CN106546674A - With the method and its special purpose device of a small amount of sample fast enriching, separation, identification and quantitative endogenous transcription factor and its compound - Google Patents
With the method and its special purpose device of a small amount of sample fast enriching, separation, identification and quantitative endogenous transcription factor and its compound Download PDFInfo
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- CN106546674A CN106546674A CN201610917464.8A CN201610917464A CN106546674A CN 106546674 A CN106546674 A CN 106546674A CN 201610917464 A CN201610917464 A CN 201610917464A CN 106546674 A CN106546674 A CN 106546674A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/08—Preparation using an enricher
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention discloses a kind of method and its special purpose device with a small amount of sample fast enriching, separation, identification and quantitative endogenous transcription factor and its compound.The characteristics of the method utilizes transcription factor and its compound be combined with sequence specific DNA element, by the nucleoprotein extracted from biological specimen or holoprotein in special purpose device TOT posts be combined with transcription factor series combination sequence (TFRE DNA, DNA baits) magnetic bead carry out the incubation of micro-scale volume, from efficiently concentrating in nucleoprotein or holoprotein, endogenous transcription factor and its compound are separated, subsequently further carry out the identification of transcription factor and its compound, quantitative.The method of the present invention is quick, stable, efficient, repeatable, can be widely applied to the aspects such as biological study, medical test, drug screening, the special purpose device application of the present invention is easy, can store, small volume, kit can be prepared into, it is applied in the fields such as clinical detection, scientific research, has a extensive future.
Description
Technical field
The invention belongs in biological technical field destination protein enrichment and separation method, more particularly to one kind only needs a small amount of
Sample can fast enriching, separations, the method and its special purpose device of identification and quantitative endogenous transcription factor and its compound with answer
With.
Background technology
About contain 1500 transcription factor encoding genes in human genome, be the second big class egg of human genome coding
In vain, 50 families can be divided into according to the difference of binding domain.Transcription factor almost participates in the regulation and control of all bioprocess, including send out
Educate, break up and signal transduction etc..Therefore, greatly concern is constantly subjected to the research of transcription factor and its compound.
Transcription factor gene expression abundance in the cell extremely low (only accounting for the 0.01-0.001% of total protein of cell), with tradition side
It is extremely difficult that method analyzes transcription factor expression spectrum in protein group level.The pure of transcription factor is carried out by traditional chromatographic process
Change and extract into hectolitre cell culture, the albumen obtained by 10,000-100,000 times of enrichment is also only enough in
The analysis of chemistry and function.It is effective hand for isolating and purifying transcription factor complex that affinity purification is carried out using specific antibody
Section, but one side antibody cost is too high, and on the other hand, there is antibody in only small amounts of transcription factor and its Molecular regulator at present,
Which greatly limits the application of antibody affinity purification method, that is to say, that this strategy is only capable of carrying out indivedual transcription factors point
Analysis, it is difficult to realize extensive concentration and separation and the identification to all transcription factors.
Additionally, using transcription factor series combination sequence (concatenated transcription factor
Response elements, catTFRE) effective enrichment of the realization to transcription factor, and transcription factor is entered with reference to mass-spectrometric technique
Row is identified and quantitative.The transcription factor series combination sequence has applied for a patent (patent publication No.：ZL 20111
0457108.X), by 2800 base compositions, the double copy core binding members comprising 100 transcription factors, each turns sequence
Double copy core binding members of the record factor are spaced 3 base-pairs (nucleotide sequence of 3 base-pairs is arbitrary).The method
The binding activity of transcription factor and DNA can be obtained, it is largely effective to transcription factor expression spectrum research, but the method needs milli
Gram level Nuclear extract extract is enriched with, and the time at least needs two days, and can not carry out high flux operation, so as to limit
Range of application and detection efficiency are made.
Therefore, system research is carried out to the binding activity of transcription factor and DNA on the level of protein group, needs one kind
Using a small amount of sample transcription factor and its compound can be carried out quick separating, purifying and identify high flux, high accuracy,
The experimental technique of high sensitivity and repeatability.
The content of the invention
First purpose of the present invention be to provide one kind only need a small amount of sample can fast enriching, separations, identify and quantitatively
The method of endogenous transcription factor and its compound.
It is provided by the present invention with a small amount of sample fast enriching, separation, identification and quantitative endogenous transcription factor and its compound
The method of thing, is first to extract nucleoprotein or holoprotein from biological specimen, then by the transcription factor tandem junction of biotin labeling
Close sequence (TFRE DNA, DNA bait) mix with the coated magnetic bead of biotin-labeled pentylamine, then by extraction nucleoprotein or holoprotein
It is incubated with the magnetic bead for being combined with transcription factor series combination sequence, obtains being combined with transcription factor series combination sequence specific
Albumen (i.e. endogenous transcription factor and its compound), then endogenous transcription factor and its compound are eluted from magnetic bead,
Then LC-MS (liquid chromatograph mass spectrography) detections are carried out to endogenous transcription factor and its compound, and uses bioinformatics work
Tool and method determine species, title and its content of endogenous transcription factor and its compound.
Transcription factor series combination sequence (the concatenated transcription factor response
Elements, catTFRE) refer to connected by the joint sequence (Linker) of 3-5bp transcription factor AP-1 1, AR, BRCA1,
CEBPA、CREB1、E2F1、ELK1、ELK4、ESR1、ETS1、EWSR1-FLI1、FEV、FOXA1、FOXC1、FOXD1、FOXF2、
FOXI1、FOXL1、FOXO3、Fra-1、GATA2、GATA3、GR、HIF1A::ARNT、HLF、HNF1B、HNF4A、HOXA5、
INSM1、IRF1、IRF2、JunB、JunD、MAX、MEF2A、MIZF、MYC::MAX、Myf、MZF1_1-4、MZF1_5-13、NF-
kappaB、NFATC2、NFE2L2、NFIC、NFIL3、NFKB1、NFYA、NHLH1、NKX3-1、NR1H2::RXRA、NR2F1、
NR3C1、NR4A2、Pax6、PBX1、PDX1、PLAG1、PPARG、PR、PXR-1:RXR-alpha、RAR-alpha、RAR-
alpha:RXR-gam、RAR-beta:RXR-alpha、REL、RELA、REST、RFX1、RFX2、RFX3、RFX5:RFXAP:
RFXANK、RORA_1、RORA_2、RREB1、RXR::RAR_DR5、RXRA::VDR、SOX10、SOX9、SP1、SPI1、SPIB、
SRF、SRY、STAT1、STAT5A、T3R-beta1、TAL1::TCF3、TBP、TEAD1、TFAP2A、TLX1::NFIC、TP53、
USF1, WT1-del2, WT1-KTS, WT1I, WT1I-del2, WT1I-KTS, XBP-1, YY1 and ZNF354C DNA binding members
In it is two or more after the artificial synthesized DNA sequence dna that obtains.General endogenous transcription used in the embodiment of the present invention because
In the transcription factor series combination sequence such as sequence table of son and its compound shown in sequence 1, the sequence does not have cell-specific, fits
For people source and all biological specimens in mouse source.Sequence 1 is by 2800 base compositions, double comprising above-mentioned 100 kinds of transcription factors
Copy core binding member, double copy core binding members of each transcription factor are spaced 3 base-pair (cores of 3 base-pairs
Nucleotide sequence is arbitrary).Transcription factor series combination sequence is introduced in also referring to Chinese patent ZL201110457108X
's.The sequence can be combined with endogenous transcription factor and its compound, so as to the transcription factor in biological sample and its compound
Thing is enriched with.
Available conventional method is marked with biotin to transcription factor series combination sequence, affine with biotin to magnetic bead
Element is coated with, biotin and biotin-labeled pentylamine can specific bond, such that it is able to transcription factor series combination sequence is combined
To on magnetic bead.After the nucleoprotein or holoprotein of extraction are incubated with the magnetic bead for being combined with transcription factor series combination sequence,
Transcription factor and its compound in nucleoprotein or holoprotein is combined with transcription factor series combination sequence specific.Remove not special
With reference to albumen (foreign protein), the albumen for obtaining being combined with transcription factor series combination sequence specific (i.e. endogenous transcription factor and
Its compound), reach the purpose for being enriched with endogenous transcription factor.The albumen that will be combined with transcription factor series combination sequence specific again
Elute from magnetic bead, reach the purpose for separating endogenous transcription factor and its compound.Then to transcription factor tandem junction
The albumen for closing sequence specific combination carries out LC-MS (liquid chromatograph mass spectrography) detections, true with bioinformatics tools and method
The species of the fixed albumen (i.e. endogenous transcription factor and its compound) combined with transcription factor series combination sequence specific, title and
Its content, so as to reach identification and quantitative purpose.
The biological sample refers to the nucleoprotein or holoprotein of extraction, refers to that consumption is only 1-250 μ g on a small quantity.Use a small amount of sample
The advantage detected by product is to save experiment material and experimental period, improves efficiency, by detectable biological sample expanded range.
The consumption of the transcription factor series combination sequence of biotin labeling is 0.5-1pmol.
The consumption of the coated magnetic bead of biotin-labeled pentylamine is that (commercialization magnetic bead, directly measures volume required 20 μ L, about contains
1.2-1.4×107Individual magnetic bead).
The nucleoprotein or holoprotein can be in special purpose device TOT posts with the magnetic bead for being combined with transcription factor series combination sequence
In carry out being incubated, the operation such as clean, separate.Therefore, second object of the present invention is to provide one kind and is used for fast enriching, divides
From, identification and the special purpose device TOT posts of quantitative endogenous transcription factor and its compound.
TOT posts (abbreviation of English TFRE on tips is enriched with reaction column) provided by the present invention, by suction nozzle, for solid
Determine the sleeve pipe and centrifuge tube composition of suction nozzle, the suction nozzle inner tail end sets membrane structure closing, built-in to be combined with transcription factor series connection
The magnetic bead of binding sequence；During use, sleeve pipe is put in centrifuge tube, fixes suction nozzle, sleeve pipe and centrifuge tube in suction nozzle plug-in-sleeve.
The membrane structure of the suction nozzle inner tail end is preferably the C18 films of 47mm.
Third object of the present invention is to provide a kind of above-mentioned special purpose device TOT posts and a small amount of sample fast enriching, divides
From, identification and the method for quantitative endogenous transcription factor and its compound.
Concrete grammar, it may include following steps：
1) by the transcription factor series combination sequence (TFRE DNA, DNA bait) and biotin-labeled pentylamine of biotin labeling
Coated magnetic bead mixing；
2) magnetic bead for being combined with transcription factor series combination sequence is added in the suction nozzle of TOT posts；
3) nucleoprotein extracted from biological specimen or holoprotein sample are added in the suction nozzle of TOT posts, incubation makes sample
In endogenous transcription factor and its compound combined with transcription factor series combination sequence specific；
4) magnetic bead is cleaned, removes the albumen (foreign protein) of non-specific bond；
5) add pancreatin enzymolysis, it is therefore an objective to the albumen that will be combined with transcription factor series combination sequence specific using pancreatin
The small peptide section that (endogenous transcription factor and its compound) is cut into length for 5-25 amino acid not etc., the molecular weight of these peptide fragments is
Peptide fingerprinting for the albumen is composed, and can be identified by LC-MS (reversed-phase liquid chromatography-mass spectrometry)；
6) by enzymolysis after the albumen (peptide fragment) combined with transcription factor series combination sequence specific and Beads enrichment；
7) albumen (peptide fragment) to being combined with transcription factor series combination sequence specific carry out LC-MS (reversed-phase liquid chromatography-
Mass spectrometry) detection obtains spectrogram, and spectrogram determination and transcription factor series combination are understood with bioinformatics tools and method
The species of albumen (i.e. endogenous transcription factor and its compound), title and its content that sequence specific is combined.
In the above-mentioned methods, the step 1) need first to synthesize life using round pcr and the primer with biotin labeling
The transcription factor series combination sequence of thing element mark, the magnetic bead is preferably the coated M280 magnetic beads of biotin-labeled pentylamine, biological
The consumption of the transcription factor series combination sequence of element mark is 0.5-1pmol, and the consumption of the coated magnetic bead of biotin-labeled pentylamine is
20μL(1.2-1.4×107Individual magnetic bead), the transcription factor series combination sequence of biotin labeling and biotin-labeled pentylamine are coated with
M280 magnetic beads 4 DEG C it is vertical mix 30min, biotin avidin can be combined with biotin, so as to transcription factor is connected
Binding sequence is attached on magnetic bead.
The step 2) be combined with transcription factor series combination sequence magnetic bead consumption be 20 μ L.
The step 3) nucleoprotein or holoprotein consumption be 1-200 μ g.
The step 4) magnetic bead is cleaned successively with NETN solution and PBS solution.
The step 5) use trypsin digestion.
The step 6) separate mode can be with the endogenous transcription factor of acetonitrile extraction acquisition and its compound peptide fragment；Also can use not
(volumetric concentration is followed successively by 6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, 35% acetonitrile solution and washes same concentration
It is de-, the peptide fragment of 9 acetonitrile concentration wash-outs is merged into into 6 component peptide fragments and (is followed successively by 6%+25%, 9%+30%, 12%+
35%th, 15%, 18%, 21%), step 7) in each component peptide fragment is detected respectively, the protein group number of depth covering can be obtained
According to.
The step 7) in LC-MS (liquid chromatograph mass spectrography) detection method be：The peptide fragment drained detection sample is used
Redissolve containing 0.1% formic acid solution and loading.Using Easy-nLC 1000nano-HPLC system (Thermo Fisher
Scientific companies) and Orbitrap Fusion mass spectrographs (Thermo Fisher Scientific companies) detected.
Liquid phase uses homemade nano-HPLC C18 post (pre-columns：3 μm,2cm×100μm ID；Analytical column：1.9 μm,
10cm × 100 μm ID) peptide fragment is separated, flow velocity 400nl/min, gradient timetable 75min, B liquid is (containing 0.1% formic acid
Acetonitrile solution) ratio rises to 30% from 5%.Ion gun uses 2000V voltages and 320 DEG C of temperature.One-level is scanned using Orbi, female
Ion scan scope 300-1400m/z, resolution ratio 120,000 (m/z 200), AGC 5e5, maximum injection length 50ms.Two grades
Using linear ion hydrazine, fragmentation energy 32%.The bioinformatics tools and method are by searching library software by the spectrogram of acquisition
2.3 (Matrix of Mascot in Proteome Discoverer 2.0 (Thermo Fisher Scientific)
Science Inc) carry out searching storehouse, searching database behaviour source National Center for Biotechnology
Information (NCBI) RefSeq Protein Data Banks (updated on 04-07-2013,32015protein
Entries), parameter is set to：Parent ion mass deviation 20ppm, product ion mass deviation 0.5Da；Allow two leakage enzyme sites；
Dynamic embellishment includes phosphor (Y, Chinese：Tyrosine residue phosphorylation modification), phosphor (ST, Chinese：Serine or Soviet Union
Histidine residue phosphorylation modification), acetyl (protein N-term, Chinese：N-terminal acetylation modification) and oxidation (M,
Chinese：Methionine residues oxidative modification)；The horizontal FDR of peptide fragment (false discovery rate) allows 1%.
The step 2)-step 6) reactant can be made to be placed in the suction nozzle inner bottom of TOT posts with the method for centrifugation.
Fourth object of the present invention is to provide a kind of a small amount of sample fast enriching, separation, identification and quantitatively endogenous turn
The kit of the record factor and its compound.
Kit provided by the present invention, including being combined with transcription factor series combination sequence (TFRE DNA, DNA bait)
Magnetic bead and special purpose device TOT posts.
The magnetic bead for being combined with transcription factor series combination sequence is placed in the suction nozzle inner bottom of TOT posts, with sealing
Film is encapsulated, and is placed in 4 DEG C of preservations.Directly take during use (detected by adding nucleoprotein or holoprotein), at three months
Shelf-life in do not have post effect reduce phenomenon.
The invention provides one kind only needs fast enriching, separation, identification and quantitative endogenous transcription factor by a small amount of sample
And its method and its special purpose device TOT posts of compound.The method can be with sequence-specific using transcription factor and its compound
The characteristics of DNA element is combined, by extract nucleoprotein or holoprotein (sample) in special purpose device TOT posts be combined with transcription because
The magnetic bead of sub- series combination sequence (TFRE DNA, DNA bait) carries out the incubation of micro-scale volume, high from nucleoprotein or holoprotein
Effect is enriched with, separates endogenous transcription factor and its compound, subsequently further carries out the identification of transcription factor and its compound and determines
Amount.
The present invention has advantages below：
1) sample size is little：Detected by only needing minimal amount of nucleoprotein or holoprotein sample, and be obtained preferably
Detection results so that the biological specimen amount for detection is substantially reduced, and is extended based on transcription factor series combination sequence (TFRE
DNA, DNA bait) endogenous transcription factors and its enrichment of compound, separation, identification and quantitative approach range of application, such as
Gastric mucosa sample that the cell of 96 orifice plate cultures, gastroscope are sampled etc. can be carried out the detection and research of transcription factor expression spectrum.
2) detection time is short：3 hours are needed after obtaining nucleoprotein only by obtain testing result.The mistake of transcription factor enrichment
Journey is easy to operate, and step is few, it is only necessary to the enzymolysis time of 1 hour, further shorten the used time of whole flow process.With tradition
TFRE methods need the test period of at least two days to compare, and the method only needs several hours, therefore is a kind of fast enriching, divides
From, identification and the method for quantitative endogenous transcription factor and its compound.
3) method has extensibility：The magnetic bead for combining transcription factor is directly put in into enzyme on miniature reverse-phase chromatographic column
Solution, is then eluted with variable concentrations acetonitrile solution, peptide fragment is divided into 6 components, is detected respectively, can obtain the albumen of depth covering
Matter group data.
4) application space is wide：In addition to the research application in laboratory, the method also has wide application space.Transcription factor
Nuclear receptor family particularly therein is the popular target spot of current drug design, and existing medicine is also tended to by activation/suppress
Transcription factor is playing its drug effect.Panorama type scans transcription factor dynamic change and especially weighs in drug action mechanism and side effect research
Will.The present invention extracts nucleoprotein or holoprotein as sample by the use of very small amount biological specimen (such as cell of 96 orifice plate cultures etc.)
Product, you can complete high throughput identification and the quantitative endogenous cellular transcription factor dynamic change under medicine irritation, this is greatly improved
The efficiency and accuracy of drug research.The 293T cell detections collected with the single hole of 96 orifice plates in the embodiment of the present invention 1 are to surpassing
Cross 200 kinds of endogenous transcription factors.
In sum, the method for the present invention is quick, stable, efficient, repeatable, can be widely applied to biological study, medical science
The aspects such as inspection, drug screening, the special purpose device application of the present invention is easy, can store, small volume, can be prepared into reagent
Box, is applied in the fields such as clinical detection, scientific research, has a extensive future.
The present invention is described in further details with reference to specific embodiment.
Description of the drawings
Fig. 1 is the flow process of the method for fast enriching of the present invention, separation, identification and quantitative endogenous transcription factor and its compound
Figure；
Fig. 2 shows the special purpose device TOT posts of the present invention：Fig. 2A is sucker structure；Fig. 2 B are the TOT posts before assembling；Fig. 2 C
For the TOT posts before and after assembling；
The photo of single TOT posts after Fig. 3 A assemblings；Fig. 3 B are photo of multiple TOT posts in centrifuge；
Fig. 4 is the XIC figures of three sections of small peptides.
Specific embodiment
In following embodiments, method therefor is conventional method if no special instructions, and concrete steps can be found in：
《Molecular Cloning:A Laboratory Manual》(Sambrook, J., Russell, David W.,
Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring
Harbor)。
The percent concentration is mass/mass (W/W, unit g/100g) percent concentration, matter if no special instructions
Amount/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percent concentration.
The acquirement approach of the various biomaterials described in embodiment is only to provide a kind of approach of experiment acquisition to reach
To specifically disclosed purpose, the restriction to biological material source of the present invention should not be become.In fact, used biomaterial
Source be it is extensive, it is any keep on the right side of the law the biomaterial that can obtain with moral ethics can according to embodiment in carry
Show that replacement is used.
The primer is synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and specific
Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.
Embodiment 1, the fast enriching of 293T cellular endogenous transcription factor and its compound, separation, identification and quantitative
As shown in figure 1, fast enriching of the present invention, separation, the method for identification and quantitative endogenous transcription factor and its compound
Comprise the following steps：
First, obtain the transcription factor series combination sequence of biotin labeling
With carry transcription factor series combination sequence plasmid vector (plasmid vector can as pUC57, pET24a+,
PGEX4T-2, pGEX4T-1, pCMV-Myc, pGH or pcDNA-Myc etc.) for template, in upstream primer：5'-
CATTCAGGCTGCGCAACTGTTG-3' and downstream primer：5'-GTGAGTTAGCTCACTCATTAGG-3'(primers are with biology
Element mark) guiding under PCR amplification transcription factor series combination sequence.100 μ L PCR amplification systems are：10×ExTaq
10 μ L of 10 μ L of Buffer, dNTPs (2.5mM/dNTP), 1 μ L of pUC57-sdTF (about 50ng), each 1 μ L of upstream and downstream primer
(1nmol), 0.5 μ L of ExTaq, H2O 87.5μL.PCR amplification conditions are：First 94 DEG C of 2min, then 94 DEG C of 45s, 60 DEG C of 45s,
72 DEG C of 2min, totally 35 circulations, then 72 DEG C of 7min, last 4 DEG C of 30min.After PCR amplifications terminate, pcr amplification product is carried out
1% agarose gel electrophoresis, reclaims the purpose fragment of 2800bp, is sequenced, in its nucleotide sequence such as sequence table shown in sequence 1,
As a result obtain the transcription factor series combination sequence (TFRE DNA, DNA bait) of the correct high-purity biotin labeling of sequence.
The DNA sequence dna of 100 kinds of transcription factor specific bonds is contained in the transcription factor series combination sequence.
2nd, the transcription factor series combination sequence of biotin labeling and the coated magnetic bead of biotin-labeled pentylamine are attached
1) take 20 μ L M280 magnetic beads (M-280streptavodin Streptavidin MagneSpheres, are purchased from
Invitrogen companies, 1.2-1.4 × 107Individual magnetic bead) to each clean EP pipe (eppendorf is managed, centrifuge tube,
In 1.5mL), multiple pipes are placed on magnetic force Ep pipe supports (referring to that unilateral wall has magnetic Ep pipe supports, purchased from Promega companies) to be inhaled
Attached magnetic bead (is referred to M280 magnetic beads by magnetic-adsorption to the magnetic Ep pipe supports side of tool, it is ensured that not to M280 when removing supernatant
Magnetic bead causes damage), then remove supernatant；
Here, it is business application with the coated magnetic of biotin-labeled pentylamine that the reason for selecting M280 magnetic beads is the magnetic bead
Pearl, and there is magnetic, diameter 280um.
2) with 50 μ L 1 × DNA Binding Buffer (DNA combination buffers, formula：10mM Tris-HCl
(pH7.5), 1mM EDTA, 2M NaCl) washing magnetic bead；
3) 0.5pmol TFRE DNA are added, articulated system is adjusted to into 1 × DNA with 2 × DNA Binding Buffer
Binding Buffer；
4) 4 DEG C vertically mix 30 minutes；
5) (contained per 100mL with BC200 solution：40 μ L of glycerine 20mL, 3M KCl 6.7mL, 0.5M EDTA, 1M
Tris (pH7.3) 2mL) wash 2 times, siphon away all supernatants.
The purpose of washing is to provide environment for the combination of transcription factor and DNA, the reason for select BC200 as detergent is
BC200 solution salt concentrations are 200mM, consistent with vivo environment.
3rd, TOT posts are prepared (abbreviation of English TFRE on tips is enriched with reaction column)
TOT posts are made up of suction nozzle, sleeve pipe (suction nozzle fixing device) and centrifuge tube (1.5mL), referring to Fig. 2A-Fig. 2 C.Suction nozzle 1
In long pyramidal structure, inside tail end, closed that (film can prevent magnetic bead from leaking down, but allow liquid to lead to using C18 films 31
Cross), the C18 films of 47mm can be used, Fig. 2A is seen；Suction nozzle 1 is used to load magnetic bead 32 and each liquid, to complete transcription factor tandem junction
Close the enrichment of sequence-magnetic bead connection, transcription factor and its compound and separate and space is provided.Sleeve pipe 2 is used to fix suction nozzle 1, inhales
It is fixed in 1 circular hole 22 that can be inserted into 2 bottom of sleeve pipe, 2 upper step 21 of sleeve pipe for fixing with centrifuge tube 3, referring to Fig. 2 B and
Fig. 2 C；Centrifuge tube 3 coordinates Design of Centrifuge, and during use, sleeve pipe 2 is put in centrifuge tube 3, so that inhaling in 1 plug-in-sleeve 2 of suction nozzle
Head and centrifuge tube are fixed, and multiple centrifuge tubes can load centrifuge to carry out centrifugally operated to the reactant in wherein suction nozzle, referring to
Fig. 3 A and Fig. 3 B.
With reference to shown in Fig. 1 and Fig. 2, following operation is carried out：
1) the C18 films (be purchased from 3M companies) of clip 47mm, be placed in suction nozzle (pipet tip, 200 μ L, it is public purchased from Axygen
Department) internal tail end (referring to the A width of Fig. 2)；
It is to ensure that liquid can flow out while retaining magnetic bead by the purpose that C18 films are placed in suction nozzle tail end.
2) in suction nozzle 100 μ L acetonitriles, 1000rpm centrifugation 1min are added to be repeated 1 times；
The purpose for adding acetonitrile is cleaning suction nozzle and C18 films, and the purpose of centrifugation is to remove the acetonitrile in suction nozzle.
3) in suction nozzle 100 μ L BC200 solution, 1400rpm centrifugation 1min are added to be repeated 1 times；
Using the purpose of BC200 solution be further remove suction nozzle in acetonitrile, it is ensured that suction nozzle internal environment be connected with
Environment residing for the coated magnetic bead of biotin-labeled pentylamine of transcription factor series combination sequence is consistent；The purpose of centrifugation is to remove to inhale
Head internal liquid, liquor capacity in suction nozzle in convenient control subsequent operation.
4) will be the magnetic bead that transcription factor series combination sequence has been connected with step 2 resuspended with 20 μ L BC200 solution, plus
Enter in TOT posts；
5) wrapped after TOT posts with sealed membrane, be placed in 4 DEG C of preservations.
Multiple TOT posts are put into into centrifuge during use, as shown in Figure 3.
4th, extract the nucleoprotein of 293T cells
Can be with nuclear and cytoplasmic extraction reagents ripe on market
(Thermo78835) kit or Dignam method (bibliography：Dignam et al.1983.Dignam J D,
Lebovitz R M,Roeder R G.Accurate transcription initiation by RNA polymerase
II in a soluble extract from isolated mammalian nuclei[J].Nucleic acids
research,1983,11(5):1475-1489.Zerivitz and Akusjarvi, 1989, Kenn Zerivitz, An imporved nuclear extract preparation method,Gene Analysis
Techniques, Volume 6, Issue 5,1989, Pages 101-109) extract nucleoprotein, the present embodiment Dignam side
Method extracts the nucleoprotein of 293T cells, and detailed process is as follows：
1) the 293T cells (15cm Tissue Culture Dish cultures) of normal culture, 4 DEG C, 1000rpm centrifugations collection in 10 minutes are taken
Cell, is precipitated with 1 × PBS re-suspended cells, 4 DEG C again, 10 minutes collection cells of 1000rpm centrifugations；
2) with hypotonic solution (10mM TrisHCl pH 7.3, the 1.5mM MgCl of 10 times of precipitation volumes2, 10mM
KCl, using front addition 10mM β-ME and 1mM PMSF) re-suspended cell precipitation, put on ice after 20 minutes, 15 are homogenized with homogenizer
Under, 4 DEG C, 4000rpm be centrifuged 15 minutes, abandon supernatant；
3) with low salt solutions (20mM TrisHCl pH 7.3, the 1.5mM MgCl of 1/2 precipitation volume2, 20mM KCl,
0.2mM EDTA, 25%glycerol, using front addition 10mM β-ME and 1mM PMSF) resuspended precipitation, and it is thin to be added dropwise over 1/2
High level salt solution (20mM TrisHCl pH 7.3, the 1.5mM MgCl of cell space product2, 1.2M KCl, 0.2mM EDTA, 25%
Glycerol, using front addition 10mM β-mercaptoethanol, 0.5 × Protein inhibitors), after mixing, 4 DEG C hang down
It is straight to mix 30 minutes, 4 DEG C, 25,000rpm is centrifuged 20 minutes；
4) supernatant BC0 solution (20mM TrisHCl pH 7.3,0.2mM EDTA, 20%glycerol, using front
Add 10mM β-ME, 1mM PMSF) equal-volume dilution, nucleoprotein is obtained, liquid nitrogen flash freezer after packing, is used, it is standby in -80 DEG C of storages.
Different biological specimens, and the different condition residing for same biological specimen, all can produce impact to testing result.Should
Extract the scheme of nucleoprotein using standardization, such as this example using 293T cells as sample, to obtain after being standardized operation
Result be defined.When the testing result of standard nucleoprotein sample is in theoretical term of reference, then the whole reality that representative sample is detected
Proved recipe case and experimental implementation process are effective.For different biological specimens, such as A549 cells, mouse tissue etc., can also answer
Nucleoprotein or holoprotein are extracted with known standard method, is not repeated one by one.
5th, the enrichment and separation of transcription factor and its compound
1) nucleoprotein (step 4 is extracted) of 50 μ g 293T cells is taken, in adding the TOT posts of the built-in magnetic bead of step 3,
After magnetic bead in nucleoprotein sample and TOT posts is mixed, stationary incubation 10min, 2300rpm centrifugations 5 minutes on ice, centrifugation is removed
Supernatant；
2) to 1) middle incubation, the operation being centrifuged of the nucleoprotein repeat step after centrifugation；
The purpose of two steps centrifugation is to remove liquid in TOT posts, and increases the bioaccumulation efficiency of transcription factor in nucleoprotein.
3) in TOT posts 100 μ L 50mM NETN solution are added (to contain per 100mL：1M Tris-HCl (pH8.0) 1mL,
200 μ L of 5M NaCl 1mL, 0.5M EDTA, 250 μ L of NP40 Nonidet P40s), after magnetic bead is mixed, 500rpm from
Heart 5min, removes supernatant；
It is repeated once step 3).The step is to remove and the non-spy of transcription factor series combination sequence using the purpose of NETN solution
The albumen of different combination, i.e., except foreign protein.
4) (PBS is filled a prescription to add 100 μ L PBS solutions in TOT posts：137mM NaCl, 2.7mM
KCl, 10mM Na2HPO4, 2mM KH2PO4), after magnetic bead is mixed, 5min is centrifuged with 500rpm, removes supernatant；
It is repeated once step 4).The effect of the step PBS solution is to remove the NETN solution that remains in TOT posts.
5) 10 μ L 50mM ammonium bicarbonate solns are added in TOT posts, and adds 0.2 μ g pancreatin, 37 DEG C stand 1 hour；
The effect of ammonium bicarbonate soln in the step is to ensure that the environmental optima in TOT posts for pancreatin reaction, the effect of pancreatin
It is that the primary structure to albumen carries out specific digestion, the small peptide section not waited for 8-25 amino acid so as to obtain length.
6) contain 50% (V/V) acetonitrile extraction of 0.1% (V/V) formic acid with 100 μ L, take extract；Repeat extraction once,
Merge extract twice the dry Protein Detection sample obtained for follow-up LC-MS detections of heat.
The effect of formic acid in the step is the reaction for terminating pancreatin, and the effect of acetonitrile is that digestion products are extracted, and is removed
Unnecessary impurity.Small peptide section containing the 5-25 amino acid for intentionally getting in extract.The dry purpose of heat is to remove extract,
The control of solution system residing for the convenient detection sample to during Mass Spectrometer Method.
Step 6) separate mode of small peptide section and magnetic bead has two kinds of selections, only needs to use 50% body in the case of quick detection
The acetonitrile solution extraction of fraction；This example uses which, obtains the Protein Detection sample of numerous small peptide sections mixing (after being used for
Continuous LC-MS detections).
When the proteome data that experiment needs depth to cover, the acetonitrile solution of variable concentrations gradient is also can use, utilized
SRP (small-sized reverse-phase chromatographic column) elutes separation respectively, such as available variable concentrations (concentration is followed successively by 6%, 9%, 12%, 15%,
18%th, 21%, 25%, 30%, the peptide fragment of 9 concentration wash-outs is merged into 6 component proteins by acetonitrile solution wash-out 35%)
Detect sample (being followed successively by 6%+25%, 9%+30%, 12%+35%, 15%, 18%, 21%) for subsequently carrying out each component egg
The detection respectively of white detection sample, can obtain the proteome data of depth covering, see embodiment 4.The detached side of the gradient elution
Formula this embody the good autgmentability of the inventive method.
6th, the identification of transcription factor and its compound and quantitative
1) LC-MS (liquid chromatograph mass spectrography) identifications
The endogenous transcription factor and its compound of 293T cells are identified with LC-MS (liquid chromatograph mass spectrography).
The Cleaning Principle of LC-MS is different material to be separated using RPLC, and mass spectrum real-time detection is simultaneously identified not
The mass-to-charge ratio of commaterial, can be used to detecting and identifying the structure of complex compound, with efficient, high accuracy, high sensitivity and
The advantage of wide dynamic range.
Detecting instrument：1000 nano-HPLC system of Easy-nLC (Thermo Fisher Scientific companies)
With Orbitrap Fusion mass spectrographs (Thermo Fisher Scientific companies)；
Liquid phase chromatogram condition：Pre-column：C18,3 μm,2cm×100μmID；
Chromatographic column:C18,1.9 μm,10cm×100μm ID；
Mobile phase：+ 0.2% formic acid of A water ,+0.2% formic acid of B acetonitriles；
Flow velocity：400nl/min；Gradient from 5%B liquid to 30%B liquid, gradient timetable 75min.
Mass Spectrometry Conditions：
Data acquisition time：75min；
Spray voltage (spray voltage) 2KV；
Collision energy (normalized collision energy) 32%；
Acquisition quality scope：300-1400Da.
LC-MS detection process is：By step 5 heat dry peptide fragment (Protein Detection sample) with multiple containing 0.1% formic acid solution
Molten and loading.Liquid phase uses nano-HPLC C18 post (pre-columns：3 μm,2cm×100μm ID；Analytical column：1.9 μm,10cm × 100 μm ID) peptide fragment is separated, flow velocity 400nl/min, gradient timetable 75min, B liquid (contain 0.1%
The acetonitrile solution of formic acid) ratio rises to 30% from 5%.Ion gun uses 2000V voltages and 320 DEG C of temperature.One-level adopts Orbi
Scanning, 300-1400m/z of precursor scans scope, resolution ratio 120,000 (m/z 200), AGC 5e5, maximum injection length
50ms.Two grades adopt linear ion hydrazine, fragmentation energy 32%.The interior of 293T cells is obtained with LC-MS (liquid chromatograph mass spectrography)
The spectrogram of source transcription factor and its compound.
2) search storehouse and quantitative
Using bioinformatics tools and method by step 1) spectrogram that obtains carries out searching storehouse.The purpose of database search is
The data of mass spectrum output are analyzed, the albumen included in the data for determining mass spectrum output.Its process is by producing to mass spectrum
Two grades of spectrograms of the parent ion in the data for going out are analyzed, the intensity point in the range of certain mass deviation to fragment ion
Cloth situation is contrasted with theoretical strength, parent ion is scored by the fragment ion situation without departing from mass deviation scope
So as to obtain the qualification result of parent ion (small peptide section).Again small peptide section and known protein amino acid sequence storehouse are carried out
Match somebody with somebody, it is determined that the Protein Information belonging to detected small peptide section, obtains the qualification result of albumen.
Bioinformatics tools and method are by searching library software Proteome Discoverer 2.0 by the spectrogram of acquisition
Mascot 2.3 (Matrix Science Inc) in (Thermo Fisher Scientific) carries out searching storehouse, is used
Searching database behaviour source National Center for Biotechnology Information (NCBI) RefSeq albumen
Matter database (updated on 04-07-2013,32015 protein entries), parameter is set to：Parent ion quality is inclined
Difference 20ppm, product ion mass deviation 0.5Da；Allow two leakage enzyme sites；Dynamic embellishment includes phosphor (Y), phosphor
(ST), acetyl (protein N-term) and oxidation (M)；The horizontal FDR of peptide fragment allows 1%.
The peptide fragment for searching storehouse determination is further carried out quantitatively.Quantitative method is nonstandard quantitative approach.Searched by database
The peptide fragment matching profile information that rope is produced is calculated to the one-level spectrogram in initial data, and the one-level for obtaining all small peptide sections is fixed
The ratio of amount result.The program that batch is calculated can use existing《Intersect the egg for returning based on high resolution mass spectrometry data peptide fragment
Bai Fengdu quantitation softwares are [referred to as：PQPCR]》1.0 (National Copyright Administration of the People's Republic of China's Copyright in Computer Software registrations of V
Book number：Soft work steps on word the 0451332nd, registration number 2012SR083269, on 09 04th, 2012 record date, copyright owner：
Beijing Proteome Research Center).
7th, testing result checking
In order to verify the feasibility and detectability of the inventive method, 3 times have been carried out using 293T cells in aforementioned manners
Repeat experiment (#1-#3 of embodiment 1), obtain about 70,000 multiple spectrograms, identify 285 accordingly respectively, 310,304 kind endogenous turn
The record factor as listed in table 1, shows that substantial amounts of endogenous transcription factor and its compound are enriched with by TOT posts in Nuclear extract, and
Nucleoprotein single experiment has only used 50 μ g.When only being tested with 1 μ g nucleoprotein, can detect that 158 kinds of transcription factors (are implemented
The #4 of example 1).
The present invention can be but identified more than 100 kinds of transcription factors using the sequence containing 100 kinds of transcription factors, this be because
For, although transcription factor binding sequence only has 100 kinds of transcription factors, but various transcription factors can be with same class binding sequence
Combine, that is to say, that transcription factor and binding sequence not one-to-one relationship.1500 or so are included in human genome
Transcription factor encoding gene, these transcription factors can be divided into 50 families, and classification foundation is that transcription factor binding domain is different,
The transcription factor of each family is tended to reference to a kind of particular sequence.Therefore the present invention selects the representativeness of each family to combine sequence
Row build transcription factor specific binding sequence and (binding sequence of each transcription factor all need not can be placed on this people
In operation row).It is have many that factor binding sequences DNA is transcribed in test, and 0.5pmol is 3.01x1011Bar DNA, therefore can be with
With reference to many transcription factors.
Table 1：The identification of endogenous transcription factor and its compound in 293T cells
Quantitative analysis is illustrated by taking transcription factor HOXB9 as an example.HOXB9 (homeobox protein Hox-B9) Jing mass spectrums
Analysis measures 3 short peptide sequences, is GEAAPGQGQAAVK, QGTPEYSLETSAGR, TWLEPAPR respectively, according to qualification result
RT (chromatographic retention) and m/z (mass-to-charge ratio), can extract its XIC (Extracted Ion Chromatogram extract
Ion stream chromatogram peak figure), see Fig. 4, its XIC size represents the quantitative values size of small peptide.Use《Based on high resolution mass spectrometry
Data peptide fragment intersects the protein abundance quantitation software for returning [referred to as：PQPCR]》The calculating of XIC peak areas is carried out to small peptide, is calculated
As a result as shown in table 2 " XIC Area " row, so as to obtain the quantitative result of small peptide.
Table 2：To transcription factor HOXB9 small peptide quantitative analysis results
| Sequence | RT(s) | m/z(Da) | XIC Area |
| GEAAPGQGQAAVK | 748.76 | 592.3066 | 7232321 |
| QGTPEYSLETSAGR | 1890.16 | 748.3543 | 15005210 |
| TWLEPAPR | 2204.84 | 485.2606 | 9507436 |
Each transcription factor small peptide can be carried out quantitatively, no longer repeating one by one using same method.Identification based on table 1 is tied
Fruit coordinates quantitative analytical data again, can be scientific research, medicine with the species and content of transcription factor in panorama type reflected sample
Screening is there is provided foundation.
The present embodiment is visible, the composite can be widely applied to the Large Scale Transcriptional factor and its compound in sample identification,
With quantitatively, when particularly sample size is less, advantage becomes apparent from specific transcription factor confirmation, and reason is that operating procedure is few, is operated
In journey, sample losses are few, and high concentration reaction system improves digestion effect.
The enrichment of the endogenous transcription factor of embodiment 2, gastric mucosa and its compound, separation, identification and quantitative
Apply the inventive method to the identification of endogenous transcription factor in clinical biochemical sample and quantitative.
Biological specimen is that with the gastric mucosa sample obtained in gastrocopy, using two gastric mucosa samples, weight in wet base is respectively
Then 7.2mg and 8.3mg, the nucleoprotein in extracting directly mucosa tissue are enriched with, and carry out reality the step of by embodiment 1
Test.73 μ g (embodiment 2-1) and 106 μ g (embodiment 2-2) nucleoprotein are extracted respectively with reference to 1 step 4 of embodiment, using TOT
Post is enriched with to transcription factor in nucleoprotein sample and its compound, is as a result identified respectively and is close to 150 kinds of transcription factors, sees
Table 1.Quantitative approach is repeated no more with embodiment 1.
This example demonstrates that the present invention is to the enrichment of endogenous transcription factor and its compound, separation, identification and quantitative side
Method not only can apply to biological study, will also be applied to the aspects such as medical test, drug screening.And the method detection time is short,
The process operation of transcription factor enrichment is easy, and step is few, it is only necessary to the enzymolysis time of 1 hour, and 3 are only needed after obtaining nucleoprotein
Testing result is obtained by hour, is the side of a kind of fast enriching, separation, identification and quantitative endogenous transcription factor and its compound
Method.
Embodiment 3, the fast enriching of endogenous transcription factor and its compound, separation, identification and quantification kit
The endogenous transcription factor of the present invention and its enrichment of compound, separations, identification and quantification kit include that detection is tried
Agent and TOT posts；
Wherein, detection reagent includes：NETN and PBS solution, detailed in Example 1 of filling a prescription.Each TOT post is each with corresponding solution
200μL。
Kit using method is referring to embodiment 1.
Embodiment 4, TOT and sRP (small-sized reverse-phase chromatographic column) separating experiment
Unless otherwise indicated, other steps are consistent with embodiment 1 for the present embodiment.
Special feature is embodiment step 5 6) using the detached mode of gradient elution, and concrete operations are：Using NETN
Magnetic bead is cleaned with PBS solution, after removing the albumen of non-specific binding, the magnetic bead in TOT posts 50mM NH is utilized into4HCO3It is molten
Liquid is transferred in the small-sized reverse-phase chromatographic column containing 1-2mg C18 fillers (3M companies), adds pancreatin, and 37 DEG C digest 1 hour.It
Afterwards with the acetonitrile solution of 9 different volumes concentration (6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, 35%) successively
Wash-out, and merge into 6 Mass Spectrometer Method components.Acetonitrile concentration gradient and merging mode are 6%+25%, 9%+30%, 12%+
35%th, 15%, 18% and 21%.
TOT enrichments are carried out using the nucleoprotein of 250 μ g 293T cell extractions, sample are detected eventually through 6 component proteins
Detection, it is total to identify 715 kinds of transcription factors, the results are shown in Table 1, it is seen that the present embodiment can obtain the protein group number of depth covering
According to.
Subordinate list：Transcription factor listed by table 1
Claims (10)
1. it is with the method for a small amount of sample fast enriching, separation, identification and quantitative endogenous transcription factor and its compound including following
Step：Nucleoprotein or holoprotein sample are extracted from biological specimen, by the transcription factor series combination sequence of biotin labeling
(TFRE DNA, DNA bait) is mixed with the coated magnetic bead of biotin-labeled pentylamine, by the nucleoprotein or holoprotein of extraction be combined with
The magnetic bead mixing of transcription factor series combination sequence is incubated, and obtains the egg combined with transcription factor series combination sequence specific
In vain (i.e. endogenous transcription factor and its compound small peptide), by endogenous transcription factor and its compound small peptide and Beads enrichment, to dividing
From endogenous transcription factor and its compound small peptide carry out LC-MS (liquid chromatograph mass spectrography) detections, and use bioinformatics
Tool and method determines species, title and its content of endogenous transcription factor and its compound.
2. method according to claim 1, it is characterised in that：The transcription factor series combination sequence is referred to by 3-5bp
Joint sequence (Linker) connect transcription factor AP-1 1, AR, BRCA1, CEBPA, CREB1, E2F1, ELK1, ELK4, ESR1,
ETS1、EWSR1-FLI1、FEV、FOXA1、FOXC1、FOXD1、FOXF2、FOXI1、FOXL1、FOXO3、Fra-1、GATA2、
GATA3、GR、HIF1A::ARNT、HLF、HNF1B、HNF4A、HOXA5、INSM1、IRF1、IRF2、JunB、JunD、MAX、
MEF2A、MIZF、MYC::MAX、Myf、MZF1_1-4、MZF1_5-13、NF-kappaB、NFATC2、NFE2L2、NFIC、
NFIL3、NFKB1、NFYA、NHLH1、NKX3-1、NR1H2::RXRA、NR2F1、NR3C1、NR4A2、Pax6、PBX1、PDX1、
PLAG1、PPARG、PR、PXR-1:RXR-alpha、RAR-alpha、RAR-alpha:RXR-gam、RAR-beta:RXR-
alpha、REL、RELA、REST、RFX1、RFX2、RFX3、RFX5:RFXAP:RFXANK、RORA_1、RORA_2、RREB1、
RXR::RAR_DR5、RXRA::VDR、SOX10、SOX9、SP1、SPI1、SPIB、SRF、SRY、STAT1、STAT5A、T3R-
beta1、TAL1::TCF3、TBP、TEAD1、TFAP2A、TLX1::NFIC、TP53、USF1、WT1-del2、WT1-KTS、WT1I、
The artificial conjunction obtained after two or more in WT1I-del2, WT1I-KTS, XBP-1, YY1 and ZNF354C DNA binding members
Into DNA sequence dna.
3. method according to claim 1 and 2, it is characterised in that：The transcription factor series combination sequence such as sequence table
Shown in middle sequence 1.
4. the method according to claim 1 or 2 or 3, it is characterised in that：The sample refers to what is extracted from biological specimen
Nucleoprotein or holoprotein, refer to that consumption is only 1-250 μ g on a small quantity；The consumption of the transcription factor series combination sequence of biotin labeling
For 0.5pmol；The consumption of the coated magnetic bead of biotin-labeled pentylamine is that 20 μ L (contain 1.2-1.4 × 107Individual magnetic bead).
5. the arbitrary fast enriching of Claims 1-4, separation, identification and quantitative endogenous transcription factor and its compound are used for
Special purpose device TOT posts used in method, are made up of suction nozzle, the sleeve pipe for fixing suction nozzle and centrifuge tube, inside the suction nozzle
Tail end sets membrane structure closing, the built-in magnetic bead for being combined with transcription factor series combination sequence；During use, sleeve pipe is put in centrifuge tube,
Fix suction nozzle, sleeve pipe and centrifuge tube in suction nozzle plug-in-sleeve.
6. special purpose device TOT posts according to claim 5, it is characterised in that：The membrane structure of the suction nozzle inner tail end is
The C18 films of 47mm.
7. special purpose device TOT posts described in a kind of use claim 5 or 6 and a small amount of sample fast enriching, separation, identification and quantitative
The method of endogenous transcription factor and its compound, comprises the following steps：
1) the transcription factor series combination sequence (TFRE DNA, DNA bait) of biotin labeling is coated with biotin-labeled pentylamine
Magnetic bead be mixed to get the magnetic bead for being combined with transcription factor series combination sequence；
2) magnetic bead for being combined with transcription factor series combination sequence is added in the suction nozzle of TOT posts；
3) nucleoprotein extracted from biological specimen or holoprotein sample are added in the suction nozzle of TOT posts, incubation is made in sample
Source transcription factor and its complex proteins and magnetic bead specific bond；
4) magnetic bead is cleaned, removes the albumen (foreign protein) of non-specific bond；
5) pancreatin enzymolysis is added, endogenous transcription factor and its complex proteins is cut into into length for 5-25 amino acid not etc. short
Peptide fragment；
6) extract peptide fragment and Beads enrichment；
7) LC-MS (liquid chromatograph mass spectrography) detections are carried out to peptide fragment and obtains spectrogram, and with bioinformatics tools and method
Spectrogram is understood, it is determined that the albumen (i.e. endogenous transcription factor and its compound) combined with transcription factor series combination sequence specific
Species, title and its content.
8. method according to claim 7, it is characterised in that：
Step 1) the transcription factor series combination sequence is the sequence mentioned in Claims 2 or 3；Using round pcr and band
There is the transcription factor series combination sequence of the primer synthesizing biotinylated mark of biotin labeling；The magnetic bead is biotin-labeled pentylamine
Coated M280 magnetic beads；In mixing the consumption of the transcription factor series combination sequence of biotin labeling be 0.5pmol, biotin parent
Consumption with plain coated magnetic bead is that 20 μ L (contain 1.2-1.4 × 107Individual magnetic bead), hybrid mode is 4 DEG C of vertical mixing 30min.
9. the method according to claim 7 or 8, it is characterised in that：
Step 2) be combined with transcription factor series combination sequence magnetic bead consumption be 20 μ L；Step 3) nucleoprotein or holoprotein use
Measure as 1-250 μ g；
Step 4) cleaned with NETN solution and PBS solution successively；
Step 6) detached mode is to obtain endogenous transcription factor and its compound peptide fragment with acetonitrile extraction；Or it is dense with different volumes
The acetonitrile solution wash-out of degree (concentration is followed successively by 6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, 35%), and by 9
The peptide fragment of individual concentration wash-out merge into 6 component peptide fragments (be followed successively by 6%+25%, 9%+30%, 12%+35%, 15%,
18%th, 21%), step 7) in each component peptide fragment is detected respectively, can obtain depth covering proteome data；
Step 7) bioinformatics tools and method be by searching library software in Protein Data Bank by the spectrogram of acquisition
Carry out searching species, the title that storehouse determines endogenous transcription factor and its compound, will be fixed using protein abundance quantitation software
Endogenous transcription factor and its compound are carried out quantitatively.
10. a kind of kit with a small amount of sample fast enriching, separation, identification and quantitative endogenous transcription factor and its compound,
The magnetic bead and special purpose device TOT for being combined with transcription factor series combination sequence referred at least including aforementioned claim
Post, the magnetic bead are packaged in the suction nozzle of TOT posts；Can also be including the NETN solution, PBS solution that refer in aforementioned claim
And/or pancreatin etc..
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