EP1622717A2

EP1622717A2 - Affinity-based enrichment of phosphorylated peptides and/or proteins

Info

Publication number: EP1622717A2
Application number: EP04729634A
Authority: EP
Inventors: Vukic Soskic; André SCHRATTENHOLZ
Original assignee: ProteoSys AG
Current assignee: ProteoSys AG
Priority date: 2003-05-06
Filing date: 2004-04-27
Publication date: 2006-02-08
Also published as: WO2004099233A2; US20070134648A1; DE10321901A1; WO2004099233A3; AU2004235906A1; JP2008500943A; CA2524522A1

Abstract

The invention relates to a substance comprising a solid carrier that is connected to a spacer by means of a linker, said spacer comprising at least two defined groups. Said substance is suitable for using as an affinity material for enriching and/or isolating phosphorylated peptides and/or proteins. The inventive substance especially enables tyrosine-phosphorylated peptides and/or proteins to be enriched and/or isolated.

Description

Affinity enrichment of phosphorylated peptides and / or proteins

The invention relates to a new material which is suitable for the enrichment and / or isolation of phosphorylated peptides and / or proteins. In addition to the material or the substance, the invention relates to an enrichment and / or isolation process for phosphorylated peptides and / or proteins and a process for producing the substance.

The phosphorylation and dephosphorylation of proteins in the cell is crucial for the function of many biological systems. The phosphorylation and dephosphorylation of proteins often transmit signals in the organism and in particular control numerous enzymatic activities. Phosphorylation is therefore a crucial factor for the signal transduction chains in living cells. Research into phosphorylated proteins is therefore of particular interest. In the context of proteome research, the term "phosphoproteome" was coined to describe the study of essentially all phosphorylated proteins in a cell. In recent years, phosphoproteome research in particular has developed further, since different enrichment protocols for phosphoproteins or phosphopeptides were optimized fractionation methods have been improved and in particular the multi-dimensional ^{"chromatography} was further developed so that phosphoproteins soft generally are present in very low concentrations, to make an analysis in the first place once accessible.

Methods used to date for the enrichment and identification of phosphorylated proteins usually include radioactive labeling with ³² P-labeled ATP and subsequent SDS polyacrylamide gel electrophoresis or thin-layer chromatography. Edman sequencing can also be used to identify the phosphorylated proteins.

A common problem in the study of proteins involved in signaling cascades, such as the phosphorylated proteins in particular, is that these proteins are generally only expressed in very small amounts and the stoichiometry of the phosphorylation is generally relatively low. Traditional methods for the investigation and in particular for the identification of these proteins are therefore often not suitable, since the amounts of protein required for this can only be provided with great difficulty.

Because of its special sensitivity, versatility and speed, mass spectrometry has proven to be a very suitable method proven for the study of phosphorylations. However, various studies have shown that the ion signal caused by phosphorylated peptides is significantly suppressed by the presence of non-phosphorylated peptides. It is therefore necessary to further enrich the phosphorylated proteins or peptides compared to the non-phosphorylated proteins or peptides in order to make the phosphorylated sites more detectable.

A conventional method of enriching phosphoproteins uses phosphospecific antibodies for affinity purification of the phosphorylated proteins or peptides. In particular, the use of antiphosphotyrosine antibodies has proven to be successful here, whereas the use of antibodies against phosphoserine or phosphothreonine-containing proteins has not yet been described so often. An alternative to antibody enrichment is to use immobilized metal affinity chromatography (IMAC). This method is based on the affinity of the negatively charged phosphate groups of the proteins to be enriched for positively charged metal ions, such as Fe ³⁺ or Ga ³⁺ , which are immobilized on a chromatographic support. IMAC has already been used in combination with "electro spray ionization" (ESI) tandem mass spectrometry (Stensballe et al., Proteomics 1 (2001), 207-222) or "matrix-assisted laser desorption / ionization" (MALDI) mass spectrometry ( MS) and alkaline phosphatase treatment for the determination of the phosphorylation sites (Raska et al., Anal. Chem. 74 (2002), 3429-3433).

The advantage of these conventional methods is that they can isolate the entire phosphoproteome of a cell. However, particular problems arise when examining phosphotyrosine-containing proteins or peptides, since the content of phosphotyrosine in proteins in, for example, vertebrate cells is only about 0.05% compared to the content of phosphoserine (about 90%) and phosphothreonine (about 10%).

Ojida et al. (J. Am. Chem. Soc. 124 (2002), 6256-6258) describe a fluorescent chemosensor which interacts with a phosphorylated peptide surface for the investigation of tyrosine-phosphorylated proteins. The authors were able to show that anthrazine derivatives with two zinc (II) dipicolylamine residues selectively bind phosphorylated peptides and thus cause a change in the fluorescence spectrum. With such a fluorescent chemosensor, phosphorylated peptides can be detected in aqueous solution. The anthrazine derivatives described here show a particular specificity for peptides containing phosphotyrosine. Mito-oka et al. (Tetrahedron Letters 42 (2001), 7059-7062) describe that similar components can bind dihistidine sequence motifs from peptides.

Since tyrosine phosphorylation plays a special role in the living cell, in particular in signal transduction and in other regulatory mechanisms, the object of the invention is to provide a method which is improved over conventional methods and with which peptides and / or proteins phosphorylated on tyrosine are enriched and / or isolated can.

This object is achieved by a substance as described in claim 1. Claim 8 describes an enrichment or isolation process which uses a corresponding substance. Claims 11, 13 and 15 deal with the use of this substance or with a corresponding affinity material. Claim 16 claims a process for producing the substance according to the invention. The dependent claims relate to various preferred embodiments of these aspects of the invention. The wording of all claims is hereby incorporated by reference into the content of the description.

According to the invention, a substance is provided which is suitable as an affinity material for the enrichment and / or isolation of phosphorylated peptides and / or proteins. This substance comprises a solid carrier which is connected to a spacer via a linker. This spacer carries at least two groups, which are described by the following general formula:

Mean in this formula

X, Y: CR ', N, S and / or O;

Z: CR " ₂ and / or (CR") ₂ ;

R \ R ": H, alkyl, halogenyl and / or O-alkyl; and

M: M A | 3+ and / or Ga .3+

For illustration, the substance of the invention is shown graphically in FIG. 1. The different letters stand for the meanings already described. This substance, and in particular the spacer with these at least two groups, has a high affinity for phosphorylated peptides and / or proteins. This substance binds corresponding peptides and / or proteins with great affinity and therefore enables these peptides and / or proteins to be enriched and / or isolated from a sample. By immobilizing this high affinity component, the substance can be used as an affinity material, which is used, for example, like a conventional column chromatography material. This substance can therefore be used with protocols, as are readily apparent to the person skilled in the art, for the enrichment and / or isolation of phosphorylated peptides and / or proteins from a sample. Zinc complexes of the substances according to the invention have proven to be particularly advantageous.

The substance according to the invention is particularly suitable for enriching or isolating phosphorylated peptides and / or proteins which are phosphorylated (pTyr) on one or more tyrosine residues. The substance or substances according to the invention can therefore also be described as synthetic pTyr receptors. In a preferred embodiment of the substance according to the invention, the group is a derivative of 2,2'-dipicolylamine. A corresponding substance according to the invention has a particularly high affinity for tyrosine-phosphorylated peptides and / or proteins.

In a preferred embodiment of the substance according to the invention, the spacer comprises one or more aromatic rings. These are in particular mono-, bi- and / or tricyclic aromatic rings. Advantageously, the spacer is a methyl dimethylbenzate, which may also have further residues. In a particularly preferred embodiment, the starting compound for the spacer is characterized by the following formula:

Here mean

Ri, R ₂ : H, alkyl, halogenyl, O-alkyl and / or mono- or bicyclic aromatic rings.

In very particularly preferred embodiments, the spacer is 2,5-, 3,4- and / or 3,5-dimethylbenzenic acid methyl ester or a corresponding derivative.

The linker between the spacer and the solid support is preferably at least one amide, ester, carbamoyl, ether and / or thioether bond. The specific design of the linker or the linker connection naturally depends on the choice of the carrier material and the composition of the spacer.

In a very particularly preferred embodiment of the substance according to the invention, the spacer is inclusive of the at least two groups 2,5-, 3,4- and / or 3,5-bis [(2,2'-dipicolylamino) methyl] benzene acid or the corresponding one Benzolsäuremethylester.

The solid support is preferably glass, silicate, gold and / or at least one organic polymer. In general, all usual carrier materials that are suitable for chromatographic applications are suitable. Chromatography materials in the form of beads, as are usually used for column chromatography, are advantageous. Preferred examples of this are Fractogel or Sepharose, in particular Sepharose 4B. However, the choice of the carrier material is not restricted to those materials which can be used for column chromatography. Rather, the invention also encompasses materials as are suitable for other affinity enrichment methods. For example, the support can be a material that is suitable for thin-layer chromatography.

The invention further comprises a method for the enrichment and / or isolation of phosphorylated peptides and / or proteins from a sample. Here, a sample is generally understood to mean a sample made of biological material. For example, this can be a cell extract, a tissue extract or the like. In particular, all proteins of cells from a cell culture can be processed for this purpose. On the other hand, a corresponding sample can also originate, for example, from the processing of biopsy material and / or from certain organ tissue. On the other hand, samples of plant origin, bacterial samples or samples from fungi can also be used in the method according to the invention. The samples can either be used without further purification, so that, for example, only the cell extract freed from cell debris is used. On the other hand, the sample can also be further purified. However, the use of samples without further purification is particularly preferred, since in this way all phosphorylated peptides and / or proteins of interest can be enriched and / or examined in more detail in this way, as is of particular interest in the field of proteome research. According to the method according to the invention, the sample is first brought into contact with a substance, as has already been described, so that interactions between the substance and the phosphorylated peptides and / or proteins can form from the sample. In a further step, unbound material, in particular non-phosphorylated peptides and / or proteins, is removed from the substance. This can be done in particular by washing with suitable buffer solutions. Furthermore, the phosphorylated peptides and / or proteins, that is to say the peptides and / or proteins interacting with the substance, are eluted, that is to say separated from the substance. This also advantageously takes place again by choosing suitable buffer conditions and / or by changing the temperature or the like. This elution step does not necessarily have to be carried out. In particular depending on the analytical method chosen or generally the objective that is sought with the method according to the invention, it can be advantageous that the phosphorylated peptides and / or proteins are not separated from the affinity material. A suitable protocol for carrying out the method and in particular the selection of suitable buffer conditions will be readily apparent to the person skilled in the art in this field.

The now enriched or isolated phosphorylated peptides and / or proteins from the sample are in the or the eluted fractions after carrying out this method. These peptides and / or proteins can further be processed or further purified as required. This can be achieved, for example, by repeatedly carrying out the method according to the invention or by other cleaning methods.

It is particularly preferred to further analyze and, if necessary, identify the phosphorylated and preferably eluted peptides and / or proteins. This can be done with conventional methods, in particular with one- and / or two-dimensional polyacrylamide gel electrophoresis and / or mass spectrometric methods. Analysis of the enriched or isolated phosphorylated peptides and / or proteins is particularly advantageous using mass spectrometric methods. An “electro spray ionization” (ESI) tandem mass spectrometer or the so-called “matrix-assisted laser desorption / ionization” (MALDI) mass spectrometry are particularly preferred. These methods in particular have already proven to be particularly suitable for the investigation of phosphorylated peptides and / or proteins. Of course, the invention also encompasses other methods of analysis as will become apparent to those skilled in the art.

The method according to the invention is preferably characterized in that the phosphorylated peptides and / or proteins to be enriched are phosphorylated one or more times on one or more tyrosine residues. The specificity of the substance according to the invention for tyrosine-phosphorylated peptides and / or proteins or for peptides and / or proteins phosphorylated elsewhere is primarily shaped by the choice of the at least two groups which are located on the spacer according to FIG. 1. In contrast to threonine and / or serine phosphorylated peptides and / or proteins, tyrosine phosphorylated peptides and / or proteins in particular are involved in signal transduction and regulation processes in the cell. They are therefore of particular biological interest. The problem of conventional investigation methods for tyrosine-phosphorylated peptides and / or proteins lies primarily in the fact that the tyrosine-phosphorylated peptides and / or proteins are only present in an extremely low concentration in the cell in comparison with peptides and / or proteins phosphorylated elsewhere. With the method according to the invention, it is now possible to specifically enrich and / or isolate these particularly rare phosphorylated peptides and / or proteins, and thus to make it accessible to an investigation. In addition, the method according to the invention makes it possible, through the specific enrichment or isolation of all tyrosine-phosphorylated peptides and / or proteins of a cell, to provide an overall overview of these very important switching points of the cell, as is the aim of phosphoproteome research in particular.

Furthermore, the invention comprises the use of the substance according to the invention, as has already been described, as an affinity material for the enrichment and / or isolation of phosphorylated peptides and / or proteins. In particular, the peptides and / or proteins to be enriched or isolated are tyrosine-phosphorylated peptides and / or proteins. With regard to further features of this use according to the invention, reference is also made to the above description.

The invention further comprises an affinity material for the enrichment and / or isolation of phosphorylated peptides and / or proteins. In this regard, too, reference is made to the above description. In particular, this affinity material is characterized in that the affinity material is a column chromatography material. This has the advantage that the process for the enrichment and / or isolation of phosphorylated peptides and / or proteins can be carried out with generally customary protocols from protein purification, the adaptation of conventional protocols to this specific affinity material being readily manageable for a person skilled in the art , In addition to column chromatography materials, the affinity material can of course also be designed in such a way that it is suitable for other affinity purifications. For example, the affinity material can be designed such that it is suitable for thin-layer chromatography or the like. In addition, the invention comprises a chromatography column which has a corresponding affinity material. The chromatography column can be designed in such a way that it is already loaded with the affinity material according to the invention. On the other hand, it can also be advantageous that the chromatography column and the affinity material are initially separate and that the column is packed in the appropriate dimension if required. With regard to this chromatography column, too, reference is made to the above description.

Finally, the invention comprises a method for producing a substance which is suitable as an affinity material for the enrichment and / or isolation of phosphorylated peptides and / or proteins. This substance has already been described in detail above. The process according to the invention for the production of this substance comprises the following process steps, these steps being illustrated schematically in FIGS. 2 and 3:

a) First, at least one compound which contains at least two methyl radicals and at least one methyl ester radical with N-bromosuccinimide (NBS), N-bromoacetamide (NBA) and / or

SO ₂ CI _{2 converted} to the corresponding bromomethyl and / or chloromethyl compound. The compound is preferably the compound Vi, which is characterized by the following formula:

in which

R-i, Rj H, alkyl, halo, O-alkyl and / or mono- or bicyclic aromatic rings.

A particularly preferred representative of this compound V-i is methyl methylbenzate. In this case the reaction product is methyl bis (bromomethyl) benzene and / or methyl bis (chloromethyl) benzene. The spacer of the substance according to the invention is formed by this compound or compounds corresponding to the formula.

b) The reaction product from process step a) is combined with alkali carbonates, bicarbonates and / or tertiary organic amines and at least one compound V ₂ according to the following formula:

in which

X, Y: CR \ N, S and / or O;

Z: CR " ₂ and / or (CR") ₂ ;

R \ R ": H, alkyl, halo and / or O-alkyl;

mean implemented. If, for example, methyl dimethylbenzenate was used in process step a), the action product of this process step bis [(V) methyl] methyl benzene. A particularly preferred representative of V ₂ is, for example, 2,2'-dipicolylamine.

c) The reaction product from step b) is then reacted with alkali metal hydroxides, carbonates, bicarbonates and / or quaternary ammonium hydroxides to give the corresponding acid. If, for example, dimethylbenzenic acid methyl ester was used initially, bis [(V ₂ ) methyl] benzene acid is formed. With a different compound Vi, a different reaction product naturally arises.

d) As a further step, a solid support material is optionally activated, on which the reaction product from process step c) is subsequently to be immobilized. Whether an activation of the carrier is necessary depends on the material selected. The time at which the carrier is activated or the carrier is made available is of course not tied to the sequence listed here.

e) The reaction product from process step c) is reacted with at least one carbodiimide, uranium and / or phosphonium salt with the carrier, which is optionally activated, so that the reaction product from process step c), for example bis [(V ₂ ) methyl ] benzene acid is immobilized on the support.

This connection of carrier and reaction product takes place via a linker, which can be, for example, an amide, ester, carbomoyl, ether and / or thioether bond.

f) Finally, the immobilized product from process step e) is loaded with metal by mixing the batch with an aqueous solution of Mn ²⁺ , Fe ³⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Al ³⁺ and / or Ga ³⁺ or the corresponding salts is treated. Zn ²⁺ or Co ²⁺ are particularly preferred. The loading with the metal according to process step f) can optionally also be carried out at another time in the reaction sequence.

Further features of the invention result from the subclaims in combination with the figures and examples. The various features can be implemented individually or in combination with one another.

In the figures shows

1 shows a schematic representation of the substance according to the invention;

2 shows a schematic representation of the reaction sequence a) to c);

3 schematic reaction sequence of process steps e) to f);

Fig. 4 Dot blot of the proteins eluted from Zn ²⁺ -BiPy and Co ²⁺ -BiPy beads. Dot 1: Sample 1; Dot 2: Sample 2; Dot 3:

Sample 3; Dot 4: Sample 4; Dot 5: Sample 5; Dot 6: Sample 6; Dot 7: Sample 7; Dot 8: Sample 8; Dot 9: Sample 9; Dot 10: Sample 10;

Figure 5 Western emblot of the proteins eluted from Zn ²⁺ -BiPy and Co ²⁺ -BiPy beads. Lane 1: whole cell extract; track

2: Sample 1 (Elution 1); Lane 3: Sample 3 (Elution 1); Lane 4: Sample 5 (Elution 1);

6 shows preferred embodiments of the substance according to the invention. A, B and C show meta, para and ortho variants of the immobilized bis [(2,2'-dipicolylamino) methyl] benzene acids; Fig. 7 Western embot analysis of pTyr proteins enriched with affinity materials A and B. The sample distribution is explained in the examples;

8 two-dimensional polyacrylamide gel electrophoresis of the enriched pTyr proteome of endothelin-stimulated fibroblasts of the rat lung.

Examples

A. Preparation of the affinity material

1. Synthesis of 2,5-. 3,4- and 3,5-di (bromomethyl) benzene acid methyl ester

19.6 g of methyl 2,5-, 3,4- or 3,5-dimethylbenzenate, 47 g of NBS and 0.5 g of benzoyl peroxide were dissolved in 120 ml of carbon tetrachloride and the reaction was carried out under reflux for 12 h. After cooling, the reaction mixture was filtered and the solvent was removed by evaporation in vacuo. The dibromo derivatives obtained were purified by recrystallization from n-hexane, so that a yield of 2,5-bis (bromomethyl) benzene acid methyl ester was obtained

21.2 g at a melting point of 71 ° C, for 3,4-bis (bromomethyl) benzene acid methyl ester a yield of 22.2 g at a melting point of 74 ° C and for 3,5-bis (bromomethyl) benzene acid methyl ester

Yield of 25.3 g at a melting point of 78 ° C gave. 2. Synthesis of 2,5-, 3,4- and 3,5-bisr (2,2'-dipicolylamino) methvπ-benzene acid methyl ester

To a solution of methyl 2,5-, 3,4- or 3,5-bis (bromomethyl) benzene (2.24 g, 8 mmol), 2,2'-dipicolylamine (3.5 g, 17.2 mmol) and K CO ₃ (4.42 g, 3.2 mmol) in 20 ml of anhydrous DMF (dimethyl fluoride), a solution of Kl (1, 3 g, 8 mmol) in DMF (8 ml) was added dropwise over 1 h 80 ° C added dropwise. After stirring for 60 min. at 60 ° C the reaction mixture was diluted with 1N HCl and washed twice with ethyl acetate. The aqueous layer was alkalized with 4N NaOH and extracted twice with diethyl ether. The combined organic layers were washed with water and brine, followed by drying over Na ₂ Sθ ₄ . After the solvent had been removed in vacuo, the residue was recrystallized from ethanol and gave a yield of 3.3 g for a 2,5-bis [(2,2'-dipicolylamino) methyl] benzene acid methyl ester at a melting point of 98 ° C. for 3 , 4- bis [(2,2'-dipicolylamino) methyl] methyl benzene gave a yield of 3.5 g at a melting point of 102 ° C. and for 3,5-bis [(2,2'-dipicolylamino) methyl] methyl benzene a yield of 4.2 g at a melting point of 111 ° C.

Synthesis of 2,5-, 3,4- and 3,5-bisr (2,2'-dipicolylamino) methyl1-benzene acid

3.0 g of methyl 2,5-, 3,4- or 3,5-bis [(2,2'-dipicolylamino) methyl] benzene in 50 ml 80% MeOH were mixed with 1.1 g Ba (OH) ₂ mixed and treated in reflux under nitrogen for 2 h. A further 0.6 g of Ba (OH) ₂ were added and the reflux was carried out for a further 2 h. After the reaction mixture had cooled, ₂ equimolar amounts of 50% H ₂ SO 4 were added with respect to Ba (OH). The precipitate obtained was separated by centrifugation and the remaining The solution was evaporated to dryness. Bis [(2,2'-dipicolylamino) methyl] benzene acids were obtained as an oily residue which was used without further purification.

4. Activation of the carrier material

4.1 Activation of Sepharose 4B with 1,4-butanediol diglycidyl ether

50 ml of suction-dried Sepharose 4B material was transferred to a 250 ml conical bottle which contained 50 ml of 0.6 M NaOH and 50 ml of 1,4-butanediol diglycidyl ether. The suspension was shaken at room temperature for 12 h. The Sepharose 4B material was washed with 2 l of deionized water and immediately used for the next reaction step.

4.2 Synthesis of amino-1, 4-butanediol ether-Sepharose 4B

10 ml of the suction-dried Sepharose 4B activated with 1,4-butanediol diglycidyl ether was transferred to a 250 ml conical bottle containing 50 ml of 2.0 M NH ₄ OH solution. The suspension was shaken at room temperature for 12 hours. The Sepharose 4B was washed with 1 l of deionized water and immediately used for the next reaction step.

4.3 Synthesis of Amino Fractogel

10 g of epoxy Fractogel material was transferred to a 250 ml conical bottle containing 50 ml of 2.0 M NH ₄ OH solution. The suspension was shaken at room temperature for 12 hours. The Fraktogel material was washed with 1 l of deionized water and immediately used for the next reaction step.

5. Immobilization

5.1 Synthesis of 2,5-, 3,4- and 3,5-bis [(2,2'-dipicolylamino) methyl] benzene acid Sepharose 4B

10 ml of amino-1,4-butanediol ether-Sepharose was washed step by step in a Buchner funnel with 50 ml of 20%, 40%, 60%, 80% and 100% DMF. The amino-1,4-butanediol ether-Sepharose beads were resuspended in 15 ml of DMF, the DMF in each case 250 mg of 2,5-, 3,4- and 3,5-bis [(2,2'-dipicolylamino) methyl ] contained benzoic acid. 20 mg of imidazole, 100 μl of pyridine and 500 μl of di-isopropylcarbodiimide were added to this suspension. The suspension was shaken at room temperature for 24 h and a further 200 μl of di-isopropylcarbodiimide was added. After 12 hours the beads were collected by filtration and washed in the following order with 50 ml each: 100%, 80%, 60%, 40%, 20% DMF and 500 ml water. The beads were then resuspended in 20 ml of 5% NaHC0 ₃ and 100 ul acetic anhydride was added. The suspension was shaken at 37 ° C for 60 min and then washed with 50 ml water, 50 ml 10% acetic acid and 500 ml water. Finally, the beads were washed in 30% EtOH and kept in the refrigerator.

5.2 Synthesis of 2,5-, 3,4- and 3,5-bis [(2,2'-dipicolylamino) methyl] benzene acid fractogel

The synthesis was carried out as in 5.1, except that the amino fractogel was used instead of Sepharose 4B. 6. Load 2.5-, 3.4- and 3,5-bisr (2,2'-dipicolylamino) methvπ-benzene acid Sepharose 4B or Fractogel with Zn ²⁺ or Co ²⁺

Columns packed with 2,5-, 3,4- and 3,5-bis [(2,2'-dipicolylamino) methyl] benzene acid-Sepharose 4B or Fractogel were washed with 5 volumes of water. Then a treatment with 3 volumes of 0.2 M solution of ZnS0 ₄ or CoCI ₂ in water followed and again 5 volumes of water. A subsequent equilibration was carried out with the corresponding buffer, which was used for the corresponding protein sample preparation.

B. Enrichment of Tyrosine Phosphorylated Proteins

Test protocol l

1. Isolation of proteins with tyrosine phosphorylation from rat liver

The following affinity materials were used in this example:

Material Type 1: 2,5-bis [(2,2'-dipicolylamino) methyl] benzene acid Sepharose 4B (25BiPy-

Sepharose 4B)

Material type 2:

3,4-bis [(2,2'-dipicolylamino) methyl] benzene acid Sepharose 4B (34BiPy-

Sepharose 4B) Material type 3:

3,5-bis [(2,2'-dipicolylamino) methyl] benzene acid Sepharose 4B (35BiPy-

Sepharose 4B) Material type 4:

3,5-bis [(2,2'-dipicolylamino) methyl] benzene acid fractogel (35BiPy fracture gel)

Table 1: Sample designation in this example

2. Sample preparation and isolation of the pTyr proteins

2.1 buffer:

Lysis buffer: 50 mM NaMOPS, 25 mM NaCI, pH 7.2, 0.5% Zwittergent 3-10, 0.5% CHAPS, 5 mM NaF, 1 mM sodium orthovanadate, 0.2 mM sodium Pervanadate, complete mini protease inhibitor cocktail (without EDTA) 1 tabl./10 ml buffer.

Wash buffer: 50mM NaMOPS, 25mM NaCl, pH 7.2, 0.1% Zwittergent 3-10, 0.1% CHAPS, 5mM NaF, 1mM sodium orthovanadate, 0.2mM sodium pervanadate.

3. Elution buffer 1: 50 mM NaMOPS, 100 mM NaCl, 50 mM phenyl phosphate (127 mg phenyl phosphate, disodium salt dihydrate per 10 ml buffer), pH 7.2, 0.1% Zwittergent 3-10, 0.1% CHAPS, 5 mM NaF, 1 mM sodium orthovanadate, 0.2 mM sodium pervanadate.

4. Elution buffer 2: 50mM NaMOPS, 100mM NaCl, pH 7.2, 0.1% Zwittergent 3-10, 0.1% CHAPS, 5mM NaF, 1mM sodium orthovanadate, 0.2mM sodium Pervanadate, 50 mM, Na ₄ EDTA.

2.2 Implementation

1. 1 g of rat liver tissue was homogenized with 15 ml of lysis buffer. The suspension was in a Sorvall SS34 rotor for 30 min. centrifuged at 18,000 rpm. The supernatant was used and the pellet was discarded. The supernatant was divided into 1.5 ml samples.

2. The samples (1.5 ml) were run through activated and equilibrated columns packed with 0.5 ml Zn ²⁺ -BiPy and Co ²⁺ -BiPy beads. 3. The columns were washed with 3.0 ml wash buffer and the first elution was eluted with elution buffer 1 (600 μl) and then with elution buffer 2 (600 μl).

3. Dot blot analysis of the proteins eluted from the Zn ²⁺ -BiPy and Co ²⁺ - BiPy beads

3.1 buffer:

1. TBS (Tris-buffered saline): 10 mM Tris-HCl pH 7.4, 170 mM NaCI, 3.4 mM KCI. 2. TBS / Tween: 0.1% Tween 20 in TBS 3. BCIP / NBT (bromochloroindolyl phosphate / nitro blue tetrazolium): NBT and BCIP stock solutions were kept in the refrigerator for several weeks. The stock solutions were prepared by dissolving 0.5 g NBT in 10 ml 70% dimethylformamide. The BCIP stock solution was obtained by dissolving

Prepared 0.33 g BCIP disodium salt in 10 ml DMF in a glass jar.

4. APB (alkaline phosphatase buffer): 100 mM NaCl, 5 mM MgCl ₂ , 100 mM Tris-HCl pH 9.5 5. APB / Tween: 0.1% Tween 20 in APB

3.2 Implementation

1. The positions of the spots on the nitrocellulose membrane were marked with a soft waterproof pen. The nitrocellulose membrane was moistened in water and almost completely dried on a clean paper.

2. The samples were applied to the marked areas, 1 μg protein per spot being applied.

3. Sufficient quenching buffer (5% BSA (bovine serum albumin) in TBS / Tween) was placed in a plastic dish to be covered so that the bottom of the dish was completely covered. The nitrocellulose blot was carefully placed on the surface of the quenching buffer so that the filter was moistened uniformly. The filter was then immersed in the quenching buffer. The incubation was carried out with agitation or shaking for at least 2 h. The solution was removed and a solution with the first antibody (1: 1,000 dilution pTyr102 antibody, Cell Signaling Technology, # 9416) in 4% BSA in TBS / Tween-

Buffer added. The incubation was carried out for 16 h at 4 ° C. with continuous agitation on a shaker. 4. The antibody solution was poured away. Without allowing the blot to dry, the blot was washed four times with 50 ml of TBS / Tween for 5 min each. washed. This washing solution was poured off again and the solution with the second antibody was added to the blot.

5. Incubation with the second antibody (1: 5000 dilution of AP-conjugated anti-mouse IgG whole molecule, Sigma 93160 in quenching buffer) was carried out for 3 hours at 4 ° C. with agitation. 6. This antibody solution was poured off and the blot four times with 50 ml TBS / Tween each for 5 min. washed.

7. The nitrocellulose blot was placed in BCIP / NBT working solution, this solution being prepared by adding 66 ml of NBT stock solution to 10 ml APB / Tween with thorough mixing and adding 33 ml of BCIP stock solution.

8. After the color developed, the reaction was stopped by washing with water and 1% acetic acid.

Figure 4 shows the results of dot blot screening. Dot 1 shows sample 1, dot 2 sample 2, dot 3 sample 3, etc.

4. Western emblot analysis of the proteins eluted from Zn ²⁺ -BiPy and Co ²⁺ - BiPy material

4.1 The polyacrylamide gel electophoresis (PAGE) was carried out according to Laemmli. In each case 20 μg protein were applied per lane of the 11% PAGE. 4.2 buffer

The same buffers were used as for dot blot screening.

4.3 Implementation

1. The electroblotting was carried out with a nitrocellulose membrane according to the manufacturer's instructions with a semi-dry electroblot apparatus from BioRad.

2. Sufficient quenching buffer (5% BSA in TBS / Tween) was placed in a plastic dish to be covered so that the bottom of the dish was completely covered. The nitrocellulose blot was carefully placed on the surface of the quenching buffer so that the filter was moistened uniformly. The filter was then immersed in the quenching buffer. The incubation was carried out with agitation or shaking for at least 2 h. The solution was removed and a solution with the first antibody (1: 1,000 dilution of pTyr102 antibody, Cell Signaling Technology, # 9416) in 4% BSA in TBS / Tween buffer was added. The

Incubation was carried out for 16 h at 4% with continuous agitation on a shaker.

3. The antibody solution was poured away. Without allowing the blot to dry, the blot was washed four times with 50 ml of TBS / Tween for 5 min. washed. This washing solution was poured off again and the solution with the second antibody was added to the blot.

4. Incubation with the second antibody (1: 5000 dilution of AP-conjugated anti-mouse IgG whole molecule, Sigma 93160 in quenching buffer) was carried out with agitation at 4 ° C. for 3 h. This antibody solution was poured off and the blot four times with 50 ml TBS / Tween each for 5 min. washed. The nitrocellulose blot was placed in BCIP / NBT working solution, this solution being prepared by adding 66 ml of NBT stock solution to 10 ml APB / Tween with thorough mixing and adding 33 ml of BCIP stock solution.

The results of the Western blot analysis are shown in FIG. 5. Track 1 shows the entire cell extract, track 2 shows sample 1 (elution 1), track 3 shows sample 3 (elution 1) and track 4 shows sample 5 (elution 1).

Test protocol II

5. Enrichment of pTyr proteins from rat brain and lung fibroblasts

5.1 buffer:

1. "Zn" lysis buffer: 10 mM imidazole-HCl, 40 mM Na-MES, 2 μM Zn (S0 ₄ ) ₂ , 0.5% Zwittergent 3-10, 0.5% CHAPS, 5 mM NaF, 1 mM Na ₃ V0 ₄ , 0.2 mM Na pervanadate, 1x PIC, pH 5.5

2. Wash buffer: 10 mM imidazole-HCl, 40 mM Na-MES, 100 mM NaCl, 2 μM Zn (S0 ₄ ) ₂ , 0.1% Zwittergent 3-10, 0.1% CHAPS, 5 mM NaF, 1mM Na ₃ V0 ₄ , 0.2mM Na pervanadate, pH 5.5

3. Elution buffer 1: 50 mM Na-MES, 50 mM Na-phenyl phosphate, 100 mM NaCl, 0.1% Zwittergent 3-10, 0.1% CHAPS, 5 mM NaF,

1mM Na ₃ V0 ₄ , 0.2mM Na pervanadate, pH 5.5 4. Elution buffer 2: 50 mM Na-MES, 50 mM Na ₂ EDTA, 100 mM NaCl, 0.1% Zwittergent 3-10, 0.1% CHAPS, 5 mM NaF, 1 mM Na ₃ V0 _4, 0 , 2 mM Na pervanadate

5.2 Implementation

Mini centrifugation columns from BioRad (# 732-6008) are filled with 600 μl of a 50% suspension of the affinity materials (beads) according to the invention. This corresponds to 300 μl of beads deposited. On the one hand, immobilized bis [(2,2'-dipicolylamino) methyl] benzene acid as the meta variant (bead A) and immobilized bis [(2,2'-dipicolylamino) methyl] benzene acid as the para variant (bead B) were used (see also Fig. 6). The columns were washed 4 times with 1 ml of water. The beads were activated with 4 × 1 ml each of 200 mM Zn (S0 ₄ ) ₂ . The mixture was then washed four times with 1 ml of washing buffer. 5-6 mg protein extract from rat brain or from lung fibroblasts of the rat was applied to the column as a protein sample. The flow was collected and reapplied. The flow was collected again. The columns were washed with 4 x 1 ml wash buffer. A first elution was first carried out with a buffer containing phenyl phosphate as a competition step. A second elution was carried out with high stringency using EDTA. For the first elution of the bound proteins, the column was eluted with 3 × 400 μl elution buffer 1. For the second elution, the column was eluted with 3 × 400 μl elution buffer 2. All eluates were collected. The eluates were concentrated using Biomax K5 (Millipore) to a final volume of approximately 40-50 μl. 6. Analysis of the political groups

A BCA assay (Pierce) was carried out for protein determination of all fractions.

6.1 SDS-PAGE

A BioRad Protean II mini gel system (BioRad, Munich) was used to carry out standard SDS-PAGE discontinuous Laemmli gels. 12% separating gels with 4% bulk gels were used for all experiments.

6.2 vest rnb lots

Samples (15 μg per sample) of the protein extract, eluate 1 and eluate 2 were separated on a 12% polyacrylamide gel (BioRad Mini) and blotted onto a PVDF membrane (BioRad).

The membrane was blocked with TBS, 0.1% Tween, 5% BSA for two hours, followed by incubation with the first antibody (anti-pTyr monoclonal antibody 4G10, Upstate) overnight at a 1: 1000 dilution in blocking. Buffer. The blot was then washed 3 times in TBS / Tween and then incubated for one hour with the secondary antibody (anti-mouse, conjugate with alkaline phosphatase, Sigma) for one hour at a dilution of 1: 1000. The blot was washed 3 times with TBS / Tween and developed using the NBT / BCIP substrate (Röche).

7. Results

The results confirm the enrichment of pTyr-P roteins by appropriate affinity materials. For this, the immobilized Zinc complexes of bis [(2,2'-dipicolylamino) methyl] benzene acid (Fig. 6) were used.

FIG. 7 shows the western embot analysis of pTyr proteins which were enriched with the affinity materials A and B (beads A and B) according to FIG. 6. Blots A and B: Western blot analysis of protein extract (CE) from rat brain, eluate 1 (E1) and eluate 2 (E2), each with the anti-pTyr affinity material A and B. The staining was carried out with anti-pTyr antibody 4G10 (Upstate ). Each lane was loaded with 15 μg protein.

Blot C from FIG. 7 shows the Western blot analysis of stimulated (EGF, ET1) fibroblasts of the rat lung, which were stained with the antibody P-Tyr 102. Affinity material A was used for this. Lane 1: whole proteome of EGF-stimulated cells, lane 2 and 3: enriched pTyr proteome of unstimulated cells; Lane 4: enriched pTyr proteome from ET1 (endothelin) stimulated cells, eluate 1; Lane 5: enriched pTyr proteome from EGF-stimulated cells, eluate 1; Lane 6: enriched pTyr proteome from ET1-stimulated cells, eluate 2; Lane 7: enriched p-Tyr proteome from EGF-stimulated cells, eluate 2. These results show that the currently most common antibody for pTyr affinity purification and detection (4G10) does not recognize some pTyr proteins in comparison with the materials according to the invention.

FIG. 8 shows the image of a two-dimensional polyacrylamide gel electrophoresis of the pTyr proteome of ET1-stimulated fibroblasts of the rat lung, which was enriched with the affinity material A according to the invention. The pattern of the proteins can be seen on the silver-stained two-dimensional gel, with many of the tyrosine-phosphorylated proteins identified by subsequent mass spectrometry. These mass spectrometric analyzes demonstrate the accumulation of various tyrosine-phosphorylated proteins, such as for example a valosin-containing protein with similarities to cdc48 (gi | 6678559) from yeast, ATPases, in particular the H ⁺ -transporting two-sector ATPase (gi | 92350), the beta chain of the ATPase synthase (gi | 114562), Actinin alpha 4 (gi | 1 1230802) and others. Furthermore, previously unknown tyrosine phosphorylated proteins were identified.

The Western blot analysis of the pTyr protein enrichment clearly shows that the affinity materials according to the invention accumulate tyrosine-phosphorylated proteins from various materials to a considerable extent. Such an enrichment could be shown particularly clearly after stimulation of lung fibroblasts with, for example, endothelin or EGF, these two substances being known to induce tyrosine phosphorylation. Furthermore, the experiments shown make it clear that the pTyr-specific antibodies currently used only cover a certain fraction of the pTyr proteome. The advantages of the affinity materials according to the invention over the conventional immunological methods therefore lie above all in the sequence-independent affinity for phosphotyrosine residues, as a result of which the entire pTyr proteome can be better covered. In addition, the affinity materials according to the invention ensure greater reproducibility and better flexibility, for example with regard to buffer conditions. In addition, an analysis can be carried out much faster and the costs of such analyzes are lower compared to conventional methods. Finally, the affinity materials according to the invention are particularly advantageously suitable for adaptation to high-throughput processes.

Claims

claims

Substance comprising a solid carrier which is connected via a linker to a spacer which carries at least two groups according to the following formula

in which

X, Y: CR \ N, S and / or O; Z: CR " ₂ and / or (CR") ₂ ;

R \ R ": H, alkyl, halyl and / or O-alkyl; M: Mn ²⁺ , Fe ³⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Al ³⁺ and / or

Ga 3+ mean.

Substance according to claim 1, characterized in that the group is formed from 2,2'-dipicolylamino.

Substance according to claim 1 or claim 2, characterized in that the spacer comprises one or more aromatic rings, in particular mono-, bi- and / or tricyclic aromatic rings.

4. Substance according to one of the preceding claims, characterized in that the spacer is formed from methyl 2,5-, 3,4- and / or 3,5-dimethylbenzenate.

5. Substance according to one of the preceding claims, characterized in that the linker comprises at least one amide, ester, carbamoyl, ether and / or thioether bond.

6. Substance according to one of the preceding claims, characterized in that the spacer with the at least two groups starting from 2,5-, 3,4- and / or 3,5-bis [(2,2'-dipicolyamino) methyl] benzene acid is formed.

7. Substance according to one of the preceding claims, characterized in that the solid support is glass, silicate, gold and / or at least one organic polymer, in particular Sepharose and / or fractogel.

8. A process for the enrichment and / or isolation of phosphorylated peptides and / or proteins from a sample, comprising the process steps:

Bringing the sample into contact with a substance according to any one of the preceding claims to form interactions between the substance and phosphorylated peptides and / or proteins in the sample, removal of non-interacting material, optionally eluting the phosphorylated peptides and / or proteins,

If necessary, analyze the phosphorylated peptides and / or proteins.

9. The method according to claim 8, characterized in that the analysis is carried out using mass spectrometric methods.

10. The method according to claim 8 or claim 9, characterized in that the phosphorylated peptides and / or proteins are tyrosine-phosphorylated peptides and / or proteins.

1 1. Use of a substance according to any one of claims 1 to 7 as an affinity material for the enrichment and / or isolation of phosphorylated peptides and / or proteins.

12. Use according to claim 11, characterized in that the phosphorylated peptides and / or proteins are tyrosine-phosphorylated peptides and / or proteins.

13. Affinity material for the enrichment and / or isolation of phosphorylated peptides and / or proteins, in particular of tyrosine-phosphorylated peptides and / or proteins, comprising at least one substance according to one of claims 1 to 7.

14. Affinity material according to claim 13, characterized in that the affinity material is a column chromatography material.

15. Chromatography column comprising an affinity material according to claim 13 or claim 14.

16. A method for producing a substance according to any one of claims 1 to 7, comprising the process steps: a) reacting at least one compound Vi of the following formula

in which

Ri, R ₂ : H, alkyl, halo, O-alkyl, mono- or bicyclic aromatic rings, with N-bromosuccinimide (NBS), N-bromoacetamide (NBA) and / or S0 ₂ CI ₂ ;

Reacting the reaction product from step a) with at least one alkali carbonate, bicarbonate and / or tertiary organic amine with at least one compound V ₂ according to the following formula

in which

X, Y: CR ', N, S and / or O;

Z: CR " ₂ and / or (CR") ₂ ; R \ R ": H, alkyl, halyl and / or O-alkyl; c) reacting the reaction product from step b) with at least one alkali metal hydroxide, carbonate, bicarbonate and / or quaternary ammonium hydroxide; d) optionally activating a solid support; e ) Reacting the reaction product from step c) with at least one carbodiimide, uranium and / or phosphonium salt with the solid support, which is optionally activated, to one on the support via amide, ester, carbomoyl, ether and / or Thioether bonds immobilized reaction product; f) loading the reaction product from step e) with at least one metal by treatment with an aqueous solution of Mn ²⁺ , Fe ³⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Al ^{3 +} and / or Ga ³ .

17. The method according to claim 16, characterized in that the compound Vi is dimethylbenzenic acid methyl ester.

18. The method according to claim 16 or claim 17, characterized in that the compound V _{2 is} 2,2'-dipicolylamine.

19. The method according to any one of claims 16 to 18, characterized in that the solid support is glass, silicate, gold and / or at least one organic polymer, in particular Sepharose and / or Fractogel.