CN109536473A - The method for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate - Google Patents
The method for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
Abstract
The invention discloses the methods for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate, the following steps are included: preparing the cell sample during entire cells transdifferentiate, it is divided into transcript profile and protein phosphorylation group, and detection is learned by group respectively and obtains every group of corresponding initial data；Initial data is pre-processed, the protein kinase of expression significant difference is screened by transcript profile, active significant difference is screened by protein phosphorylation group and regulates and controls the protein kinase of significant difference；Integrate it is all there are the protein kinase of significant difference, it is comprehensive to measure, obtain key protein matter kinases.The method of the invention can provide small-scale candidate kinase for the Mechanism Study of cells transdifferentiate, research for cells transdifferentiate mechanism provides direction, high degree reduces screening range, improve the accuracy of Efficiency and experiment, it is covert to shorten the R&D cycle, experiment success rate is improved, mechanism of cell differentiation research cost is reduced.
Description
Technical field
It is closed the present invention relates to technical field of cell biology, in particular to integrate in multiple groups data-speculative cells transdifferentiate
The method of key protein kinase.
Background technique
With the development and perfection of the technologies such as body-cell neucleus transplanting, cell fusion and cells transdifferentiate, majority is had been demonstrated that
The nucleus of zooblast has totipotency, and the destiny of well differentiated cell is also not irreversible.Specific cell class
Type suitably reprogramming transcription factor induction under can transdifferentiation be another cell type, transdifferentiation technology be mankind's cell disease
Model and molecule replace therapy to provide brand-new thinking.In recent years, cell reprogramming makes a breakthrough, and screens for the first time
The cell reprogramming factor obtains inductive pluripotent stem cells, it was demonstrated that the reprogramming based on transcription factor compares with known technology more
The conversion ratio of cell type can be improved.
Cells transdifferentiate, which refers to, does not pass through stem cell or progenitor cells state for a kind of mature body cell, is directly translated into another
The biological process of kind mature cell.Cells transdifferentiate is that the conversion of research cell fate and clinical solution organ or tissue supply are dilute
Few important channel has great researching value and application value.In the past few years there are many researchers to complete cell to turn to divide
The research of change, but the research of the regulation and its core mechanism during its cells transdifferentiate, since research difficulty is higher, researcher
It is less, also fail to apparent progress.
The Mechanism Study of cells transdifferentiate is usually by the way of Large-scale Screening at present, and this filtering mode is at high cost
High, without specific purpose, the period is longer, and success rate is extremely low, to many biological experiments without obvious practical significance and help,
This seriously constrains the research and development of cells transdifferentiate mechanism.
Summary of the invention
In view of this, integrating key in multiple groups data-speculative cells transdifferentiate the object of the present invention is to provide a kind of
The method of protein kinase, to solve the above problems.
The present invention is achieved through the following technical solutions: being integrated key protein matter in multiple groups data-speculative cells transdifferentiate and is swashed
The method of enzyme, comprising the following steps:
(1) prepare the cell sample during entire cells transdifferentiate, be divided into transcript profile and protein phosphorylation group,
And detection is learned by group respectively and obtains every group of corresponding initial data；
(2) every group of detected initial data of group is pre-processed, expression significant difference is screened by transcript profile
Protein kinase, the protein kinase and regulation significant difference of active significant difference are screened by protein phosphorylation group
Protein kinase；
(3) all expression are integrated, activity, regulate and control to have the protein kinase of significant difference, it is comprehensive to measure each albumen
The difference of matter kinases obtains the key protein matter kinases during cells transdifferentiate.
The working principle of the technical scheme is as follows cells transdifferentiate mainly passes through the transcription factor for being overexpressed some keys,
Inducing cell the turn of the wheel, thus the data of transcript profile level can more intuitively observe the difference of gene expression in cell.
Meanwhile protein phosphorylation participates in almost all creatures process as one of most important protein post-translational modification, it can
Regulating and controlling effect can be played during cells transdifferentiate.Therefore, it can be analyzed by protein phosphorylation group according to Chi-square Test
Obtain the activity difference of protein kinase and regulation difference in cell.Then by one group of number of transcript profile by way of group detection
The comprehensive analysis of Key kinases during cells transdifferentiate is carried out according to two groups of data with protein phosphorylation group.Inspection is learned using group
The mode of survey can help researcher faster more directly to find the core regulatory factor in bioprocess, and the mode of single group is examined
The aspect of survey is limited, and there are certain limitations for itself, and the mode of multiple groups then can more provide a comprehensive observation and assessment.
In order to which method of the present invention is better achieved, further, the cell sample in the step (1) includes also
The cell of material time point in derived cell, conversion end and successful transformed cells and the conversion process not converted,
Wherein the cell of material time point includes beginning state cell, intermediate state cell, maturity state cell, stable state cell.
In order to which method of the present invention is better achieved, further, which is characterized in that described in the step (2)
Preprocessing process is carried out to the initial data of transcript profile are as follows: using Tophat or Cufflink software to the initial data of transcript profile
It carries out reading disconnected matching and quantify.
In order to which method of the present invention is better achieved, further, expression significant difference is screened by transcript profile
Protein kinase detailed process are as follows: according to the initial data for the transcript profile that pretreatment is completed, analyze each protein kinase and exist
The differential expression of different samples, setting threshold value are p < 0.01, the significant protein kinase of screening differential expression.
It is further, described to protein phosphorus in the step (2) in order to which method of the present invention is better achieved
The initial data of acidification group carries out preprocessing process are as follows: carries out searching library and quantitative analysis using MaxQuant software, obtains each egg
The data and its strength information of white matter kinase phosphorylation sites, and swashed using the upstream of each phosphorylation site of iGPS software prediction
Enzyme.
In order to which method of the present invention is better achieved, further, in the step (2), pass through protein phosphoric acid
Change group screens the detailed process of active significant difference protein kinase are as follows: the original completed by the pretreatment of protein phosphorylation group
Beginning data add up phosphorylation site intensity of each protein kinase in corresponding cell sample, it follows that all
Intensity of the protein kinase in corresponding cell sample analyzes the otherness of protein kinase intensity in different samples, setting
Threshold value is p < 1*10-5, to screen the significant protein kinase of activity difference.
In order to which method of the present invention is better achieved, further, in the step (2), pass through protein phosphoric acid
The detailed process of change group screening regulation significant difference protein kinase are as follows: the original completed by the pretreatment of protein phosphorylation group
Beginning data comb the relationship of protein kinase and phosphorylation site, then construct kinase regulatory network, and by the tune of each kinases
It controls network split and lowers two sub-networks at site up-regulation and site, up-regulation net is calculated separately with the variation multiple of phosphorylation site
Network and the total value for lowering network, difference of the check analysis kinases in upper downward network, setting threshold value is p < 0.01, to sieve
Select the significant protein kinase of differential expression.
In order to which method of the present invention is better achieved, further, the activity of the protein kinase or regulation are equal
It is obtained by Chi-square statistic analysis.
In order to which method of the present invention is better achieved, further, in the step (3), by ballot method come comprehensive
The difference for measuring each protein kinase is closed, thus the key protein matter kinases during obtaining cells transdifferentiate.
Compared with prior art, the present invention have the following advantages that and the utility model has the advantages that
(1) method of the present invention can provide small-scale candidate kinase for the Mechanism Study of cells transdifferentiate, be
The research of cells transdifferentiate mechanism provides direction, and high degree reduces screening range, improves the standard of Efficiency and experiment
True property, it is covert to shorten the R&D cycle, experiment success rate is improved, mechanism of cell differentiation research cost is reduced；
(2) method of the present invention observes cells transdifferentiate process using the method that group learns detection, is analyzed by calculating
Speculate the important kinases during cells transdifferentiate, the Large-scale Screening of blind sieve is avoided to work, integration includes transcript profile and albumen
The data of multiple groups level including matter phosphorylation group speculate to multi-angle stage construction Key kinases, more comprehensively and reliable；
(3) method of the present invention has great directive function and help to the research of cells transdifferentiate mechanism, removes
It can be applied to other than biological experiment, moreover it is possible to it plays a role in promoting to the development of many biological engineerings, while
New opportunity to develop is provided for the research mechanism of cells transdifferentiate.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And it can be implemented in accordance with the contents of the specification, and in order to allow above and other objects of the present invention, feature and advantage can
It is clearer and more comprehensible, the followings are specific embodiments of the present invention.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described.It should be appreciated that the following drawings illustrates only certain embodiments of the present invention, therefore it is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the flow diagram of the method for the invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Usually
The component for the embodiment of the present invention being described and illustrated herein in the accompanying drawings can be arranged and be designed with a variety of different configurations.Cause
This, is not intended to limit claimed invention to the detailed description of the embodiment of the present invention provided in the accompanying drawings below
Range, but it is merely representative of selected embodiment of the invention.Based on the embodiment of the present invention, those skilled in the art are not doing
Every other embodiment obtained under the premise of creative work out, shall fall within the protection scope of the present invention.
It should also be noted that similar label and letter indicate similar terms in following attached drawing, therefore, once a certain Xiang Yi
It is defined in a attached drawing, does not then need that it is further defined and explained in subsequent attached drawing.
Embodiment 1:
The method for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate is disclosed in detail in the present embodiment,
Detailed process, as shown in Figure 1, this method collects the various cell samples in cell differentiation procedure based on a group data for detection,
By the expression of the protein kinase in multiple samples, activity and the significant difference for regulating and controlling three indexs, synthesis obtains cell
Key protein matter kinases in transdifferentiation.
Detailed process is as follows:
(1) prepare cell sample, cell sample is uniformly divided into transcript profile, protein phosphorylation group, to every group respectively into
Row group learns detection, obtains corresponding initial data；
Wherein, cell sample includes the derived cell not converted, conversion terminates and successful transformed cells, Yi Jizhuan
Beginning state cell, intermediate state cell, maturity state cell during change, stable state cell.
The acquisition of transcript profile initial data: the original image of the DNA sequence dna of transcript profile cell sample is obtained by sequenator
File, the DNA for then obtaining transcript profile cell sample after base identifies and analyzes conversion for the raw image files are original
Sequencing sequence, the DNA primitive sequencer sequence are the initial data of transcript profile.The DNA sequencer used can be abi prism
310 type Genetic Analysers (pe abi company, the U.S.).
The acquisition of protein phosphorylation group initial data: the protein peptide of each cell sample is directly obtained by mass spectrometer
The mass spectrogram of section, which is the initial data of protein phosphorylation group.The mass spectrometer can be LTQ Orbitrap
XL ETD mass spectrograph (silent winged scientific and technological (China) Co., Ltd of generation that of match).
(2) initial data of the cell sample of transcript profile, protein phosphorylation group is handled respectively；
Transcript profile original data processing: using Tophat, Cuffiink to the progress read matching of transcript profile data and quantitatively.
Wherein, the use of software Tophat, Cufflink is generally used after Tophat compares completion using default parameters
Cufflinks carries out the assembling of transcript and quantifies, this step can take both of which, i.e.-G mode and-g mode, the former
New transcript is not assembled, and can ignore not structurally compatible with any reference
The comparison of transcript, and the latter is assembled using RABT (Reference Annotation Based Transcript), meeting
Obtain new transcript and new gene, since the former does not assemble new transcript, can all reads all with known transcript ratio
It is right, cause the two quantitative differences；In addition, the R packet also having without assembling, counts the number of corresponding reads, directly to calculate
RPKM value.Wherein, RPKM value refers in the reads on every 1,000,000 map on map to every 1,000 bases of exon
Reads number.
Protein phosphorylation group original data processing: the mass spectrum picture and text input for the protein peptide fragment that mass spectrograph is obtained
MaxQuant software carries out protein peptide fragment using the default parameters of MaxQuant software and searches library, to obtain protein phosphorus
The quantitative data of polyadenylation sites, data and its strength information including phosphorylation site, the protein that mass spectrograph will be detected
Phosphorylation site sequence inputting to iGPS software, using the threshold value of iGPS software default carry out prediction phosphorylation site it is possible on
Kinases is swum, and it is recorded.It the use of mass spectrograph can be LTQ Orbitrap XL ETD mass spectrograph (match Mo Feishier section
Skill (China) Co., Ltd).
(3) pass through transcript profile screening expression significant difference protein kinase: use the result of Cufflink software as
Data are finally entered, the quantitative values FPKM under samples different in transcript profile is subjected to gene expression with Cuffdiff software, is obtained
In the protein kinase for transcribing upper differential expression.
For the significant protein kinase of screening differential expression, threshold value p has to < 0.05, therefore transcribes in the present embodiment
The threshold value p < 0.01 of expressed differential proteins matter kinases in group.
(4) protein kinase of active significant difference is screened by protein phosphorylation group: determining that corresponding upstream is swashed first
The intensity of its all phosphorylation site, to each kinases a, is carried out phase in each sample by the intensity of enzyme in the sample
Add, obtains intensity of the kinases in this sample.The intensity value of kinases in all samples is added to obtain kinases total intensity value A work
For the comparison data for finally carrying out activity difference analysis；Kinases total intensity value A such as following formula:
PK is kinases total intensity value A.
Then, using Chi-square Test analysis kinases intensity in different samples otherness, given threshold p < 1*10-5,
Example is as follows:
It is a in the intensity of sample 1 such as to kinases 1, the intensity in sample 2 is b, and the kinases overall strength of sample 1 is A,
The kinases overall strength of sample 2 is B, carries out Chi-square Test to intensity of each kinases in different samples using 2 × 2 contingency tables,
It is as follows:
| Certain kinases intensity | Other kinases overall strengths | It amounts to | |
| Sample 1 | a | A-a | A |
| Sample 2 | b | B-b | B |
| It amounts to | a+b | A+B-a-b | A+B |
Thus the kinases of active significant difference is filtered out.
(5) pass through the protein kinase of protein phosphorylation group screening regulation significant difference: firstly, with protein kinase phosphorus
Acidizing protein substrate, the amino acid of phosphorylation are phosphorylation site.Using protein kinase and protein substrate as point, kinases
Regulation protein substrate constructs kinase regulatory network as side, and by the regulated and control network of each kinases split into site up-regulation and
Lower two sub-networks in site；
Then, up-regulation network is calculated separately with the variation multiple of phosphorylation site and lowers the total value of network.For example, sample
1 compares with sample 2, phosphorylation site sample 2 intensity value divided by the intensity value in sample 1, be more than or equal to 1 for up-regulation subnet
Network lowers sub-network less than 1.
Finally, the difference using Chi-square Test analysis kinases in upper downward network, is used as threshold value using p value < 0.01,
The kinases of screening regulation significant difference.
(6) kinase expression difference, activity difference and the difference kinases for regulating and controlling three levels of difference are integrated, it is comprehensive using ballot method
The difference for measuring each kinases is closed, the Key kinases during cells transdifferentiate are finally obtained.
(9) Key kinases are analyzed: the difference for integrating kinase expression difference, activity difference and regulation three levels of difference swashs
Enzyme.
It is specific as follows:
By differential expression, activity difference and regulation difference obtain the difference kinases of three levels, and each level is 1 ticket,
Kinases more than poll is more crucial.For example, the kinases A in sample 1, there are differential expressions, but there is no poor activity exclusive or to regulate and control
Difference is then 1 ticket, and the kinase b in sample 2, there are activity differences, but is then also 1 there is no differential expression or regulation difference
Ticket.Kinase c in sample 1 exists simultaneously differential expression, activity difference and regulation difference, is then 3 tickets, kinase c is obviously more to close
The kinases of key.
In the description of the present invention, it is also necessary to which explanation is unless specifically defined or limited otherwise, term " setting ",
" connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected.It can be
Mechanical connection, is also possible to be electrically connected.It can be directly connected, two can also be can be indirectly connected through an intermediary
Connection inside element.For the ordinary skill in the art, above-mentioned term can be understood in the present invention with concrete condition
In concrete meaning.
In the description of the present invention, it should be noted that the orientation or positional relationship of the instructions such as term "left", "right" is base
In orientation or positional relationship shown in the drawings or the invention product using when the orientation or positional relationship usually put, only
It is that for the convenience of describing the present invention and simplifying the description, rather than the device or element of indication or suggestion meaning must have specifically
Orientation is constructed and operated in a specific orientation, therefore is not considered as limiting the invention.In addition, term " first ", " the
Two " etc. are only used for distinguishing description, are not understood to indicate or imply relative importance.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate, which is characterized in that including
Following steps:
(1) prepare the cell sample during entire cells transdifferentiate, be divided into transcript profile and protein phosphorylation group, and divide
It Tong Guo not group detection every group of corresponding initial data of acquisition；
(2) every group of detected initial data of group is pre-processed, the egg of expression significant difference is screened by transcript profile
White matter kinases screens the protein kinase of active significant difference by protein phosphorylation group and regulates and controls the albumen of significant difference
Matter kinases；
(3) all expression are integrated, activity, regulate and control to have the protein kinase of significant difference, synthesis is measured each protein and swashed
The difference of enzyme obtains the key protein matter kinases during cells transdifferentiate.
2. the method according to claim 1 for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate,
It is characterized in that, the cell sample in the step (1) include the derived cell not converted also, conversion terminate and it is successful
The cell of material time point in transformed cells and conversion process.
3. the method according to claim 2 for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate,
It is characterized in that, the cell of material time point includes initial state cell, intermediate state cell, mature shape in the conversion process
State cell, stable state cell.
4. described in any item key protein matter of integrating in multiple groups data-speculative cells transdifferentiate swash according to claim 1~3
The method of enzyme, which is characterized in that in the step (2), the initial data to transcript profile carries out preprocessing process are as follows: uses
Tophat or Cufflink software is carried out reading disconnected matching and be quantified to the initial data of transcript profile.
5. the method according to claim 4 for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate,
It is characterized in that, screening the protein kinase detailed process of expression significant difference by transcript profile are as follows: completed according to pretreatment
The initial data of transcript profile analyzes each protein kinase in the differential expression of different samples, and setting threshold value is p < 0.01,
Screen the significant protein kinase of differential expression.
6. described in any item key protein matter of integrating in multiple groups data-speculative cells transdifferentiate swash according to claim 1~3
The method of enzyme, which is characterized in that in the step (2), the initial data to protein phosphorylation group carries out pretreated
Journey are as follows: carry out searching library and quantitative analysis using MaxQuant software, obtain each protein kinase phosphorylation site data and its
Strength information, and use the upstream kinases of each phosphorylation site of iGPS software prediction.
7. integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate according to claim 6 is described in any item
Method, which is characterized in that in the step (2), active significant difference protein kinase is screened by protein phosphorylation group
Detailed process are as follows: the initial data completed by the pretreatment of protein phosphorylation group, by each protein kinase corresponding thin
Phosphorylation site intensity in born of the same parents' sample adds up, it follows that all proteins kinases is strong in corresponding cell sample
Degree analyzes the otherness of protein kinase intensity in different samples, and setting threshold value is p < 1*10-5, to screen poor activity
Different significant protein kinase.
8. integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate according to claim 6 is described in any item
Method, which is characterized in that in the step (2), regulation significant difference protein kinase is screened by protein phosphorylation group
Detailed process are as follows: the initial data completed by the pretreatment of protein phosphorylation group combs protein kinase and phosphorylation position
Then the relationship of point constructs kinase regulatory network, and the regulated and control network of each kinases is split into site up-regulation and site downward
Two sub-networks, calculate separately up-regulation network with the variation multiple of phosphorylation site and lower the total value of network, and check analysis swashs
Difference of the enzyme in upper downward network, setting threshold value is p < 0.01, to screen the significant protein kinase of differential expression.
9. the side according to claim 7 or 8 for integrating key protein matter kinases in multiple groups data-speculative cells transdifferentiate
Method, which is characterized in that the activity of the protein kinase or regulation are obtained by Chi-square statistic analysis.
10. described in any item key protein matter of integrating in multiple groups data-speculative cells transdifferentiate swash according to claim 1~3
The method of enzyme, which is characterized in that in the step (3), the difference for measuring each protein kinase is integrated by ballot method, from
And obtain the key protein matter kinases during cells transdifferentiate.
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Application publication date: 20190329 |