CN1658892A - Method for isolating subpopulations of proteins that engage in protein-protein interactions - Google Patents

Abstract

The invention provides a method for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a chemically reactive supporting matrix to isolate proteins that in turn non-covalently bind other proteins. The supporting matrix is isolated, and the non-covalently bound proteins are subsequently released for analysis. Because the proteins are accessible to chemical manipulation at both the binding and release steps, identification of the non-covalently bound proteins yields information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The method has the advantage of screening the entire proteome simultaneously, unlike two-hybrid systems or phage display methods which can only detect proteins binding to a single bait protein at a time. The method is applicable to the study of protein-protein interactions in biopsy and autopsy specimens, to the study of protein-protein interactions in the presence of signalling molecules, pharmacological agents or toxins, and for comparison of diseased and normal tissues or cancerous and untransformed cells.

Description

Participate in the separation method of the protein subgroup of protein protein interaction

Invention field

Invention relates to the research of protein protein interaction, and estimates to use it for biochemical signals transduction, protein group, drug discovery, toxicology and diagnostic field.

Background of invention

Protein protein interaction is the basis of various physiological processes.Cell processes such as neuron signal, cell development, grow and duplicate and all depend on protein-protein and the interactional complex network of protein-micromolecule in the cell.These interactions can be categorized as that composing type interacts as between the hemoglobin subunit and the interaction of signal dependency as between cAMP-deopendent protein kinase subunit or the conjugated protein subunit of GTP-.The complexity of studying these interaction tasks has some idea of from the potential quantity of protein interaction: screen 15,000 binary in the protein comprehensively and interact and need test to surpass 2 * 10 ⁸Plant paired protein combination.This complexity means that conventional biochemical method purposes is limited.Although sufficient research is arranged, still there is not gratifying method to be used for studying the protein interaction of mammalian cell or other protein complex mixture.

Some technology are used to study independent protein protein interaction, comprise protein cross [1,2,3], green fluorescent protein [4,5], phage display [6,7], two-hybrid system [8], protein array [9], optical fiber evanescent wave pick off [10,11], chromatographic technique [12] and fluorescent energy resonance transfer [13,14].Yet these methods generally are used for only screening 1 bait albumen at every turn.Brief overview is referring to [15].

Report expands to these methods on suitably extensive and identifies body internal protein-protein interaction.A kind of method comprises the fusion rotein that produces proteins of interest matter, and it has " tandem affinity purification " (TAP) labelling [16].Divide isolated fusion protein and form the protein of complex with it by two step affinity purification methods based on the TAP affinity labeling, the protein that links is identified by gel electrophoresis and mass spectral combination.The method of other report is used very similar strategy, with an epitope labelling and a step affinity purification method [17].These two kinds of methods all show " high flux " by brute force method (brute force), are included in to process in the previous case above 1,700 gene with greater than 725 genes in 1,000 independent yeast expression clone and the latter event.

Rappsiber etc. [18] have also finished the affinity purification of protein complex, follow crosslinked related protein.

Said method is prepared and is expressed the restriction of the demand of independent fusion rotein, to the reorganization expressive host dependency seriously limited the cell category multiformity that can study.The TAP labeling method also shows to resist and is lower than the proteinic deflection of 15kDa, and TAP and epitope labelling can both disturb normal protein matter-protein interaction.

Had high throughput automated to assist, two-hybrid system also can be adapted to the protein group screening.In these experiments [19,20], the Gal4 storehouse of 600 yeast proteins is assigned in the independent hole of 96 orifice plates, the screening of relative activation domain storehouse, each hole, and producing nearly, 4,549 kinds of possible yeast proteins interact.

Unfortunately this technology can not be used to screen mammalian proteins matter, because this can cause the non-specific interaction of vast scale.Mammalian proteins matter is extensively modified after translation in the tissue specificity mode at the subcellular organelle official and the specific cells kind apoplexy due to endogenous wind normal expression of multiple separation.There is not technology to can be used for double cross analysis bank tissue samples.Therefore, with physiological process as study or the relevant native protein of Alzheimer interacts or can not use this technical research by the interaction of signal generation.

Summary of the invention

The invention provides the method for the protein protein interaction that is used to screen composing type and Signal Regulation.The inventive method has some advantages:

(1) this is a kind of technology based on protein, does not need to clone but use to be in native protein natural, folded state, and these protein are suitably modified after translation.

(2) interaction energy influences by chemical operation, and this can easily study interesting protein protein interaction subgroup, as the Ca-dependent protein protein interaction.

(3) also can screening of medicaments reagent or the protein protein interaction interference that causes of toxin or the difference between cancer and no transformed cells;

(4) if use suitable matched group, only observe the protein interaction of natural generation.From analyze, remove non-physiology and interact, because this non-physiology of two groups interacts identical.

This method uses activatory solid support non-covalent in conjunction with other proteinic protein to separate.Holder is gel preferably, is more preferably agarose.The existence activation holder of the proteinic chemical reactivity of energy covalent bond functional group.The agarose of cyanogen bromide-activated (TM) is preferred holder.Because interacting proteins is subjected to experimental environmental effect, the mass spectrum identification of protein can produce the information about the specific interaction kinds of protein, relies on or substrate dependence protein matter-protein interaction as calcium.This allows to select and the isolated protein subgroup from the complex mixture on the specific interaction standard base.

The method can be screened complete protein group simultaneously, and unlike two-hybrid system or phage display, they only can detect in conjunction with the proteic protein of single bait at every turn.Because only observing the proteinic natural generation that is in native state interacts, the method can be widely used in the protein interaction in research organization's sample and the obduction sample, be used to screen protein protein interaction that signaling molecule, pharmaceutical agent or toxin cause disturbs and screening cancer and no transformed cells between difference.

On aspect the most widely, invention provides the method for isolated protein subgroup from protein mixture, and this subgroup is made up of the protein that participates in protein protein interaction in fact.Method comprises step: (a) under the condition that allows protein covalent bond holder and protein protein interaction mixture is contacted with the chemical reactivity holder; (b) the protein covalent bond holder in the permission mixture; (c) with holder and any not bonded with it Separation of Proteins; (d) holder is placed under the condition of destroying protein protein interaction; (e) is with holder and any not bonded with it Separation of Proteins at last.The protein that step (e) discharges is non-covalent in conjunction with other proteinic protein under the condition of step (a).

The chemical reactivity holder can comprise can be in the proteinic any chemical reactivity of covalent bond in suitable environment functional group.Preferred chemical reactivity partly includes but not limited to cyanate group, isocyanates group, isothiocyanate group, activatory carboxyl, activatory sulfonyl, aldehyde radical, epoxy radicals, sulfydryl.Preferred especially cyanate group.

Holder can be can with any substrate of reactant mixture physical separation.Holder is preferably with the form of microgranule or pearl.Holder preferably includes optional cross linked polymer or gel.Preferred support material includes but not limited to polystyrene, agar, agarose, polyacrylamide, dextrose, hydroxylation polyvinyl and carboxylation polyvinyl.Particularly preferred holder comprises agarose, for example the Sepharose of selling with trade name agarose (TM).

Method of the present invention also is used for the protein microarray analysing protein and interacts.In conventional microarray applications, the whole protein group is applied to the microarray of fixing protein with research protein protein interaction [21,22].Yet any interested specific bond albumen is competed with the excessive greatly specific protein interacting proteins that do not participate in.These excessive protein can non-special absorbing on the microarray and/or by their higher concentrations in mixture be competed binding site.Equally, the protein of big relatively quality needs proportional big solubilising buffer volume, and this reduces the concentration of proteins of interest matter.By concentrating effect, fluorescent quenching, competition and dilution, a large amount of irrelevant proteinic existence can significantly reduce from the ratio of the relative noise of protein microarray gained signal.

The present invention reduces these problems by preselected protein subgroup, and this is on the basis of they and other protein target interaction ability.This subgroup accurately comprises the protein of possibility specific bond arrays of immobilized protein.Before being applied to protein microarray, the method for invention can reduce in the protein group at least 75% different proteins sum.The protein of removing is the protein that does not participate in specific protein-protein interaction, and comprising may non-special absorption or sink into protein on the array.

If desired, but the protein that labelling keeps (for example with biotin or suitable fluorescent dye) and be applied to protein microarray is present used identical in mode and existing the application.Do like this, from not needing proteinic potential noise greatly to reduce, protein microarray gained result's quality is significantly improved on the protein microarray.The application of another kind method on microarray comes from that the easily separated tool of method is special, the ability of the protein subgroup of required biochemical property.For example, but practitioner's using method is selected protein, and these protein interactions depend on the existence of the thunderous handkerchief mycin of calcium, cAMP, specific DNA sequence or pharmaceutical agent.The control washing condition can be selected the protein of the low relatively or relative high-affinity of tool.When method of the present invention was used for preselected proteins of interest matter, microarray more may successfully be identified the participation interacting proteins.The method can be tested researcher and need do not made up specific microarray or other costliness or indirect method in addition and reaches similar results.

Therefore, the method of analysing protein mixture comprises the array that makes mixture contact fixing protein, the improvement that the inventive method provides is to separate subgroup from protein mixture, and subgroup is made up of the protein that participates in protein protein interaction in fact, subsequently the subgroup contact array.

Method of the present invention is described in publication [23].

The accompanying drawing summary

Figure 1A shows the 6 kind possible outcomes of bimolecular protein complex (AB) in conjunction with agarose (TM) support microgranule.

Figure 1B describes the non-covalent protein that links of eluting from agarose (TM) microgranule of Figure 1A.

Fig. 2 is the Western blotting of SDS-PAGE gel, relatively uses CNBr-agarose (TM) method (left side) and the isolating calmodulin, CaM of calmodulin, CaM affinity chromatograph (right side)-conjugated protein.

Fig. 3 is the two-phase gel of the Coomassie blue stain of rat brain extract, and it is by method enrichment Ca-dependent protein protein interaction of the present invention.Can see on this gel to be less than 200 points, indicate selected protein subgroup.In 23 maximum points, 12 indication points are identified by mass spectrum.

Fig. 4 is with the two-phase gel of 8M carbamide from the rat brain extract of CNBr-agarose release.

Fig. 5 shows Ca ²⁺Dependence protein matter-protein interaction is selected the calmodulin, CaM point (left side) in the back two-phase gel, with calmodulin, CaM point (right side) comparison in the nonoptional gel.The gross protein of same amount (100 μ g) is applied to a gel.Calmodulin, CaM point concentrates about 50 times.

A kind of potential application of Fig. 6 display packing is used for studying the study dependent change of protein interaction.Plate is to train (left hurdle) and train the proteinic two-phase polyacrylamide gel of (right hurdle) rat hippocampus extract corresponding region in water maze.Two kinds of protein (central authorities or left hurdle) are the candidates of study-special change in the protein protein interaction.

Detailed Description Of The Invention

Abbreviation:

CHAPS:3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid

EGTA: ethylene glycol bis (the amino ether of 2-)-N, N, N ', N '-tetraacethyl

MARCKS: the rich alanine C of myristoylation kinase substrate

The relevant interaction of non-physiology is the important potential problems in numerous protein-protein interaction research, comprises two-hybrid system.In the method, when the tissue sample homogenize, except the protein interaction that processing causes, non-specific protein interacts and takes place between also under normal circumstances discontiguous each other protein.For example, can produce between film and nucleoprotein or the interaction between astrocyte and neuronal protein.For preventing to produce wrong interaction, need to use and handle and control sample only thinks that also the difference of two kinds of sample rooms represents potential important interaction.Because any non-special XNOR physiology protein interaction that takes place after the cell homogenize in contrast and the processed group is identical, can distinguish normal, physiology related protein-protein interaction and artificial interaction easily.Contrast identical with the non-special interaction of processed group, and detecting by taking place in vivo of causing of processing in conjunction with changing easily.

As an example of this variation analysis, the method can be used to study issuable protein interaction after rat water maze task [24, the 25] learning by association.Training group of swimming and the not training control group of swimming are used in this experiment in no platform groove in containing the tank that seals platform.Use CNBr-agarose (TM) method to separate separately the interacting protein of Hippocampus extract in each group and on the two-phase polyacrylamide gel, analyze subsequently.Any protein interaction difference that study produces is reflected as a little, and the some intensity of training group is different from the intensity of matched group corresponding protein particle.Fig. 6 proves this result of experiment, and the study-specificity that has two kinds of protein may show protein interaction increases (the upward middle part on left hurdle).In case these protein are identified by mass spectrum or other method, are easily identified its binding partners.This provides a kind of useful method to identify the new signal approach relevant with physiological process.

Other example of this variation analysis is tested the protein interaction difference between normal and cancerous cell, determines the interior effect of body of pharmaceutical agent or toxin.Any difference between two groups can not produce the artificial interaction that takes place in sample processing, but the body internal protein interaction difference that produces is handled in representative.

When the outer interaction protein of chorista depends on the group of some pharmaceutical agents existence as being subjected to Ca-dependent interacting proteins group or interaction, calcium or testing drug reagent add buffer in each step, all related proteins of concentration sufficient to guarantee are combined closely.In elution step, to wash chromatographic column by buffer and come elute protein, rather than use 8M carbamide, buffer is identical in all respects, except omitting pharmaceutical agent.The ionic strength of two kinds of buffer and pH should be identical to prevent any protein since pH or ion concentration change and eluting.

In this case, not strict relatively two the independent samples that need produce in test tube because protein of interest matter interacts.

Although the method can enrichment 50 times interacting protein, if some non-interacting proteins are higher than protein affinity each other to the affinity of agarose (TM), then also can detect.Although protein may be absorbed in agarose (TM) [26], effect is generally less, can remove by adding matched group as mentioned above.Do not observe non-specific bond in the experiment described herein with agarose (TM).

Form by relating to proteinic raw information with the data that the method produces, these protein can with unidentified protein or some other protein interactions.Although this itself is valuable information and makes problem space reduce several magnitude, still need to confirm the protein interaction of inferring.In case the evaluation target protein is easily found its binding partners, use routine techniques such as affinity chromatograph [27] or double cross analysis.For complete understanding interacts, also need to confirm to infer the generation that interacts with some other methods.Certainly, other screening technique such as dna microarray, phage display or two-hybrid system also need to determine observed variation.Because this method is only measured the total protein that is in interacting state, also need to confirm to interact to determine the biochemical basis of interaction level increase, this may, abundance bigger generation higher by the target protein affinity, or even reduces by activator activation or some interaction inhibitor levels in deadly condition and produce.

This technology also can be revised, and adds cross-linking step and sulfydryl agarose (TM) and replace CNBr-agarose (TM) after initial flush.This allows interacting protein to separate being good for two sulfur of agarose (TM) by cutting connection protein, crosslinking protein is verified can separate and be accredited as single unit.

Except the Ca-dependent protein interaction, there is the example of many protein protein interactions of regulating by GTP, cAMP, protein phosphorylation, zymolyte or other biochemical phenomenon.This method can be used to study these protein interaction kinds, for example comparison protein phosphatase or the nucleotide phosphohydrolase pattern that exists or produce when lacking.

New method has the advantage of screening the whole protein group simultaneously, and unlike other method, they only can detect in conjunction with the proteic protein of single bait at every turn.In addition, the method need not cloned, but the protein-protein interaction that separating natural produces, protein is in natural, folded state, suitably modification after translation.Protein is influenced by chemical operation also, can select the protein subgroup on the specific interaction standard base from the complex mixture.Method not only is used to study protein protein interaction, also identifies the action site of low molecular weight compound such as xenobiotic or pharmaceutical preparation.In the past, determined that whether xenobiotic influences protein protein interaction was a difficult task, unless known a kind of target protein.With present method, can screen the whole protein group rapidly to identify the protein that interacts and influenced by molecules of interest, produce specific target and be used for further research.

Difunctional cross-linking reagent adds the compound protein extract and generates unmanageable common insoluble aggregation mixing, wherein comprises multiple proteins.This infers that the cross-linking reagent by the micromolecule size causes, this cross-linking reagent make its in a plurality of positions in conjunction with two kinds of interacting partners.Cross-linking agent also makes common incoherent protein random incorporation each other.In the method for the invention, these problems disappear, because use chemical reactivity holder rather than cross-linking agent.

A pair of interacting protein A and B covalent bond chemical reactivity holder can produce 6 kinds of possible kinds as a result, and this depends on that a kind of still two kinds of protein are in conjunction with holder.Figure 1A shows the particular sketch map, and wherein holder is agarose (TM) microgranule.Described 6 kinds of possible outcomes are:

1. a-protein is free in conjunction with two kinds of microgranules and PROTEIN B,

2. a-protein is free in conjunction with a kind of microgranule and PROTEIN B,

3. a-protein and PROTEIN B be in conjunction with different microgranules,

A-protein in conjunction with a kind of microgranule and PROTEIN B in conjunction with two kinds of microgranules,

5. a-protein and PROTEIN B be all in conjunction with two kinds of microgranules,

6. a-protein and PROTEIN B are all in conjunction with a kind of microgranule.

Each bar line is represented one or more being good among the figure, and the supposition of two kinds of agaroses (TM) microgranule is commutative.Have 3 kinds of other results, wherein A and B conversion.Because particle size is big relatively, get rid of the result (in Figure 1A 1,3,4 and 5) of protein complex, because mechanical pressure surpasses valence bond in conjunction with different microgranules.As long as activated group density is not too high on the microgranule, two kinds of companions should be rare relatively in conjunction with the result (6) of identical microgranule.This only stays result (2), and wherein a kind of protein covalent bond microgranule and another kind are non-covalent with it to link to each other.

For selecting the protein of noncovalent interaction, the agarose of protein mixture and cyanogen bromide-activated (TM) reacts, and used mode makes 50% protein covalent bond.Remain on the agarose (TM) with the remaining protein flush away or by noncovalent interaction with the covalency conjugated protein.After washing in suitable buffer, main component is in the bonded protein in single site (result 2), and wherein 1 companion's covalency invests microgranule and its non-covalent interacting partner that adheres to by keeping with protein-bonded affinity.Wash the non-covalent attachment protein matter of eluting (Figure 1B) by 8M carbamide subsequently.In addition, can revise elution buffer, interact as substrate dependency or Ca-dependent to detect specified protein-protein interaction type.Protein [28,29] with suitable method such as two-phase gel electrophoresis or capillary tube LC-MS analysis eluting.

The method proves that as the ability that the feasibility of triage techniques detects Ca-dependent protein protein interaction by its Ca-dependent protein protein interaction relates to calmodulin, CaM.Rat cerebral even slurry CHAPS extract is in conjunction with CNBr-activated agarose (TM), and agarose (TM) is washed with the Tris-acetate buffer, interacting protein EGTA eluting.EGTA uses with the lucky half strength of Tris-acetate, thereby carboxyl or amino group concentration do not change, and can change elute protein by ionic strength when converting EGTA to.Concentrate the protein of eluting, desalination, unidirectional SDS-polyacrylamide gel electrophoresis is separated, and trace is transferred the antibody staining of protein dependent kinase I, calmodulin-dependent kinases II, MARCKS or protein phosphatase 2A with anticalcium on nitrocellulose.Western blot analysis (Fig. 2, left side swimming lane) shows that whole 4 kinds of caldesmons of surveying (CaM kinases I and II, MARCKS and protein phosphatase 2A) can detect with the method.

The independent sample of brain homogenate extract is in conjunction with the calmodulin, CaM affinity column of routine preparation, the EGTA eluting, and the protein of eluting is with above-mentioned western blotting analysis.Result's (Fig. 2, right side swimming lane) is comparable, and it illustrates that method of the present invention can separation of C a ²⁺The dependency caldesmon.Blank (" blk " swimming lane) from sample, wherein brain extract is splined on agarose (TM), by presenting inertia with the Tris-HCl reaction.

For example, describe inventive method and be used to study the Ca-dependent protein interaction.The method is applied to the rat brain extract reveals the Ca-dependent protein interaction with option table protein.In 12 protein that mass spectrum is identified, 8 is known calbindin or the protein that known Ca-dependent protein interaction is arranged, prove this method can be on specified protein interacts the kind basis from very complicated mixture the enrichment protein subgroup.

Be the research method specificity, rat brain extract sample such as preceding in conjunction with CNBr-agarose (TM), the protein of EGTA eluting separates by the two-phase polyacrylamide gel electrophoresis.Fig. 3 shows the Coomassie blue stain gel of this experiment.When all interacting proteins during with 8M carbamide eluting, but the sum of measurement point (about 172) is put (not shown) less than 300-400 visible point (Fig. 4) for conventional visible 1000-1200 in the non-selected extract.23 points that institute's detected intensity is bigger on two-phase gel Trypsin matter enzymic digestion, the gained peptide is analyzed with LC-MS/MS.With the LC-MS/MS data with from the peptide and the fragment quality matches of Protein Data Bank sequence, 12 the positive evaluations in 23 protein of analyzing.In 12 identification of protein, known 8 protein bound calcium or rely on mode and other protein interaction with calcium.

Identify that 11 a little bigger trials of residue are unsuccessful among Fig. 3, comprise 10kDa, pI7.3 and 52kDa, pI4.8's is a little bigger.Although obtain many peptides, analyze mass spectrometric data do not produce with the data base in any proteinic coupling.

The protein that abundance is the highest on the two-phase gel (point 1) is accredited as calbindin calmodulin, CaM (table 1 and Fig. 3).This point also can detect in crude extract, but micro-relatively component (Fig. 5).Most of residue can identify and a little comprise atp synthase, mitochondrial ATP enzyme inhibitor and heterogeneity nuclear ribonucleoprotein matter A2 (hnRNP A2), be also referred to as calbindin or with the protein [30,31,32,33,34] of calbindin direct interaction.Also detect the peptide from another kind of calbindin S-100, S-100 is subjected to many Ca-dependent protein interactions [35].Although M is identical with S-100 with pI, because observed peptide quantity is little in the digestion, mass spectrum is identified and is not reached statistically significant.

Table 1 has been summarized the protein that mass spectrum is identified.Except hemoglobin, citrate synthase and carbonic anhydrase, the protein of all evaluations is the fine definite protein of known calbindin or Ca-dependent protein interaction feature.For example, tropomyosin matter links to each other with the actin matter of knowing-troponin matter-myosin matter complex.Ca ²⁺Make troponin mass-energy in conjunction with tropomyosin matter and mobile with combining of troponin matter from the myosin matter binding site of actin matter.There is not Ca ²⁺When existing, troponin matter no longer can be in conjunction with tropomyosin matter, and tropomyosin matter is blocked the myosin matter binding site on the actin matter once more.Tropomyosin matter is also in conjunction with calbindin calcyclin matter [36].Similarly, the Rho GDP inhibitor that dissociates is strong with combining of the conjugated protein rho of low-molecular-weight GTP-, and rho participates in actin cell plastid skeleton reorganization [37] with calbindin cadherin matter.Flesh calcium sample albumen (Calponin) also is rho-kinase substrate [38].

Table 1

The protein that the Ca-dependent protein interaction is arranged

Period Mr pI coating ratio identifying species

1 17,420 4.25 37 calmodulin, CaM calcium are in conjunction with 2 52,480 4.86 2-mannosidase calcium are in conjunction with 35,370 4.93 21 S100 β chain calcium are in conjunction with 4 10,100 7.66 63 hemoglobin alphas 1-6 60,220 5.52 48 atp synthase calcium are in conjunction with 11 9,770 4.42 9 atpase inhibitor calcium are in conjunction with 12 8,750 4.45 18 atpase inhibitor calcium are in conjunction with 17 61,660 6.28 12 dihydropyrimidinases relevant-26 26,980 7.67 26 carbonic anhydrases 2-28 36,470 8.18 2 heterogeneities nuclear RNP A2 calmodulin, CaM is in conjunction with 31 31,330 5.23 28 tropomyosin matter calcyclin matter are in conjunction with 32 30,480 5.80 41 Rho GDI-1 through Rho in conjunction with cadherin matter 35 44,510 9.061 6 citrate synthases-

In the above example, the protein of the total inspection above 30% is calmodulin, CaM, and it is to rely on mode in conjunction with many other proteinic calbindins [39] with calcium.4 kinds of other protein (two kinds of forms of atp synthase, atpase inhibitor and S100) also is known calbindin, links closely with calbindin and 3 kinds in addition (tropomyosin matter, Rho GDP dissociate inhibitor and heterogeneity nuclear ribonucleoprotein matter A2 (hnRNP A2)) is also known.Should point out that method of the present invention is not only the method that detects calbindin.On the contrary, the special detection of the method relies on mode in conjunction with some other proteinic protein subgroups with calcium.This comprises some calbindins, but also comprises its target, and as Ca-dependent kinases and signal-proteins (as rho and rab), they rely on mode with calcium and calbindin interacts.

Also detecting 3 kinds of inexpectancy protein is hemoglobin, carbonic anhydrase 2 and citrate synthase.Although hemoglobin depends on Fe with combining of other hemoglobin subunit ²+ and O ₂Do not know that it is in conjunction with calcium; Yet hemoglobin can be in conjunction with skein cell film [40] when calcium existed, and pointing out it may be embrane-associated protein companion in conjunction with calcium.Citrate synthase and carbonic anhydrase can link with the protein of not determining feature when similarly, calcium existed.

Methods described herein are applied to study the protein protein interaction in the mammalian tissues.For example, prompting Alzheimer and other neurodegenerative disease cause [41,42] by the pathologic protein protein interaction.Similarly, the complex network of protein protein interaction is depended in cell signal, synaptic plasticity, study and growth.The method expection is used to separate the macro-molecular protein complex, as the part of protein group screening sequence, is used to related protein-protein interaction of identifying that further institute is used.

Experiment

Titration CNBr agarose (TM): before the use, the agarose of cyanogen bromide-activated (TM) 4B (Pharmacia) rehydration also washes with water 3 times.By the extract of not commensurability CNBr agarose (TM) incubated at room fixed amount, CNBr rat brain extract titration.After 1 hour, centrifugal sample and the not conjugated protein dyestuff of using are measured in conjunction with measuring [43], calculate the CNBr agarose (TM) of protein concentration minimizing 50% is measured.

Separate interacting proteins: rat brain passes through at 10mM NaHCO ₃, ultrasonic degradation comes homogenize among the pH7.7, wherein contains 5%CHAPS, 0.1mM benzene mesyl chloride and 1mM CaCl ₂, 100, centrifugal 20 minutes of 000g.Adding is enough in conjunction with 50% proteinic rehydration CNBr agarose (TM) amount sample room temperature vibration 1 hour.The Tris-acetate adds to 0.1M subsequently with sealing unreacted CNBr, hatches to continue 30 minutes again.Mixture goes to little chromatographic column also with containing 1mM CaCl ₂100mM Tris-acetate fully wash.A when eluate ₂₈₀Reach at 0 o'clock, protein 50mM EGTA eluting by Ca-dependent interaction maintenance, desalination also concentrates in the Centricon-3 ultrafiltration apparatus, mix at 1: 1 with IEF sample buffer (8.5M carbamide, 2M thiourea, 0.4%CHAPS, 0.5%IPTG buffer (Amersham) and 0.01% bromophenol blue), be applied to Immobiline pH 3-10 polyacrylamide isoelectrofocusing bar, carry out flat (flatbed) two-phase polyacrylamide gel electrophoresis (ExcelGel 12-14) with same solution rehydration.

The gel Coomassie blue stain is downcut the visible point of 23 maximums and is carried out the enzymic digestion of Trypsin matter and the LC-MS/MS analysis.Wherein, 12 are passed through following SEQUEST and the evaluation of Mascot software.

Affinity chromatograph: rat brain extract 50mM NaHCO ₃, pH7.7 and 1mM CaCl ₂In 2cm3 calmodulin, CaM-agarose (TM) 4B incubated at room 15 minutes.Mixture goes to post, with containing 1mM CaCl ₂Tris-HCl wash, up to A ₂₈₀Can not detect.Caldesmon also concentrates in the Centricon-3 ultrafiltration apparatus with 50mM EGTA eluting, desalination, and the 4-20%SDS-polyacrylamide gel electrophoresis is separated, and trace is to nitrocellulose membrane.

Western blot analysis: analytic sample is by electrophoresis on 4-20% acrylamide gradient sds gel, and then trace is to nitrocellulose membrane, and antibody is surveyed, and develops with nitroblue tetrazolium and 5-bromo-4-chloro-3-indole phosphoric acid.

Mass spectrum: with (in-gel) method in the described glue of Association of Biomolecular Resource Facilities [44], downcut dyeing protein spots and the enzymic digestion of Trypsin matter from the two-phase gel.The enzymic digestion of Trypsin matter is spent the night at 37 ℃ and is finished, and peptide is from gel extraction to 5% formic acid: the acetonitrile (1: 1), extract 5% formic acid for the second time: in the acetonitrile (5: 95).Collect extract, reduce volume by traditional vacuum, final volume to 10 microlitre wherein has 0.1%TFA.Remove impurity salt and microgranule, this is in conjunction with C by peptide ₁₈(Millipore, MA), 0.1%TFA washes-ZipTip, is eluted to 10 microlitre 0.1%TFA: in the acetonitrile (1: 1).Analyze the peptide of Trypsin matter enzymic digestion by series connection liquid chromatography/mass spectrometry (LC-MS/MS).Phase chromatography-use Michrom Magic HPLC system carries out, and the constant pressure diverter is arranged so that the flow velocity by post reduces to 400nl/ minute.By the reversed phase chromatography isolated peptides, use Vydac C ₁₈, 5 micron particle, fill in 300 dust holes.The molten silicon capillary tube of the column filling to 75 of about 5cm micron I.D. (PicoFrit, New Objective Inc., Woburn MA).Peptide separates from the 2-85% buffer B with linear gradient that (buffer A: 5% acetonitrile in the water has 0.5% acetic acid and 0.005%TFA; Buffer B: 80% acetonitrile, 10%n-propanol, 10% water have 0.5% acetic acid and 0.005%TFA).The direct EFI of LC effluent is mapped to LCQ DECA spectrometer sample well, and (Thermo Finnigan CA), uses micro-electrojet interface to adapt to [45].Operation LCQ DECA is used for collecting the MS/MS spectrum in data dependence mode, and 4 the strongest ions that surpass preset threshold value divide and analyze.The MS/MS data that analyze to produce, determine with NCBI-Non-redundant data storehouse in the coupling of protein sequence (mammal subgroup), use SEQUEST[46] and MASCOT[47] program.

Sequence Identification is based on Mowse value [48] (10 * log (P), wherein P is a probability, the observation of Mascot software discovery coupling is random event).Protein value greater than 60 is o'clock remarkable in p＜0.05.In each situation, the prediction M of evaluation _rWith the pI and the M that observes _rMate in ± 5% with the pI value.

Computer analysis: image is quantitative, and point is arranged and molecular weight estimates to use image analysis program tnimage[49] finish, this program can obtain oneself ( Http: //) entropy.brni-jhu.org/tnimage.html).

List of references

1.S.E.Benashski with S.M.King.Investigation of protein-protein interactions withinflagellar dynein using homobifunctional and zero-length crosslinking reagents.Methods, 22:365-371 (2000).

2.C.T.Rollins etc., Aligand-reversible dimerization system for controlling protein-protein interactions.Proc.Natl.Acad.Sci.USA, 97:7096-7101 (2000).

3.S.A.McMahan with R.R.Burgess.Use of aryl azide cross-linkers to investigateprotein-protein interactions:An optimization of important conditions as applied toEschercia coli RNA polymerase and localization of a sigma 70-alpha corss-link to theC-terminal region of alpha.Biochemstry, 33:12092-12099 (1994).

4.S.Kiessig, J.Reissmann, C.Rascher, GKullertz, A.Fischer and F.Thunecke.Application of a green fluorescent fusion protein to study protein-protein interactionsby electrophoretic methods.Electrophoresis, 22:1428-1435 (2001).

5.S.H.Park with R.T.Raines.Green fluorescent protein chimeras to probeprotein-protein interactions.Methods Enzymol, 328:251-261 (2000).

6.G.P.Smith.Filamentous?fusion?phage：novel?expression?vectors?that?displaycloned?antigens?on?the?virion?surface.Science，228：1315-1317(1985).

7.S?Rossenu，D.Dewitte，J.Vandekerckhove，and?C.Ampe.A?phage?display?techniquefor?a?fast，sensitive，and?systematic?investigation?of?protein-protein?interactions.J.Protein?Chem.，16：499-503(1997).

8.S.Fields with O.Song.A novel genetic system to detect protein-protein interactions.Nature, 340:245-247 (1989).

9.G.MacBeath with S.L.Schreiber.Printing proteins as microarrays for high-throughput function determination.Science, 289:1760-1763 (2000).

10.M.N.Kronick with W.A.Little.A new immunoassay based on fluorescenceexcitation by internal reflection spectroscopy.J.Immunol.Methods, 8:235-240 (1995).

11.J.D.Andrade，D.E.Van?Wagenen，D.E.Gregonis，K.Newby，and?J.N?Lin.Remotefiber?optic?biosensors?based?on?cvanescent-excited?fluoroimmunoassay:concepts?andprogress.IEEE?Trans.Elec.Dev.，ED-32：1175-1179(1985).

12.S.Beeckmans.Chromatographic?methods?to?study?protein-protein?interactions.Methods，19：278-305(1999).

13.A.K.Kenworthy.Imaging?protein-protein?interactions?using?fluorescenceresonance?energy?transfer?microscopy.Methods，24：289-296(2001).

14.N.Mochizuki，S.Yamashita，K.Kurokawa，Y.Ohba，T.Nagai，A.Miyawaki，andM.Matsuda.Spatio-temporal?images?of?growth-factor-induced?activation?of?ras?andrap1.Nature，411：1065-1068(2001).

15.A.Kumar and M.Snyder, Nature 415:123-124 (2002).

16.A.-C.Gavin etc., Functional organization of the yeast proteome by systematicanalysis of protein complexs.Nature 415:141-147 (2002).

17.Y.Ho etc., Systematic identification of protein complexs in Saccharomycescerevisiae by mass spectrometry.Nature 415:180-183 (2002).

18.J.Rappsilber，S.Siniossoglou，E.Hurt，M.Mann.A?generic?strategy?to?analyze?thespatial?organization?of?multi-protein?complexes?by?cross-1inking?and?massspectrometry.Anal.Chem.，72：267-275(2000).

19.P.Uetz etc., A comprehensive analysis of protein-protein interactions insaccharomyces cerevisiae.Nature, 403:623-627 (2000).

20.T.Ito，T.Chiba，R.Ozawa，M.Yoshida，M.Hattori，and?Y.Sakaki.A?comprehensivetwo-hybrid?analysis?to?explore?the?yeast?protein?interactome.Proc.Natl.Acad.Sci.USA.，98(8)：4569-4574(2001).

21.C.Paweletz etc., Reverse phase protein microarrays which capture diseaseprogression show activation of pro-survival pathways at the cancer invasion front.Oncogene 20 (16): 1981-1989 (2001).

22. P.Wagner etc., Arrays of protein-capture agents and methods of use thereof. United States Patent (USP) 6,329,209 (2001).

23.T.Nelson, P.Backlund, Jr., A.Yergey and D.Alkon.Isolation of ProteinSubpopulations Undergoing Protein-Protein interactions. Mol.Cell.Proteomics, 1:253-259 (2002).

24.R.Morris.Developments?of?a?water-maze?procedure?for?studying?spatiallearning?in?the?rat.J.Neurosci.Methods，11(1)：47-60(1984).

25.R.Brandeis，Y.Brandys，and?S.Yehuda.The?use?of?the?morris?water?maze?in?thestudy?of?memory?and?learning.Intern.J.Neurosci.，48：29-69(1989).

26. J.C.Tercero and Diaz-Maurino.T.Affinity chromatography of fibrinogen on lensculinaris agglutinin immobilized on CNBr-activated sepharose:study of the activegroups involved in nonspecific absorption.Anal Biochem, 174 (1): 128-136 (1988).

27.?S.Beeckmans，ref.12.

28.?M.P.Washburn，D.Wolters，J.R.Yates?III.Large-scale?analysis?of?the?yeastproteome?by?multidimensional?protein?identification?technology.NatureBiology19：242-247(2001).

29. L.Li etc., High Throughput Peptice Identification from Protein Digests UsingData-Dependent Multiplexd Tandem FTICR Mass Spectrometry Coupled withCapillary Liquid Chromatography.Anal Biochem, 73:3312-3322 (2001).

30.M.J.Hubbard with N.J.McHuhh.Mitochondrial ATP synthase F1-beta subunit is acalcium-binding protein.FEBES Lett, 391 (3): 323-329 (1996).

31.N.Arakaki，Y.Ueyama，M.Hirose，T.Himeda，H.Shibata，S.Futaki，K.Kitagawa，andT.Higuti.Stoichiometry?of?subunit?e?in?rat?liver?mitochondrial?H(+)-ATP?synthase?andmemberance?topology?of?its?putative?Ca(2+)-dependent?regulatory?region.Biochim.Biophys.Acta，1504(2-3)：220-228(2001).

32.E.W.Yamada with N.J.Huzel.The calcium-binding ATPase inhibitor protein frombovine heart mitochondria.purification and properties.J.Biol.Chem., 263 (23): 11498-11503 (1988).

33.B.W.Schafer with C.W.Heizmann.The S100 family of EF-hand calcium-bindingprotein.Trends in Biochem, 21:134-139 (1996).

34.R.Bosser, M.Faura, J.Serratosa, J.Renau-Piqueras, M.Pruschy and O.Bachs.Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins a2 and c can bemodulated by calmodulin.Mol.Cell.Biol, 15 (2): 661-670 (1995).

35.S.Treves，E.Scutari，M.Robert，S.Groh，M.Ottolia，GPrestipino，M.Ronjat，andF.Zorzato.Interaction?of?S100A1?with?the?Ca2+release?channel(ryanodine?receptor)ofskeletal?muscle.Biochemistry，36(38)：11496-11503(1997).

36.N.L.Golitsina，J.Kordowska，C.L.Wang，and?S.S.Lehrer.?Ca2+-dependentbinding?of?calcyclin?to?muscle?tropomyosin.Biochem?Biophys?Res?Commun，220(2)：360-365(1996).

37.N.K.Noren，C.M.Niessen，B.M.Gumbiner，and?K.Burridge.Cadherin?engagementregulates?rho?family?GTPase.J.Biol.Chem.，276(36)：33305-33308(2001).

38.T.Kaneko，M.Amano，A.Maeda，H.Goto，K.Takahashi，M.Ito，and?K.Kaibuchi.Identification?of?calponin?as?a?novel?substrate?of?rho-kinase.Biochem.Biophys.Res.Commun.，273(1)：110-116(2000).

39.H.Weinstein with E.L.Mehler.Ca2+ binding and structural dynamics in thefunctions of calmodulin.Ann.Rev.Physiol., 56:213-236 (1994).

40.E.Friedrichs, R.A.Farley and H.J.Meiselman.Influence of calciumpermeabilization and membrane-attached hemoglobin on erythrocyte deformability.Am.J.Hematol., 41 (3): 170-177 (1992).

41.M.Sudol，Sliwa?K，and?Russo?T.Functions?of?WW?domains?in?the?nucleus.FEBES?Lett.，490(3)：190-195(2001).

42.J.Q.Trojanowski with V.M.Lee.Fatal attractions of proteins.a comprehensivehypothetical mechanism underlying alzheimer ' s disease and other neurodegenerativedisorders.Ann.N.Y.Acad.Sci., 924:62-67 (2000).

43.M.M.Bradford.A?rapid?and?sensitive?method?for?the?quantitation?of?microgramquantities?of?protein?utilizing?the?principle?of?protein-dye?binding.Anal.Biochem.，72：248-254(1976).

44.Association?of?Biomolecular?Resource?Facilities.Representative”In-Gel”Digestion?Protocol?for?Proteins?in?SDS?PAGE?Gel.(1977).

(http：∥)www.abrf.org/ABRF/ResearchCommittees/intprotseqrescomm.html

45.M.T.Davis, D.C.Stahl, S.A.Hefta and T.D.Lee.A microscale electrosprayinterface for on-line, capillary liquid chromatography/tandem mass spectrometry ofcomplex peptide mixture.Anal.Chem., 24:4549-4556 (1995).

46.J.K.Eng，A.L.McCormack，and?J.R.III.Yates.An?approach?to?correlate?tandemmass?spectral?data?of?peptides?with?amino?acid?sequences?in?a?protein?database.J.Amer.Soc.Mass?Spec.，5：976-989(1994).

47.?D.N.Perkins，D.J.Pappin，D.M.Creasy，and?J.S.Cottrell.Probability-based?proteinidentification?by?searching?sequence?databases?using?Mass?spectrometry?data.Electrophoresis，18：3551-3567(1999).

48.D.Pappin, P.Hojrup and A.Bleasby.Rapid identification of proteins bypeptide-mass fingerprinting.Curr.Biol.3 (6): 327-332 (1993).

49.The?tnimage?program?is?available?at( http：∥)entropy.brni-jhu.org/tnimage.html.

Claims (7)

1. method of from protein mixture, separating subgroup, this subgroup is made up of the protein that participates in protein protein interaction in fact, it is characterized in that described method comprises:

(a) allowing (i) protein covalent bond holder, and under the condition of (ii) protein protein interaction mixture is being contacted with the chemical reactivity holder;

(b) the protein covalent bond holder in the permission mixture;

(c) with holder and any not bonded with it Separation of Proteins;

(d) holder is placed under the condition of destroying protein protein interaction; With

(e) with holder and any not bonded with it Separation of Proteins.

2. the method for claim 1; it is characterized in that described chemical reactivity holder comprises the chemical reactivity part that is selected from cyanate group, isocyanates group, isothiocyanate group, activatory carboxyl, activatory sulfonyl, aldehyde radical, epoxy radicals, sulfydryl.

3. method as claimed in claim 2 is characterized in that, described chemical reactivity holder comprises the cyanate group.

4. as each described method among the claim 1-3, it is characterized in that described holder comprises optional cross linked polymer or gel.

5. method as claimed in claim 4 is characterized in that, described holder comprises the material that is selected from polystyrene, agar, agarose, polyacrylamide, dextrose, hydroxylation polyvinyl, carboxylation polyvinyl.

6. method as claimed in claim 5 is characterized in that described holder comprises agarose.

7. analysing protein mixture method, comprise described mixture contact fixing protein array, improvement is formed by separate subgroup from protein mixture, subgroup is made up of the protein that participates in protein protein interaction in fact, described subsequently subgroup contacts described array, it is characterized in that the method for separating subgroup comprises:

(a) allowing (i) protein covalent bond holder, and under the condition of (ii) protein protein interaction mixture is being contacted with the chemical reactivity holder;

(b) the protein covalent bond holder in the permission mixture;

(c) with holder and any not bonded with it Separation of Proteins;

(d) holder is placed under the condition of destroying protein protein interaction; With

(e) with holder and any not bonded with it Separation of Proteins.

Images