fragment is arranged to 7 in step (4).
Wherein, mass spectrum, and fold change >=1.5or≤0.67, P in triplicate after step (5) data analysis<0.01,
Obtain differential protein.
By the way that compared with prior art, technical scheme has advantages below：
1st, fast colorectal cancer cell is attacked using the migration of Transwell technology screenings, has carried out the screening of eight wheel cellses, obtained
Invasive ability stronger colon-cancer cell HCT116-I8 and RKO-I8 are arrived, invasive ability can improve more than ten times, and can be more permanent
Ground keeps the characteristic of high invasion and attack.Existing existing screening is only to proceed to 1~3 wheel, and cell invasion ability, which has, somewhat to be improved, and not
The characteristic of the high invasion and attack of holding that can be for a long time.And when a kind of invasive ability difference simultaneously can keep considerably long one section to a certain extent
During the time, some potentially molecules related to transfer can be just identified.(such as LI B, XU W W, LAM AK Y, et
al.Significance of PI3K/AKT signalingpathway in metastasis of esophageal
squamous cell carcinoma and its potential as atarget for anti-metastasis
therapy[J].Oncotarget,2017,8(24):38755-66. 3 wheels are only screened)
2nd,, therefore, will because the invasive ability of HCT116 and RKO cells is all weaker in the screening process to intestinal cancer
The serum-concentration of lower room increases to 20%, after three-wheel screens, by the concentration of serum be reduced to 10% continue screening and progressively
Shorten the time (- 1 day -2 days 3 days -36h-24h-12h) of screening.It is small that the invasive ability of cell was finally shortened to 12 by three days
When.Finally the invasive ability of the cell is greatly promoted and (improves more than 50 times as shown in Figure 3).
3rd, step sizing is carried out to single tumor cell line with Transwell method, the hereditary back of the body can be avoided well
The problem of scape and Tumor Heterogeneity, but the foundation of existing high invasion and attack cell model (mice foot-pad inoculation) can not avoid tumour
The problem of heterogeneous, different portion's cell protein type and quantity of same tumour have very big difference, so traditional is swollen
Tumor metastasis cell model can not still overcome Tumor Heterogeneity problem and genetic background difference problem very well.
4th, protein science is repeated poor, and the present invention utilizes two kinds of cells of Mass Spectrometric Identification HCT116 and HCT116-I8
128 differential proteins be the albumen that all identifies in mass spectrum three times repeats to test, substantially increase the difference that identification obtains
The reliability of M-band.The p value that the differential protein identified repeats three times is respectively less than 0.01, than in general standard (p<0.05) more
To be strict.
5th, SILAC technology for detection scope is wide, and still undiscovered low abundance proteinses can also be identified before；Detection essence
Degree is high, and carrying out the work such as peptide fragment matching, quantitative with reference to the protein that bioinformatics means identify to mass spectrum can be accurately
Reflect changes of contents of a certain protein in different conditions cell.This kind of technology can disposably identify 10,000 kinds with setting egg(s)
White matter, and traditional immunoblot assay can only once identify some protein, detection range compares limitation, and precision is not high.
6th, technical scheme combines being total to for Transwell tumor models and quantitative proteomicses technology
With using, the heterogeneity of tumour had both been avoided that, can also high flux, high depth, high-precision identification have been carried out to all proteins, keep away
Prior art is exempted from the defects of cell transfer research.Can be found on a large scale on the problem of avoiding Tumor Heterogeneity and
The related protein molecular of cancer metastasis.Many ignored albumen can be detected, make cell transfer invasion and attack research
Powerful support.
Brief description of the drawings
Fig. 1 is Transwell tumor models flow charts.
Fig. 2 is the high invasion and attack Cytomics modified flow figure of proteomics identification.
Fig. 3 is Transwell detection colorectal cancer cell screening effects.
Fig. 4 is Western blot detections HCT116, HCT116-I8 and RKO-I8, RKO-I8 EMT indexs.
Fig. 5 is the result of the Quantitative Western that mass spectrum is got to three times.
Fig. 6 is the detection of Cdc42 signal paths activity.
Fig. 7 is to suppress Cdc42 signal paths activity to significantly inhibit the invasive ability of cell.
Fig. 8 is that Western blot detections suppress the EMT indexs after Cdc42 signal paths.
Fig. 9 is the block diagram of mRNAs and albumen of the Cdc42BPA in I8 cells and wild-type cell expression.
Figure 10 is the race close-burning fruit of mRNAs and albumen of the Cdc42BPA in I8 cells and wild-type cell expression.
Figure 11 is Cdc42BPA interference effect.
Figure 12 is that suppression Cdc42BPA expression can significantly inhibit the invasive ability of colorectal cancer.
Figure 13 is the EMT indexs after Western blot detection Cdc42BPA interference.
Embodiment
With reference to specific embodiment, the present invention is further illustrated.
Human colorectal cancer cells HCT116and RKO (ATCC, Rockville, MD, USA) are used respectively contains 10% serum
RPMI 1640and DMEM (the Thermo FisherScientific, San of (Thermo Fisher Scientific)
Jose, CA, USA) cultivate.
Embodiment 1
The I8 that this example screens high invasive ability with Human colorectal cancer cells HCT116 and RKO by Transwell methods is thin
Born of the same parents combine SILAC quantitative proteomicses technology and IPA analysis of biological information finds Cdc42 signal paths and protein
Cdc42BPA attacks the influence of transfer to colorectal cancer.
Transwell screens cell
(1) with 1 × PBS rinse cell wild type HCT116 and RKO cell twice, digested and collected with 0.05% pancreatin
Get off, centrifugation removes supernatant, and cell is resuspended with serum free medium (1640)；
(2) cell count, according to formula：Cell density (individual/mL)=(4 big lattice sum/4) × 104× extension rate；
(3) 20 × 10 are taken4Individual cell adds upper chamber, volume difference between filling up group with plasma-free DMEM medium；
(4) nutrient solution that 600 μ L contain 20% serum is added below in cell；
(5) 37 DEG C, 5%CO are placed in272h is incubated in cell culture incubator；
(6) when having moved to 72h, the cell pancreatin through cell is digested, continues to cultivate, cell concentration reaches one
After determining degree, repeat step 1, after often taking turns, the time can be gradually shortened, and the concentration for the serum that lower room contains gradually decreases；
(7) after screening the 8th wheel, Transwell and Western blot method detects to the cell of screening,
It was found that the I8 cell invasion abilities screened are greatly improved (Fig. 3, Fig. 4).
SILAC marks cell
(1) operate under sterile conditions, every bottle of culture medium in SILAC Kit is poured out into 50mL, adding 50mL dialysis
The hyclone crossed；
(2) 50mg 13C6L-Lysine-2HCl and 50mg L-Arginine-HCl are thoroughly dissolved in above 10mL and matched somebody with somebody
In the culture medium put；
(3) it is the amino acid dissolved is degerming with 0.22 μm of membrane filtration；
(4) filtered amino acid is added in the 490mL configured culture medium, mixed；
(5) " heavy " is marked on bottle, 4 DEG C of preservations；
(6) 50mg L-Lysine-2HCl and 50mg L-Arginine-HCl are dissolved with 10mL culture mediums again, with 0.22 μm
Membrane filtration it is degerming, be then added in 490mL culture medium, mix, on bottle mark " light ", 4 DEG C preservation；
(7) HCT116 cells are cultivated with heavy chain culture medium and light chain culture medium respectively, 9 instead of after, for other experiments；Mark
Note efficiency reaches more than 90% after testing, can carry out next step experiment.
Protein sample in-solution digestion
The I8 cells and wild-type cell marked respectively to heavy chain using cell pyrolysis liquid is carried out cracking and obtains protein, it
BCA method detectable concentrations are used afterwards, 200 micrograms of protein are respectively taken further according to the concentration surveyed, and are mixed.Digested.Comprise the following steps that：
(1) protein cleavage will be carried out by the method for protein cleavage by control group and experimental group cell, takes 1mg albumen to carry out
In-solution digestion.
(2) the 8M Urea of appropriate volume are added, the final concentration of sample is more than 4M.
(3) 1M DTT (1mL are prepared：154.25mg), appropriate volume is added, it is 50mM to make its working concentration, and sample is placed in 37
1h in DEG C water-bath.
(4) configuration concentration is 1M IAA (1mL IAA：184.96mg), appropriate volume is added in sample cell makes its work
Make concentration in the range of 120-150mM, room temperature lucifuge reaction 30min.
(5) a new flat super filter tube is taken, with 50mM TEAB rinses bottom, 12000g centrifuges 10min and all filtered to liquid
Under, notice that each centrifugation time of super filter tube does not exceed 15min, often more than 15min, change and centrifuge angle.
(6) sample is added to the super filter tube after rinse, pays attention to not exceeding 400 μ L per pipe volume, with 12000g from
Mental and physical efforts centrifuge 15min.
(7) add 100 μ L 8M Urea rinse super filter tubes, be repeated twice, centrifuged every time with 12000g.
(8) plus 100 μ L 50mM NH4HCO3 rinses 2-5 time, 12000g centrifugations, according to the complexity tune above centrifuged
Whole centrifugation number, be more difficult to from sample, centrifugation number it is more.
(9) change a new collecting pipe and collect albumen, sequentially add the 50mM TEAB and trypsase of appropriate volume, two
The volume sum of person is in the range of 120-130 μ L, and be vortexed concussion 1min.Notice that trypsin solution can be configured to 0.6 μ g/ μ L, press
According to 1:30–1:50 ratio addition.
(10) super filter tube is encased with sealed membrane, is placed in 37 DEG C of incubators and is incubated overnight, enzymolysis time 12h
(11) peptide fragment digested is collected by centrifugation in 12000g, adds 70 μ L mass spectrums dedicated waters to be centrifuged after being vortexed.
(12) filtrate centrifuged is shifted in new centrifuge tube and freezed.
(13) the A liquid that the sample freezed adds 102 μ L HPLC is resuspended, and pre-separation is carried out after the concussion that is vortexed.
(14) freeze.
(15) dissolved with 35-40 μ L loading A liquid (98% mass spectrum dedicated water, 2%ACN, the configuration of 0.1% formic acid form),
And carry out component separation it is lyophilized after, take proper volume to enter loading pipe, notice that loading pipe can not have bubble.
(16) loading.
Mass spectrometric data bioinformatic analysis searches storehouse parameter setting
(1) wiff the and wiffscan files for taking each component are copied from the mass spectrographic computer of connection, it is soft to import maxquant
Part.
(2) the full storehouses of fasta (reviewed) of newest people are downloaded on uniprot websites.
(3) " modifications＆labels " page is selected, " multiplicity " selects 2, and " H labels " choose
" lys8 " and " arg10 ", variable modification：In addition to choosing oxidative modification and acetylation modification, then choose " Gln->pyro-
Glu”.Protease selects Trysin.Peptide fragment is arranged to 7.
(4) " MS/MS＆sequence " page is selected, addition fasta storehouses, chooses " Re-quantify ", " I=L ".Selection
Appropriate CPU core number starts to search storehouse.
Differentially expressed protein bioinformatic analysis
Mass spectrum in triplicate after data analysis, the differential protein got to take common factor, and fold change >=1.5or≤
0.67, P<The albumen of 0.01 differential expression is imported in IPA (Ingenuity Pathway Analysis) software, carries out function
With structure enrichment and path analysis, differential protein is obtained.
I8 cells and wild-type cell are compared, if albumen has differences in I8 cells and wild-type cell,
It is dynamic change during cell invasion then to illustrate this albumen, then implies that this albumen may participate in cell transfer and invade
The regulation and control attacked.Carry out repeating to test three times, protein science data find that mass spectrum identifies 3161 Quantitative Westerns (figure altogether three times
5).Change more than 1.5 times P<The albumen (table 1) of 0.01 difference has 128 kinds, and in these differential proteins, major part is all
It has been reported that the regulation and control of cell-invasion process are participated in, in addition to the albumen that those have been reported, it was found that and Cdc42 signals
The relevant protein C dc42BPA of path up-regulated expressions in I8 cells.This prompting Cdc42BPA be found pass through Cdc42
Signal path participates in the protein of cell transfer invasion and attack.
Table 1:I8 cells and wild-type cell compare more than 1.5 times of differential protein
Mass Spectrometric Identification is carried out after enzymolysis, the albumen of quantitative information is obtained by bioinformatics MaxQuant software analysis
The differential protein of matter and more than 1.5 times difference.These differential proteins are subjected to IPA analyses, the higher network path of score is as follows
(table 2).IPA also indicates that these differential proteins are main and cell movement, propagation, cycle, apoptosis etc. relevant (table 3) simultaneously.As a result
Show that the motion of these differential proteins and cell, death etc. are relevant and illustrate that protein science data are reliable.Wherein CDC42BPA exists
Identified in mass spectrum three times, choose the CDC42BPA and path CDC42pathway related to it and further studied.
Table 2：The network that the differential protein that I8 cells and wild-type cell relatively obtain is participated in leads to
Table 3：The differential protein function of I8 cells and wild-type cell
Next prove that Cdc42 signal paths are living really in I8 cells using the method for Cdc42 activity detection kits
Property higher (Fig. 6), and by MT141 suppress the path activity can significantly suppress colorectal cancer invasive ability (Fig. 7,
Fig. 8).Its experimental result indicates the reliability of Mass Spectrometric Identification data.
Next prove that Cdc42BPA protein is expressed in I8 cells using Western Blot and qRT-PCR method
It is whether consistent with Mass Spectrometric Identification.Experiment proves that Cdc42BPA protein contents significantly raise in I8 cells, with the horizontal phase of Mass Spectrometric Identification
Unanimously (Fig. 9).
Function test confirms that interference Cdc42BPA expression can suppress the invasive ability (Figure 10) of colorectal cancer cell,
EMT molecular indexes also confirm that the relation (Figure 11) of itself and invasion and attack.Prove that Cdc42BPA participates in the invasion and attack transfer of regulation colon-cancer cell.
It is to study having for cancer metastasis invasion and attack mechanism that above example, which is proved using Transwell models and SILAC technologies,
Effect means, more deeply it can widely probe into the signal path and protein molecule related to cell invasion.This is also to probe into
Transcellular mechanism is provided convenience in the diseases such as cancer, can largely reduce tumor recurrence, has important application
Value.
The implementation of the present invention is not limited to this, according to the above of the present invention, is known using the ordinary skill of this area
Knowledge and customary means, under the premise of the above-mentioned basic fundamental thought of the present invention is not departed from, the present invention can also make other a variety of shapes
Modification, replacement or the change of formula, all fall within rights protection scope of the present invention.
Claims (7)
- A kind of 1. method for detecting protein group in colorectal cancer, it is characterised in that comprise the following steps：(1) with Transwell methods screening cell；(2) with SILAC methods mark cell；(3) super filter tube enzymolysis protein sample solution is used；(4) mass spectrometric data bioinformatic analysis；(5) bioinformatic analysis differentially expressed protein.
- 2. according to the method for claim 1, it is characterised in that the detailed step of step (1) includes：(i) with 1 × PBS rinse cell wild type HCT116 and RKO cell twice, digested and collected down with 0.05% pancreatin Come, centrifugation removes supernatant, and cell is resuspended with serum free medium 1640；(ii) cell count, according to formula：Cell density (individual/mL)=(4 big lattice sum/4) × 104× extension rate；(iii) 20 × 10 are taken4Individual cell adds the upper chamber of Transwell experimental provisions, between filling up group with plasma-free DMEM medium Volume difference；(iv) 600 μ L nutrient solutions are added in the lower room of Transwell experimental provisions；(v) 37 DEG C, 5%CO are placed in272h is incubated in cell culture incubator；(vi) when migrating 72h, the cell pancreatin through upper chamber is digested, continues to cultivate, cell concentration reaches 20 × 104Afterwards, Repeat step (i), referred to as 1 wheel；After often taking turns, the time can be gradually shortened, and the concentration for the serum that lower room contains gradually decreases；(vii) after cell screening is taken turns to the 2nd~8, the phenotype checking of protein is done.
- 3. according to the method for claim 2, it is characterised in that step (iv), which adds, contains 20% serum free culture system liquid；Step (vii) cell screening 8 is taken turns, and obtains I8 cells.
- 4. according to the method for claim 1, it is characterised in that the detailed step of step (2) includes：(i) operate under sterile conditions, every bottle of culture medium in SILAC Kit is poured out into 50mL, adding what 50mL dialysed Hyclone；(ii) 50mg 13C6L-Lysine-2HCl and 50mg L-Arginine-HCl are thoroughly dissolved in into 10mL steps (i) to match somebody with somebody In the culture medium put；(iii) 13C6L-Lysine-2HCl and L-Arginine- dissolved with 0.22 μm of membrane filtration sterilization step (ii) HCl；(iv) 13C6L-Lysine-2HCl and L-Arginine-HCl that have filtered are added to the step (i) that 490mL configured Culture medium in, mix, 4 DEG C preservation, obtain heavy chain culture medium；(v) used again with culture medium dissolving 50mg L-Lysine-2HCl and the 50mg L-Arginine-HCl of 10mL steps (i) 0.22 μm of membrane filtration is degerming, is then added in 490mL culture medium, mixes, and 4 DEG C of preservations, obtains light chain culture medium；(vi) HCT116 cells are cultivated respectively with heavy chain culture medium and light chain culture medium.
- 5. according to the method for claim 4, it is characterised in that step (vi) cultivates 8 generation HCT116 cells.
- 6. according to the method for claim 1, it is characterised in that peptide fragment is arranged to 7 in step (4).
- 7. according to the method for claim 1, it is characterised in that mass spectrum, and fold in triplicate after step (5) data analysis Change >=1.5 or≤0.67, P<0.01, obtain differential protein.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109799350A (en) * | 2019-01-17 | 2019-05-24 | 暨南大学 | Albumen thermostabilization measurement combines two-way stable isotope labelling protein science screening medicine target calibration method and application |
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Non-Patent Citations (6)
| Title |
|---|
| CHUN-I WANG: "Quantitative Proteomics Reveals a Novel Role of Karyopherin Alpha 2 in Cell Migration through the Regulation of Vimentin−pErk Protein Complex Levels in Lung Cancer", 《J PROTEOME RES》 * |
| JIAN PAN: "ATP synthase ecto-a-subunit: a novel therapeutic target for breast cancer", 《JOURNAL OF TRANSLATIONAL MEDICINE》 * |
| JUAN MARTINEZ-AGUILAR: "Quantitative mass spectrometry for colorectal cancer proteomics", 《PROTEOMICS CLIN APPL》 * |
| MARTA MENDES: "Mapping the spatial proteome of metastatic cells in colorectal cancer", 《PROTEOMICS》 * |
| RODRIGO BARDERAS: "In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion, Migration, and Invasion", 《MOLECULAR & CELLULAR PROTEOMICS》 * |
| XIAO ZENG: "Quantitative secretome analysis reveals the interactions between epithelia and tumor cells by in vitro modulating colon cancer microenviroment", 《JOURNAL OF PROTEOMICS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799350A (en) * | 2019-01-17 | 2019-05-24 | 暨南大学 | Albumen thermostabilization measurement combines two-way stable isotope labelling protein science screening medicine target calibration method and application |
| CN109799350B (en) * | 2019-01-17 | 2022-02-25 | 暨南大学 | Method for screening drug target by combining protein thermal stability measurement with bidirectional stable isotope labeling proteomics and application |
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