CN109839303A - A kind of crosslinking peptide fragment enrichment method and its application in protein interaction research - Google Patents
A kind of crosslinking peptide fragment enrichment method and its application in protein interaction research Download PDFInfo
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Abstract
The present invention relates to a kind of crosslinking peptide fragment enrichment method and its applications in protein interaction research.Intracellular protein complex is crosslinked and is digested with the crosslinking agent containing o-dihydroxy group on reaction active groups and linking arm using two sides, selective enrichment and efficiently release are then carried out to crosslinking peptide fragment using boron affinitive material.This method has the advantages that operation is easy and efficient, high-throughput, high confidence level and the analysis for being applied to protein interaction.
Description
Technical field
The present invention relates to a kind of crosslinking peptide fragment enrichment method and its applications in protein interaction research.
Background technique
Being crosslinked mass-spectrometric technique is the new technology to grow up over the past decade, it utilizes chemical cross-linking agent by intercellular spaces
It is connected apart from two amino acid that are close enough, being reacted with crosslinking agent with covalent bond, then using based on mass spectrum skill
The proteomics of art analyzes cross-linking products, to realize the space structure and protein-protein interaction to protein
The parsing (Sinz, A., Expert Rev.Proteomics., 2014,11 (6): 733-743.) of mode.
However, being required using tradition crosslinking mass-spectrometric technique Mass Spectrometric Identification very high.Since the practical crosslinking in each site is imitated
Rate is often far below 100%, so crosslinking peptide fragment abundance is relatively low, spectrogram number is few for crosslinking, signal difference, identifies band to crosslinking spectrogram
Come difficult.In addition, into before mass spectral analysis, needing to carry out digestion to it for complex proteins complex sample.It is crosslinked egg
In different location digestion, which occurs, for white matter can generate three kinds of products: a. is crosslinked single peptide of agent modification, i.e. water occurs for crosslinking agent one end
Solution, only one end is reacted (Type-0 type peptide fragment) with peptide fragment；B. peptide fragment internal crosslinking, i.e., crosslinking agent both ends binding site is same
On one peptide fragment (Type-1 type is crosslinked peptide fragment)；C. it is crosslinked between peptide fragment, i.e., crosslinking agent both ends binding site is respectively in two peptide fragments
Upper (Type-2 type is crosslinked peptide fragment).Since the reactive group hydrolysis rate of cross-linking reagent two sides is fast, Type-0 type peptide fragment content is remote
Peptide fragment is crosslinked higher than Type-1 type needed for protein interaction analytic and Type-2 type.
For the abundance for improving crosslinking peptide fragment, has been developed be enriched with group (such as alkynyl-nitrine, life containing different type at present
Object element-Avidin etc.) cross-linking reagent, however, due to containing crosslinking agent in three kinds of products, using containing conventional enrichment base
The cross-linking reagent of group can not separate crosslinking peptide fragment with Type-0 type peptide fragment.Further, since the reason of special groups is added,
General brachium is longer than simple crosslinking agent, this declines cross-linking efficiency, the distance between obtained crosslink sites
Restriction effect can also die down.Meanwhile macromolecule group be introduced into will lead to crosslinking peptide fragment fragmentation is more difficult in mass spectrum.
Summary of the invention
In order to overcome conventional chemical cross-linking method to cannot achieve to crosslinking peptide fragment selective enrichment, and can be on crosslinking peptide fragment
The deficiencies of introducing modification group, the present invention provides a kind of crosslinking agent using containing o-dihydroxy group on linking arm will be intracellular
Protein complex is crosslinked and is digested, and is then crosslinked peptide fragment using boron affinitive material selective enrichment and is efficiently discharged.It should
Method have the advantages that operation is easy and efficient, high-throughput, high confidence level and be applied to protein structural analysis and protein it is multiple
The analysis of zoarium interaction binding site.
To achieve the above object, the technical solution adopted by the present invention are as follows:
(1) preparation of protein/cell sample solution and cross-linking agent solution: pancreatin digestion or cell scraper are digested to obtain
Living cells sample, the use of pH is the ammonium hydrogen carbonate buffer salt solution of 7.1-10, phosphate buffered saline solution, 4- hydroxyethyl piperazine second
One of sulfonic acid buffer salt solution or trishydroxymethylaminomethane buffer salt solution or two kinds or more, and be free of and crosslinking used
The buffer for the group that reactive group reacts in agent washes off culture solution and cell is made to suspend；For single protein sample or mix
Hop protein sample, the protein solution for the use of water or buffer being 1 μ g/mL-100mg/mL at concentration；Use water, buffer
Or it is 1 μ that the organic solvent of one of acetonitrile, organic alcohols, organic acid, DMF or DMSO or two kinds or more, which is configured to concentration,
The cross-linking agent solution of M-1M, the crosslinking agent two sides used contain succinimide, halogenated aryl hydrocarbon, imidoether, maleimide,
On 2- mercaptopyridine, thiosulfonate, haloacetyl, carbodiimide, isocyanates, hydrazides, aziminobenzene, double ethylene imines etc.
One or two kinds of reactive groups are stated, and contain o-dihydroxy group on linking arm；
(2) cross-linking reaction: cross-linking agent solution being added into cell sample or protein sample and is reacted, for cell sample
Product, cell concentration is 10 in reaction system6-109A/mL, for protein sample, protein concentration is 1nM-1mM in reaction system,
The concentration of crosslinking agent is 10nM-100mM；The reaction condition is 15-40 DEG C of reaction 10min-2h or 0-10 DEG C of reaction
10min-10h；
(3) removal of extra crosslinking agent: for cell sample, after centrifugation discards the reaction solution in cell sample, to cell
Middle addition lysate carries out cell cracking using one of mechanical lysis or high temperature incubation method or two kinds of cell cracking methods, obtains
The protein example that must be crosslinked；For protein sample, be added reaction terminating liquid into reaction system, or by dialysis, filter membrane or
Gel removes reaction solution, obtains crosslinking protein sample, reacts 10min-2h at room temperature or reacts 10min-10h on ice；
(4) dissolution, denaturation and reduction of protein example: use pH for the formic acid of 1-6.5, trifluoroacetic acid, trichloroacetic acid
Or the acidic buffer solutions such as acetic acid or pH be 7.5-14 alkaline buffer solution prepare dissolved with surfactant or organic solvent
Lysate carry out soluble protein quality sample, one of reducing agents such as DTT, TCEP or beta -mercaptoethanol or two kinds or more are added,
It is incubated for 1min-10h under 40-100 DEG C of water-bath, while carrying out the denaturation and reduction of protein example；
(5) one of iodo acetic acid or iodoacetamide or two kinds the alkylation of protein example and enzymatic hydrolysis: are added to egg
After white matter sample is alkylated reaction, trypsase, Proteinase K, pepsin, elastin laminin are added into protein example
One of enzyme, carboxypeptidase, chymotrypsin, intracellular protein enzyme Lys-C/N, protein incision enzyme Glu-C/N, Asp-C/N or
Two kinds or more, when using two kinds or more, while use or sequence use；
(6) into the product of step (5) be added with monomer be 2- Carboxybenzeneboronic acid, 5- carboxyl -2- methylol phenyl boric acid,
One of 4- formylphenylboronic acid, amino phenyl boric acid or 5- amino -2- methylol phenyl boric acid etc. or two kinds or more of boron are affine
The enrichment material of the organic/inorganic materials such as the Ago-Gel ball of group, silicon ball, polymer drops preparation, and and example reaction；
(7) peptide fragment of non-specific adsorption on enrichment material is washed away using eluents such as salting liquid or organic solvents；
(8) peptide fragment being bonded on enrichment material is released using acid solution, desalination, freeze-drying, which is laid equal stress on, dissolves into row mass spectrum
Analysis and data retrieval.
(9) above-mentioned sample pretreating method is used for protein phase in body signal path and protein structure parsing field
The research of interaction.
The invention has the following advantages that
1, easy to operate.Experimental procedure is simple, and the enrichment of crosslinking peptide fragment and rate of release are fast.
2, enrichment selectivity is high.Boron affinitive material selects the crosslinking peptide fragment of the group containing o-dihydroxy with efficiently concentrating
Property.
3, the rate of recovery is high.Solution system is replaced into acid solution, crosslinking peptide fragment can be high from the affine enrichment material of boron
Effect release.
4, with a high credibility.Non-crosslinked peptide fragment interference is greatly lowered, and crosslinking peptide fragment abundance increases substantially, and friendship is greatly improved
The identification sensitivity for joining peptide fragment, to improve the confidence level of qualification result.
Specific embodiment
Embodiment 1
1. the cross-linking reaction of protein example
It is pure that 10 μ g ox bloods are dissolved using the 20mM 4- hydroxyethyl piperazineethanesulfonic acid buffer salt solution (HEPES) that pH is 7.4
Protein sample (BSA), final protein concentration 1mg/mL, the double ambers for the use of dimethyl sulfoxide (DMSO) compound concentration being 25mM
Imide tartrate (DST), crosslinking agent, which is added into BSA solution, makes its final concentration of 1mM, reacts 1h at room temperature.
2. the removal of extra crosslinking agent
Ammonium bicarbonate soln (ABC) is added into the reaction solution in step 1, it is anti-to terminate crosslinking to make its final concentration of 50mM
It answers.
3. the dissolution of protein example, denaturation and reduction
Urea and DTT are added into the BSA solution after crosslinked in step 2, makes the final concentration of 8M of urea, DTT in solution
Final concentration of 10mM, 37 DEG C of water-bath 30min.
4. alkylation and the enzymatic hydrolysis of protein solution
Iodo acetic acid is added into the BSA solution in step 3, and makes its final concentration of 20mM, is protected from light 30min.Alkane
After glycosylation reaction, sample solution is diluted with water 4 times, and 1 μ g serine protease Lys-C, 37 DEG C of water-baths are added
4h.After reaction, 2 μ g trypsase are added into sample solution, 37 DEG C of water-baths are stayed overnight.Enzymolysis product is subjected to desalination
And it is lyophilized.
5. being crosslinked the enrichment of peptide fragment
It is using 50mM ammonium bicarbonate soln (ABC, pH 10) that enzymolysis product weight is molten, and phenyl boric acid silicon ball is added, at room temperature
React 2h.
6. the removal of non-specific adsorption peptide fragment
Use the peptide fragment of the non-specific adsorption in 50mM ABC solution (pH 10) cleaning silicon ball.
7. the release of peptide fragment
It is added eluent (acetonitrile: water: trifluoroacetic acid=50:49:1), reacts 1h at room temperature.
8. the determination of crosslink sites
After the crosslinking peptide fragment freeze-drying that release is obtained, it is dissolved in 0.1% formic acid solution again, into mass spectral analysis.
Qualification result
| VTKCCTESLVNR(3)-CASIQKFGER(6) |
| LCVLHEKTPVSEK(7)-CASIQKFGER(6) |
| LAKEYEATLEECCAK(3)-VTKCCTESLVNR(3) |
| KVPQVSTPTLVEVSR(1)-HKPKATEEQLK(4) |
| LAKEYEATLEECCAK(3)-CASIQKFGER(6) |
| CCTKPESER(4)-SLGKVGTR(4) |
| VHKECCHGDLLECADDR(3)-ALKAWSVAR(3) |
| FKDLGEEHFK(2)-DTHKSEIAHR(4) |
| CASIQKFGER(6)-SLGKVGTR(4) |
| RHPYFYAPELLYYANKYNGVFQECCQAEDK(16)-GACLLPKIETMREK(7) |
| KVPQVSTPTLVEVSR(1)-CASIQKFGERALK(6) |
| DTHKSEIAHR(4)-LVTDLTKVHK(7) |
| TCVADESHAGCEKSLHTLFGDELCK(13)-RDTHKSEIAHR(5) |
| LVTDLTKVHK(7)-ALKAWSVAR(3) |
| LKECCDKPLLEK(2)-FPKAEFVEVTK(3) |
| YICDNQDTISSKLK(12)-FPKAEFVEVTK(3) |
| LAKEYEATLEECCAK(3)-ALKAWSVAR(3) |
| CASIQKFGER(6)-ALKAWSVAR(3) |
| NYQEAKDAFLGSFLYEYSR(6)-ALKAWSVAR(3) |
| DTHKSEIAHR(4)-SLGKVGTR(4) |
| ATEEQLKTVMENFVAFVDK(7)-KVPQVSTPTLVEVSR(1) |
| CASIQKFGER(6)-LSQKFPK(4) |
| CCTKPESER(4)-LSQKFPK(4) |
| SHCIAEVEKDAIPENLPPLTADFAEDK(9)-CCTKPESERMPCTEDYLSLILNR(4) |
| CCTKPESERMPCTEDYLSLILNR(4)-GACLLPKIETMR(7) |
| SHCIAEVEKDAIPENLPPLTADFAEDK(9)-YICDNQDTISSKLK(12) |
| RHPYFYAPELLYYANKYNGVFQECCQAEDK(16)-ECCDKPLLEK(5) |
| ECCKDPLLEKSHCIAEVEK(10)-ADLAKYICKNQDTISSK(5) |
Embodiment 2
The cross-linking reaction of 1.BSA protein example；The removal of extra crosslinking agent；The dissolution of protein example is denaturalized and goes back
Former, alkylation digests, the same embodiment of release steps of the enrichment of crosslinking peptide fragment, the removal and peptide fragment of non-specific adsorption peptide fragment
1。
2. the classification of enrichment crosslinking peptide fragment and the determination of crosslink sites
The crosslinking peptide fragment that release obtains in step (1) is subjected to desalination, freeze-drying, using Cation exchange separation to sample into
Row classification, desalination are lyophilized and are analyzed by mass spectrometry using 0.1% formic acid solution weight is molten.
Qualification result
Embodiment 3
1. the cross-linking reaction of protein example
10 μ g rabbit creatine kinase protein samples (CK), egg are dissolved using the 50mM phosphate buffered saline solution (PBS) that pH is 7.4
The final concentration of 1mg/mL of white matter, the DST for the use of dimethylformamide (DMF) compound concentration being 25mM, crosslinking agent is added to CK
Make its final concentration of 1mM in solution, reacts 1h at room temperature.
2. the removal of extra crosslinking agent
Trishydroxymethylaminomethane buffer solution (Tris) is added into the reaction solution in step 1, keeps its final concentration of
50mM is to terminate cross-linking reaction.
3. the dissolution of protein example, denaturation and reduction
Urea and DTT are added into the CK solution after crosslinked in step 2, makes the final concentration of 8M of urea in solution, DTT is whole
Concentration is 10mM, 37 DEG C of water-bath 30min.
4. alkylation and the enzymatic hydrolysis of protein solution
Iodo acetic acid is added into the CK solution in step 3, and makes its final concentration of 20mM, is protected from light 30min.Alkyl
Change after reaction, sample solution is diluted with water 4 times, and 1 μ g serine protease Lys-C, 37 DEG C of water-bath 4h is added.
After reaction, 2 μ g trypsase are added into sample solution, 37 DEG C of water-baths are stayed overnight.Enzymolysis product is subjected to desalination simultaneously
Freeze-drying.
5. being crosslinked the enrichment of peptide fragment
It is using 50mM ammonium bicarbonate soln (ABC, pH 7) that enzymolysis product weight is molten, and phenyl boric acid magnetic ball is added, at room temperature
React 2h.
6. the removal of non-specific adsorption peptide fragment
Use the peptide fragment of the non-specific adsorption on 50mM ABC solution (pH 7) cleaning magnetic ball.
7. the release of peptide fragment
It is added eluent (acetonitrile: water: trifluoroacetic acid=50:49:1), reacts 1h at room temperature.
8. the determination of crosslink sites
After the crosslinking peptide fragment freeze-drying that release is obtained, it is dissolved in 0.1% formic acid solution again, into mass spectral analysis.
Qualification result
| SIKGYTLPPHCSR(3)-IEEIFKK(6) |
| VISMEK(3)-FCVGLQKIEEIFKK(7) |
| VISEKGGNK(5)-GGVHVKLAHLSK(6) |
| LAHLSKHPK(6)-LQKR(3) |
| TGKSIKGYTLPPHCSRGER(3)-LSVEALNSLTGEFKGK(2) |
| LNYKSEEEYPDLSK(4)-AVEKLSVEALNSLTGEFK(4) |
| GKYYPLK(2)-VISMEKGGNMKEVFRR(6) |
| LNYKSEEEYPDLSK(4)-VLTPDLYKK(8) |
| GGVHVKLAHLSK(6)-LQKR(3) |
| GGDDLDPHYVLSSR(9)-VISEKGGNK(5) |
| GGVHVKLAHLSK(6)-LEKGQSIDDIPAQK(3) |
The present invention using the crosslinking agent containing o-dihydroxy group on linking arm to the protein example of cell or extraction into
Row crosslinking, after protein example is digested, is crosslinked peptide fragment using between boron affinitive material selective enrichment peptide fragment, due to alkalinity
Under the conditions of carboxyl in Type-0 type peptide fragment o-dihydroxy and boron atom on crosslinking agent can be prevented to act on, therefore may be implemented pair
The high-selectivity enrichment of Type-1 and Type-2 type crosslinking peptide fragment.Meanwhile solution system is replaced into acidic environment, it is crosslinked peptide fragment
It can efficiently be discharged from boron affinitive material, modification group will not be introduced.Gained crosslinking peptide fragment is entered into mass spectral analysis, can be obtained
Take protein structure and protein interaction information.This method have operation is easy and efficient, high-throughput, high confidence level it is excellent
Put and can be applied to the structural analysis of protein and the analysis of protein complex interaction binding site.
Claims (10)
1. a kind of method for being crosslinked peptide fragment enrichment, it is characterised in that: use the friendship containing o-dihydroxy group on linking arm
Connection agent is crosslinked cell or the protein example of extraction, after crosslinking protein quality sample is digested, using the affine material of boron
Expect that selective enrichment is crosslinked peptide fragment, to obtain protein structure and protein interaction information；
It specifically includes:
(1) for cell sample, culture solution is washed off using buffer and cell is made to suspend；For the albumen sample extracted
Product, using water or buffer at solution；For crosslinking agent, solution is configured to using water, buffer or organic solvent；
(2) cross-linking agent solution is added into cell sample solution or protein sample solution and is reacted；
(3) for cell sample, centrifugation discards the reaction solution in reaction system, then cracks, must be crosslinked to cell sample
Protein sample；For protein sample, reaction terminating liquid is added into reaction system, or anti-by dialysis, filter membrane or gel removal
Liquid is answered, crosslinking protein sample is obtained；
(4) alkaline buffer solution for using pH to be 7.5-14 for the acidic buffer solution of 1-6.5 or pH is prepared living dissolved with surface
Property agent or the lysate of organic solvent dissolve crosslinking protein sample, reducing agent, high temperature incubation is added, while carrying out protein sample
The denaturation and reduction of product；
(5) after addition alkylating reagent is alkylated reaction to the protein example after denaturation and reduction, to protein example
Middle addition protein enzyme solution is digested, and enzymolysis product is carried out desalination and is lyophilized；
(6) enrichment material for having boron affinity groups is added into the product of step (5)；
(7) peptide fragment of non-specific adsorption on enrichment material is washed away using eluent；
(8) peptide fragment being bonded on enrichment material is released using acid solution, desalination, molten, progress mass spectrum point of laying equal stress on is lyophilized
Analysis and data retrieval.
2. preprocess method described in accordance with the claim 1, it is characterised in that: buffer described in step (1) is that pH is
Ammonium hydrogen carbonate buffer salt solution, phosphate buffered saline solution, 4- hydroxyethyl piperazineethanesulfonic acid buffer salt solution or three hydroxyls of 7.1-10
One of aminomethane buffer salt solution or two kinds or more, and without can be reacted with reactive group on crosslinking agent used
Group；
Cell sample described in step (1) is pancreatin digestion or the living cells sample that cell scraper digests；In step (1)
The protein sample, is the mixed protein sample of single protein sample or two kinds or more albumen, the albumen quality of preparation with it is molten
The volume ratio of liquid is 1 μ g/mL-100mg/mL；Crosslinking agent described in step (1) contains the group reacted with albumen for two sides,
And the crosslinking agent containing chemical disruption group on linking arm, the crosslinker concentration of preparation are 1 μM of -1M；
Organic solvent described in step (1) is acetonitrile, organic alcohols, organic acid, dimethylformamide (DMF) or diformazan
One of base sulfoxide (DMSO) or two kinds or more.
3. preprocess method according to claim 1 or 2, it is characterised in that: the crosslinking agent has for two sides and egg
The chemicals of the active group of white upper amino reaction, active group is succinimide group, halogenated aryl hydrocarbon group, imidoether
One of group or two kinds；Or there are the chemicals of the active group reacted with sulfydryl on albumen for two sides, active group is
One of maleimide base group, 2- mercaptopyridine group, thiosulfonic acid group, haloacetyl group or two kinds；It or is two
Side has the chemicals of the active group reacted with carboxyl on albumen, and active group is carbodiimide group, in isocyanate group
One kind or two kinds；Or there are the chemicals of the active group reacted with sugar chain on albumen for two sides, active group is hydrazide group
Group, one of amino group or two kinds；Or there are the chemicals of the active group reacted with group any on albumen for two sides,
Active group is one of aziminobenzene group, double ethylene imine groups or two kinds；The crosslinking agent, two sides active reaction
Group is respectively any of the above-described reactive group；The crosslinking agent, the chemical enrichment group on linking arm are o-dihydroxy base
Group.
4. preprocess method described in accordance with the claim 1, it is characterised in that: cell in reaction system described in step (2)
Concentration is 106-109A/mL, final concentration of protein 1nM-1mM, the final concentration of 10nM-100mM of crosslinking agent；The reaction item
Part is 15-40 DEG C of reaction 10min-2h or 0-10 DEG C of reaction 10min-10h.
5. preprocess method described in accordance with the claim 1, it is characterised in that: reaction terminating liquid described in step (3) is tool
There is the substance for the group that can be reacted with crosslinking agent two sides reactive group；The reaction condition is 15-40 DEG C of reaction 10min-2h
Or 0-10 DEG C of reaction 10min-10h.
6. preprocess method described in accordance with the claim 1, it is characterised in that: cell lysing methods described in step (3) are
One of cleavage methods such as mechanical lysis or high temperature incubation method or two kinds or more；
Specially protease inhibitors is added in the mixture of extracting solution of protein and biological sample in above-mentioned mechanical disruption methods
Afterwards, tissue or cell sample is cracked using homogenizer, Ultrasound Instrument, mortar or tissue mashing machine etc.；
Above-mentioned protease inhibitors be one of following substance or two kinds or more: 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride,
Aprotinin, aminopeptidase inhibition, sodium pyrophosphate, trans- epoxy succinyl base-L- leucyl amido (4- guanidine radicals) butane, ethylenediamine tetrem
One of acid disodium, leupeptin or Pepstatin A, this mesyl fluoride or two kinds or more；
Every kind of protease inhibitors is in the concentration range in extracting solution of protein respectively between 1-200mg/mL in above-mentioned substance；
The mixture of extracting solution of protein and biological sample is specially incubated for by above-mentioned high temperature incubation method under 40-100 DEG C of water-bath
1-60min。
7. preprocess method described in accordance with the claim 1, it is characterised in that: the dissolution of protein example described in step (4)
In liquid, pH is the acidic buffer solution of 1-6.5, is formic acid, trifluoroacetic acid, trichloroacetic acid or acetum；The pH is
The alkaline buffer buffer solution of 7.5-14 is ammonium hydrogen carbonate buffer salt solution, phosphate buffered saline solution, 4- hydroxyethyl piperazine second sulphur
One of acid buffering salting liquid or trishydroxymethylaminomethane buffer salt solution or two kinds or more；
In the protein lysates, the surfactant includes:
1) dodecyl sodium sulfate, NaTDC, alkyl glycosides, 100 (Triton X-100) (Triton X-
100), 3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt (CHAPS), RapiGest SF or the poly- second two of ethylphenyl
One of alcohol (NP-40d) or two kinds or more, mass-volume concentration by surfactant quality (as unit of g) and buffering
The ratio between volume (as unit of mL) of solution is calculated as 0.1%-30%；
2) cationic portion is imidazoles, pyridines, quaternary amines or the quaternary phosphine cationoid that alkyl chain part contains 2 or more carbon
One of or two kinds or more；Anion part is halide ion, NO3 -、ClO4 -、AlCl4 -、BF4 -、PF4 -、CF3COO-、
CF3SO3 -、(CF3SO2)2N-Or SbF6 -One of or two kinds or more of ionic liquid；Mass-volume concentration is by ionic liquid
Quality (as unit of g) and the ratio between the volume (as unit of mL) of alkaline buffer solution are calculated as 0.1%-30%；
3) one of detergents such as urea, thiocarbamide or guanidine hydrochloride or two kinds or more, molar concentration by the substance of detergent amount
0.1-20M is calculated as with the ratio between the volume of buffer solution；
The organic solvent is one of organic alcohols or organic acid or two kinds or more, volumetric concentration by organic solvent with
The ratio between volume of buffer solution is calculated as 0.1%-100%.
The reducing agent is in the reducing agents such as dithiothreitol (DTT) (DTT), three (2- carboxyethyl) phosphines (TCEP) or beta -mercaptoethanol
One or two or more kinds, molar concentration is calculated as 0.1- by the ratio between the amount of the substance of reducing agent and the volume of alkaline buffer solution
1000mM。
The high temperature incubation method specially incubates the mixture of extracting solution of protein and biological sample under 40-100 DEG C of water-bath
Educate 1min-10h.
8. preprocess method described in accordance with the claim 1, it is characterised in that: alkylating reagent described in step (5) is iodine
For one of acetic acid or iodoacetamide or two kinds, the molar concentration after being dissolved in above-mentioned alkaline buffer solution is 1-200mM；
The protease is trypsase, Proteinase K, pepsin, elastoser, carboxypeptidase, chymotrypsin, born of the same parents
One of interior protease lysine-C/N, protein incision enzyme Glu-C/N, Asp-C/N or two kinds or more, use two kinds or more
When, the enzyme of selection can be used simultaneously or sequence uses, and the mass ratio of protease and protein is 1:500-500:1.
9. preprocess method described in accordance with the claim 1, it is characterised in that: the list of boron affinity groups described in step (6)
Body is 2- Carboxybenzeneboronic acid, 5- carboxyl -2- methylol phenyl boric acid, 4- formylphenylboronic acid, amino phenyl boric acid or 5- amino -2- hydroxyl
One of methylphenylboronic acid etc. or two kinds or more；
The matrix of the enrichment material is the organic/inorganic materials such as Ago-Gel ball, silicon ball, polymer drops；
Eluent described in step (7) is one of following: concentration is the chlorination of the sodium chloride solution of 0.1-8M, 0.1-8M
Potassium solution, the sodium carbonate liquor of 0.1-1M, the urea liquid of 2-10M, the guanidine hydrochloride solution of 1-8M or 10-1000mM bicarbonate
Ammonium salt solution；Acetonitrile, methanol, isopropanol；Dodecyl sodium sulfate, Triton X-100, Chaps, Tween；
Crosslinking peptide fragment release solution described in step (8) is the acid solution that pH is 1-6.5, and acid solution is formic acid, three
One of fluoroacetic acid, trichloroacetic acid or acetum or two kinds or more.
10. preprocess method described in accordance with the claim 1, for egg in body signal path and protein structure parsing field
The application of white matter repercussion study.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112083081A (en) * | 2019-06-12 | 2020-12-15 | 中国科学院大连化学物理研究所 | In-situ analysis method for intracellular protein complex and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112083081A (en) * | 2019-06-12 | 2020-12-15 | 中国科学院大连化学物理研究所 | In-situ analysis method for intracellular protein complex and application thereof |
| CN112083081B (en) * | 2019-06-12 | 2022-02-08 | 中国科学院大连化学物理研究所 | In-situ analysis method for intracellular protein complex and application thereof |
| CN112986569A (en) * | 2019-12-02 | 2021-06-18 | 中国科学院大连化学物理研究所 | Method for removing single-end cross-linked peptide and application of method in analysis of protein complex cross-linking sites |
| CN112986569B (en) * | 2019-12-02 | 2022-05-06 | 中国科学院大连化学物理研究所 | Method for removing single-end cross-linked peptide and application of method in analysis of protein complex cross-linking sites |
| CN112379008A (en) * | 2020-09-30 | 2021-02-19 | 中国药科大学 | Asp for detecting Prx2 protein in erythrocyte46Method for the degree of racemization |
| CN114574525A (en) * | 2020-11-30 | 2022-06-03 | 中国科学院大连化学物理研究所 | Transport carrier for intracellular targeting mitochondria and application thereof |
| CN114577557A (en) * | 2020-11-30 | 2022-06-03 | 中国科学院大连化学物理研究所 | Application of catalytic enzyme activation-based targeting cross-linking agent in protein analysis |
| CN114574525B (en) * | 2020-11-30 | 2024-06-11 | 中国科学院大连化学物理研究所 | Transport carrier for intracellular targeted mitochondria and application thereof |
| CN114459848A (en) * | 2021-01-17 | 2022-05-10 | 谱度众合(武汉)生命科技有限公司 | Universal proteomics sample preparation method and kit |
| CN114459848B (en) * | 2021-01-17 | 2023-02-03 | 谱度众合(武汉)生命科技有限公司 | Universal proteomics sample preparation method and kit |
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