WO1999038012A1

WO1999038012A1 - Assay for detection of phosphate or carbohydrate groups on serine residues

Info

Publication number: WO1999038012A1
Application number: PCT/AU1999/000043
Authority: WO
Inventors: John Andrew Paul Rostas; John Edgar Thomas Corrie; Adam Mccluskey; Andrew James Delaney
Original assignee: The University Of Newcastle Research Associates Limited
Priority date: 1998-01-21
Filing date: 1999-01-21
Publication date: 1999-07-29
Also published as: AUPP148098A0; EP1057034A1

Abstract

The present invention provides a method for demonstrating the presence of phosphate and/or carbohydrate bound to serine residues in proteinaceous material which method comprises: providing the proteinaceous material immobilised on a solid support; derivatising the proteinaceous material with a nucleophile under conditions which selectively replace at least some of the phosphate and/or carbohydrate groups present on serine residues in the proteinaceous material with the nucleophile to form a derivatised preoteinaceous material comprising an adduct formed by the nucleophile; and detecting the derivatised proteinaceous material by recognition of the adduct formed by the nucleophile.

Description

_ASSAY _{FOR DETECTION OF PHO}S_PHATE OR CARBOHYDRATE GROUPS ON SERINE RESI^DUE^S

TECHNICAL FIELD The present invention relates to methods for the detection of phosphate or carbohydrate groups on serine residues in proteins, polypeptides and/or peptides, and to uses of those methods, as well as anti- MBA antibodies .

BACKGROUND ART

Phosphorylation and glycosylation are both ubiquitous post-translational modifications of proteins used by all pro aryotic and eukaryotic cells.

Phosphorylation

The cellular consequences of receptor activation by hormones, neurotransmitters and drugs, as well as changes in the intracellular concentration of a number of substances are usually brought about by activating or inhibiting one or more enzymes that phosphorylate or dephosphorylate proteins. Approximately 90% of protein phosphorylation occurs on serine or threonine residues; less than 10% occurs on tyrosine residues. The traditional way to study protein phosphorylation is by isotopic labelling. Although this is sensitive and can readily identify dynamic changes in phosphorylation in a research laboratory setting, this approach is limited by cost, problems of occupational health and safety and waste disposal and the impracticability of radioactive labelling in whole animal experiments or clinical testing.

The development of antibodies to phosphotyrosine revolutionised the study of tyrosine phosphorylation because it eliminated the need for isotopes for most experiments and could readily distinguish phosphorylation at tyrosine residues from phosphorylation at serine or threonine residues. Unfortunately, phosphoserine and phosphothreonine are only poorly antigenic and the use of antibodies to these amino acid residues has not yet proved to be a viable, non-isotopic method for detecting serine and threonine phosphorylation in most applications. Other non-isotopic methods are of limited applicability because they require advanced protein and peptide chemistry skills and usually cannot be used with mixtures of proteins .

Glycosylation

Patterns of protein glycosylation serve a wide range of biological functions including: serving as a biological clock by, for instance, determining the half life of plasma proteins; directing intracellular trafficking of proteins; proclaiming the identity of a cell or a basement membrane by acting as a recognition site for protein ligands and mediating cell surface interactions between cells. Patterns of protein glycosylation on the cell surface can be used diagnostically for several purposes including to distinguish between cell types or identify malignant transformations in cells.

Carbohydrate groups of many different kinds can be N-linked to proteins through the amino acid asparagine or O-linked through the amino acids serine or threonine. Sophisticated and highly specialised methods have been developed for determining the structure of the carbohydrate groups but these are not suitable for routine screening, diagnostic testing or use with mixtures of proteins such as cell extracts, cultures, clinical samples, etc.

Lectin binding assays provide a simple and versatile method for obtaining a limited amount of information about the structure of the carbohydrate group attached to proteins and this forms the basis of some diagnostic tests. Because the chemical composition of N- and O- - 3 -

linked carbohydrates is different, the binding of some lectins can be used to detect the presence of these two types of linkages. However, identifying whether a particular carbohydrate group is N-linked or O-linked usually requires analysis of the sensitivity of the group to hydrolysis by one or more purified endoglycosidases or strong alkali and usually is not done with mixtures of proteins. Furthermore there is no simple method for determining whether an O-linked carbohydrate group is linked to serine or threonine.

DESCRIPTION OF THE INVENTION The present invention provides a simple and versatile technique for demonstrating the presence of phosphate or carbohydrate bound to serine residues in proteins, polypeptides and/or peptides present in crude mixtures or in purified proteins, polypeptides and/or peptides .

The method involves the specific and quantitative chemical modification of phosphorylated or glycosylated serine residues in proteins under conditions which can demonstrate both groups at once, or alternatively, which can be selective for just one.

The chemical modifications can be carried out in solution or on a solid phase support.

In a first aspect the present invention provides a method for demonstrating the presence of phosphate and/or carbohydrate bound to serine residues in proteinaceous material which method comprises : providing the proteinaceous material immobilised on a solid support; derivatising the proteinaceous material with a nucleophile under conditions which selectively replace at least some of the phosphate and/or carbohydrate groups present on serine residues in the proteinaceous material with the nucleophile to form a derivatised proteinaceous material comprising - 4 -

an adduct formed by the nucleophile; and detecting the derivatised proteinaceous material by recognition of the adduct formed by the nucleophile. In a second aspect the present invention provides a method for demonstrating the presence of phosphate and/or carbohydrate bound to serine residues in proteinaceous material which method comprises : providing the proteinaceous material in solution; derivatising the proteinaceous material with a nucleophile under conditions which selectively replace at least some of the phosphate and/or carbohydrate groups present on serine residues in the proteinaceous material with the nucleophile to form a derivatised proteinaceous material comprising an adduct formed by the nucleophile; retrieving the derivatised proteinaceous material ; and detecting the derivatised proteinaceous material by recognition of the adduct formed by the nucleophile.

Typically the nucleophile is chosen such that the adduct formed does not present a group for recognition that occurs naturally in proteinaceous material, because adducts which present components for recognition that do occur naturally in proteins will not be distinguishable from those components in the proteinaceous material under investigation, without further processing, particularly where the proteinaceous material is of unknown composition, thereby preventing identification of false positives.

The methods of the invention rely in part on the ability of phosphate and carbohydrate groups linked to serine, threonine and tyrosine, to undergo β-elimination at greatly different rates under strongly alkaline - 5 -

conditions, and the fact that this rate can be modified by the chemical conditions employed. Under normal strongly alkaline conditions only phosphate and carbohydrate groups linked to serine groups undergo β-elimination and the dehydroalanyl intermediate produced can be attacked by a suitable nucleophile to produce a specific adduct. Under appropriately modified reaction conditions phosphate and carbohydrate groups linked to threonine and phosphate linked to tyrosine can also be made to undergo β-elimination and suitable nucleophiles can then form adducts with the intermediates formed.

The proteinaceous material may be one or more peptides or polypeptides or proteins present in a crude mixture or in purified form. Where mixtures are present the method typically includes the separation of the peptide(s), polypeptide (s) and/or protein(s) present sufficient to permit the identification of the different components of the mixture. Usually the method is carried out in solution in accordance with the second aspect of the invention only when the proteinaceous material consists of a single protein, peptide or polypeptide.

The nucleophile is preferably p-mercaptomethylbenzoic acid (MMBA) or a derivative of M BA. Other suitable nucleophiles include other compounds with a suitable nucleophilic group and portion of the molecule that can be detected either directly by its colour or fluorescence or its radioactivity or indirectly by binding to another molecule (e.g. biotin- avidin or DNA-DNA) that can be detected either directly (by its colour or fluorescence or radioactivity) or indirectly by being covalently coupled to: an enzyme which can produce a coloured or fluorescent product and a signal that can be amplified; or a fluorescent chemical group, or by binding to another molecule which can be detected directly or indirectly. Suitable nucleophilic groups include thiols, amines, amides or alkanols, in an alkaline environment. While radioactivity does, in general, present safety issues which can limit its use, where a nucleophile is radioactively labelled it can be used on patient or animal samples . Under the conditions employed in the methods of the invention the amount of radioactivity required is less than in prior art methods and the isotope used can be a lower energy one than ³²P (which is used in traditional methods for measuring phosphorylation) .

Derivatives of MMBA include chemically altered versions of the molecule in which the attributes of the molecule as a nucleophile and as a detectable molecule are retained.

It will be understood that the nucleophiles to be used in accordance with the present invention must be of sufficient solubility to permit the adduct formation to occur at a sufficient rate.

Initial tests indicate that the methods of the invention can detect derivatised proteins in the nanogram range when using MMBA as the nucleophile and an antibody to MMBA to detect the adduct. This makes the method comparable in sensitivity to standard radioisotopic methods of detection. When the methods are used with nucleophiles with greater water solubility and a detection method with higher affinity than antibodies and/or utilising a fluorophore with a high extinction coefficient or an amplifiable enzymatic step, the sensitivity of the methods can be made several orders of magnitude greater.

Recognition of the adduct formed by the nucleophile may be achieved by a variety of methods, including immunochemically, colorimetrically or by fluorescence or chemiluminescence. It may involve the use of a separate detection reagent which recognises the adduct. Recognition may be achieved by chemical reaction or by non-covalent binding.

In one preferred embodiment the nucleophile is MMBA and the detection reagent comprises an antibody which - 7 -

recognises MMBA. Typically, the anti-MMBA antibody is detected with a commercially available secondary antibody. The secondary antibody typically comprises a label such as alkaline phosphatase or horseradish peroxidase, or another reporter enzyme, together with one or more appropriate colour or light producing reagents, or a fluorescent label (e.g. FITC) .

In another preferred embodiment the nucleophile is a biotinylated nucleophile and the detection reagent comprises avidin or streptavidin. In this embodiment a preferred detection reagent also comprises a label such as alkaline phosphatase or horseradish peroxidase, or another reporter enzyme together with one or more appropriate colour or light producing reagents, or a fluorescent label (e.g. FITC) . Because of the high binding affinity between avidin and biotin, this nucleophile is preferred in cases where maximum sensitivity is required. Another preferred nucleophile is a nucleophilic group coupled to a variable sequence and length of DNA nucleotides. In this case, the adduct can be detected as before by a fluorescent group or enzyme coupled to the DNA molecule or by hybridisation with another DNA molecule comprising the appropriate complementary sequence of nucleotides, coupled to a fluorescent group or enzyme. In this way the same (or different) nucleophilic group (s) can be coupled with multiple DNA molecules with different base sequences that could be detected by DNA molecules with the appropriate complementary sequences coupled to different detection systems (fluorophores and/or enzymes) . Because of the specificity of hybridisation between complementary pairs of different DNA sequences, this embodiment offers the possibility of detecting multiple groups simultaneously. This possibility is of use in distinguishing phosphate from carbohydrate bound to serine and also in distinguishing serine bound phosphate and/or carbohydrate from threonine bound phosphate and/or - 8 -

carbohydrate and/or tyrosine bound phosphate.

There are two types of colour detection reagent which can be used: soluble reagents giving insoluble products which precipitate at the required spot on a support; or soluble reagents giving soluble products to be detected by colorimetry for ELISA assay type applications where the proteinaceous material is immobilised onto a microtitre plate or tube or if the assay is done in solution.

The solid phase method of the first aspect of the invention typically involves the following steps:

(1) a sample comprising a mixture of protein (s) polypeptide (s) and/or peptide(s) is treated to separate the protein(s), polypeptide (s) and/or peptide(s) by a separation technique, such as SDS-PAGE or a chromatographic step (e.g HPLC) , and the separated protein (s), polypeptide (s) and/or peptide(s) are transferred to a support such as sheets or strips of Nylon 66 or polyvinyl difluoride

(PVDF) . Any support which: survives the alkaline conditions under which the assay is carried out, at 55°C for the length of time required; does not have a chemical substituent on it which can undergo nucleophilic attack by the nucleophile; and has suitable sites for attachment of the proteinaceous material, can be used. Alternatively purified protein (s) , polypeptide (s) and/or peptide(s) can be applied directly to the support. The transfer of protein (s ) , polypeptide (s) and/or peptide(s) to the support can be performed electrophoretically, by diffusion or by spotting from a pipette or column eluate ;

(2) the protein(s), polypeptide (s) and/or peptide(s) are crosslinked to the support using a cross-linking reagent which may be an aldehyde such as glutaraldehyde . Typically, any cross linking reagent is suitable provided it does not react with the support to produce a high background in the detection part of the method;

(3) unreacted chemical groups on the cross link forming agent are blocked by reaction with a suitable chemical reagent; in the case of glutaraldehyde the aldehyde groups are blocked with a low molecular weight source of free amino groups, such as ethanolamine ; (4) protein(s), polypeptide (s) and/or peptide(s) on the solid support are derivatised by incubation at approximately 55°C for 30 to 60 minutes in a solution comprising p-mercaptomethylbenzoic acid (MMBA) at a pH of at least 12.5 and preferably at a concentration of at least 20mg/ml. Preferably, the solution comprises 12-17% ethanol, 34-42% DMSO made up to pHl2.9 with NaOH (e.g. concentrated NaOH) or a Group II metal hydroxide, especially Ba(OH)₂ [e.g. saturated Ba(OH)₂]. It will be understood that the choice of NaOH or Group II metal hydroxide can be used to favour reaction with carbohydrate or phosphate respectively;

(5) after removal of unreacted MMBA (typically by washing with solvent minus MMBA) , the MMBA derivatised protein(s), polypeptide (s) and/or peptide(s) are detected using an antibody prepared against the MMBA adduct using standard immunodetection methods used for western blotting and immunodetection reagents as described above. The solution based method of the second aspect of the invention typically comprises the following steps: 1) protein (s) , peptide(s) and/or polypeptide (s) are derivatised by incubation at approximately 55°C for 30 to 60 minutes in a solution comprising p-mercaptomethylbenzoic acid (MMBA) at a pH of at least 12.5 and preferably at a - 10 -

concentration of at least 20mg/ml. Preferably, the solution comprises 12-17% ethanol, 34-42% DMSO made up to pH12.9 with NaOH (e.g. concentrated NaOH) or a Group II metal hydroxide, especially Ba(OH)₂ (e.g. saturated Ba(OH)₂) It will be understood that the choice of NaOH or Group II metal hydroxide can be used to favour reaction with carbohydrate or phosphate respectively. ;

2) removal of unreacted excess MMBA by gel filtration. Depending on the nature of the protein (s), polypeptide (s) and/or peptide(s) involved this step could also be done by dialysis or selective extraction with organic or alkaline solvents (e.g. sodium bicarbonate solution) as long as the proteinaceous material is not soluble under these conditions and can still be redissolved afterwards. Alternatively this step could be performed by filtration through a membrane eg PVDF or Nylon 66) to which the proteinaceous material would bind and on which the subsequent detection reaction could be done. This format lends itself to semi automated or automated analysis using a microtitre plate or similar ;

3) after removal of the excess MMBA, the MMBA derivatised protein is detected using an antibody prepared against the MMBA adduct using standard solution based immunodetection techniques. The anti-MMBA antibodies can be polyclonal antibodies raised in rabbits against either MMBA derivatised bovine submaxillary gland mucin or MMBA derivatised phosvitin. Alternatively they can be monoclonal antibodies raised against either of these MMBA derivatives. In another alternative, any protein containing O-linked carbohydrate or phosphate could be used to make the MMBA derivative. The derivative is most effective when the protein is a good natural antigen (e.g. mucin) . - 11 -

Typically purified bovine submaxilliary mucin or egg white phosvitin is derivatised in solution with MMBA as described above. Protein and unreacted MMBA are separated by gel filtration on Sephadex G25 (Pharmacia Laboratories) . Protein fractions are pooled, neutralised and dialysed against water and freeze dried.

The immunisation protocol is standard, utilising intramuscular injections of each of the proteins (in separate rabbits) emulsified in Freund's complete adjuvant followed by boosters in Freund's incomplete adjuvant .

The rate of β-elimination of phosphate groups for serine is 100 times faster than carbohydrate groups in the presence of a Group II metal hydroxide, especially Ba(OH)₂ whereas the rate of elimination of the two groups is similar in the presence of NaOH. Therefore, the difference between the derivatisation patterns in the presence of these counter ions can be used to differentiate between phospho- and glyco-serine residues. Consequently, in a third aspect the present invention provides a method for differentiating between phospho- and glyco serine residues in a proteinaceous material selected from the group consisting of proteins, polypeptides and peptides or mixtures thereof present in crude mixtures, and purified proteins, polypeptides or peptides, which method comprises: derivatising the proteinaceous material with at least one nucleophile under conditions which selectively replace at least some of the phosphate and/or carbohydrate groups present on serine residues in the proteinaceous material with the nucleophile (s) to form a derivatised proteinaceous material comprising at least one adduct formed by the nucleophile (s) in separate reactions in the presence of a Group II metal hydroxide (especially

Ba(OH)₂) and NaOH; and - 12 -

detecting the derivatised proteinaceous material by recognition of the adduct (s) formed by the nucleophile (s) .

Typically the nucleophile (s) are chosen such that the adduct (s) formed do not present a group for recognition that occurs naturally in proteinaceous material, because adduct (s) which present components for recognition that do occur naturally in proteins will not be distinguishable from those components in the proteinaceous material under investigation, without further processing, particularly where the proteinaceous material is of unknown composition, thereby preventing identification of false positives . When the method is conducted in the solid phase, the proteinaceous material is immobilised on a solid support .

Typically this method when conducted in the solid phase involves the steps specified above with respect to the first aspect of the invention.

When the method is performed in solution it additionally comprises retrieving the derivatised proteinaceous material prior to detection.

The use of the two reaction conditions (Group II metal hydroxide versus NaOH) applied to parallel samples provides two patterns of derivatisation - preferential labelling of phosphoserine versus labelling of both phospho- and glyco-serine, from which the phospho- and glyco- serine containing molecules can be deduced by difference. Alternatively, the two derivatisation conditions can be employed sequentially. When applied for parallel reaction with two samples of the same proteinaceous material the pattern produced by the Group II metal hydroxide and the nucleophile upon recognition of the adduct will give a detectable pattern which is preferential for phosphoserine. The parallel sample can be reacted under the same Group II metal hydroxide - 13 -

conditions but providing a "silent" adduct (i.e. one that cannot be detected by the detection technique used) and the reaction conditions then changed to the NaOH conditions so that the pattern produced will be selective for glycoserine. In an alternative form of sequential reaction two different detectable adducts can be formed in the same sample; one under Group II metal hydroxide conditions for preferential detection of phosphoserines and the second under NaOH conditions for preferential detection of glycoserines . In this way phospho and glyco serines can be distinguished in a single sample. Where a silent adduct is required the first reaction can be performed with a nucleophile such as mercaptoethanol, ethanethiol etc, which forms an adduct that is not identified by the detection technique employed (eg fluorescence) . If different nucleophiles are used in the two rounds of β-elimination and adduct formation both of which can be detected (eg nucleophiles which contain different fluorophores coupled to them or DNA molecules with different nucleotide sequences) the preferential labelling of the phospho- and gyco-serines in the two stages of the reaction can be visualised simultaneously in the same sample. Other preferred and typical characteristics of the reagents used for the method of the third aspect are as described above with respect to the first and second aspect provided that this does not conflict with specific features of the method of the third aspect of the invention set out above. In a fourth aspect the invention provides an anti-MMBA antibody. The anti-MMBA antibody may be a polyclonal or a monoclonal antibody.

In a fifth aspect the present invention provides a method for investigating phosphorylation changes in tissues from intact animals after drug or other treatment by comparing the phosphorylation before and after treatment using a method of the first, second or third - 14 -

aspect of the invention.

In a sixth aspect the present invention provides a method for investigating phosphorylation changes in biopsy or blood samples from human patients or animals as a diagnostic aid by comparing the phosphorylation in different samples using a method of the first, second or third aspect of the invention.

Diagnostic applications of the analysis of protein phosphorylation have been largely precluded in the past by the need for ³²P radioactivity.

In a seventh aspect the present invention provides a method for differentiating between serine and threonine or tyrosine phosphorylation on individual proteins in a mixture . The methods of the first, second and third aspects of the invention detect all serine linked phosphate regardless of its rate of turnover. Isotopic labelling (depending on how the labelling is done) favours labile or high turnover phosphate groups . If isotopic labelling of a protein shows a phosphorylation site which does not label with a method of the first, second or third aspect of the invention this indicates that the residue is threonine or tyrosine. However, it should be noted that there will be low turnover threonine sites which will not be labelled isotopically or by the methods of this invention.

Until now there has been limited methodology available to perform this analysis.

For the detection of serine linked carbohydrate the method of the invention may be used in conjunction with other selective probes for carbohydrates such as lectins or antibodies directed against carbohydrate epitopes. Whereas lectins and antibodies differentiate between carbohydrate groups on the basis of their sugar composition, sequence or structure, the methods of this invention can distinguish between carbohydrate groups on the basis of the type of amino acid to which they are - 15 -

linked. In particular, the present invention permits, for the first time, differentiation between serine and threonine glycosylation on individual proteins in a mixture; until now this could only be done by direct chemical analysis of purified proteins . Therefore used in combination with lectins, antibodies and other selective carbohydrate stains based on sugar content, the methods of this invention greatly extend the capacity to analyse the glycosylation of proteins. Patterns of glycosylation of cell surface proteins can be used to distinguish between cells from different species or strains of organisms or to identify differences due to genetic polymorphisms (e.g. like blood types in humans) or changes in cellular differentiation (e.g. during development or oncogenesis) . Therefore screening tests based on such techniques have potential application in any industry where recognition of wanted or unwanted strains is important such as food, brewing and wine industries. The availability of simple, cheap, versatile analysis of glycosylation patterns in proteins would also have application in the biotechnology industry for the development of procedures to produce recombinant glycosylatyed proteins and in quality control .

Detection of serine linked carbohydrate by the methods of the invention also is of use in screening clinical samples to look at genetic mutations in glycoproteins and thus extends the capacity of such screening by providing an additional tool for use in that screening. Under appropriately modified reaction conditions phospho- and glyco- threonine and phosphotyrosine will also be able to undergo β-elimination and adduct formation by nucleophilic attack of the intermediate formed. Because these conditions will be very different from those required to derivatise the phospho- and/or glyco-serines, application of these conditions to samples that have already been treated to derivatise the phospho - 16 -

and/or glycoserines will allow either simultaneous detection of these groups using multiple nucleophiles or selective detection if the serine derivatisation is done with a nucleophile that produces a silent adduct as described above with respect to the third aspect of the invention.

In an eighth aspect the present invention provides a method for differentiating between serine and threonine glycosylation on individual proteins in a mixture . The selectivity of the methods of the invention lies in not detecting the threonine linked carbohydrate. Thus combining an assay of the first or second aspect with other selective assays which distinguish carbohydrate sites based on sugar sequences (e.g. lectins), permits discrimination of serine linked from threonine linked carbohydrate .

Under appropriately modified reaction conditions glyco- threonine will also be able to undergo β- elimination and adduct formation by nucleophilic attack of the intermediate formed. Because these conditions will be very different from those required to derivatise the glyco-serines, application of these conditions to samples that have already been treated to derivatise the glycoserines will allow either simultaneous detection of these groups using multiple nucleophiles or selective detection if the serine derivatisation is done with a nucleophile that produces a silent adduct as described above with respect to the third aspect of the invention. The methods of the first, second and third aspects of the invention could also be incorporated into protein sequencing technology to detect phosphorylated or glycosylated peptides .

In a ninth aspect the present invention provides a way of selectively purifying peptide(s), polypeptide (s) or protein (s) from mixtures based on their content of phospho- or glyco-serine. In this aspect the nucleophile used to derivatise the phospho or glycoserine - 17 -

has an attached chemical group that can be bound to another molecule attached to a support (such as gel chromatography beads) with an affinity suitable for its use for purification by affinity chromatography. One preferred example is a nucleophile containing a DNA molecule consisting of a polydA sequence which will be able to bind to polydT DNA sequences coupled to gel chromatography beads and be eluted from them with solutions containing high salt concentrations or chaotropic agents that break the hydrogen bonds formed during hybridisation. In this way phosphorylated or glycosylated protein (s), peptide(s) or polypeptide (s) can be purified from a crude mixture and the affinity purification can be made more or less selective for phospho- or glyco-serine containing protein (s), polypeptide (s) or peptide(s) by employing various derivatisation conditions in one or more sequential steps as outlined above.

The methods of the invention can also be carried out on tissue sections, and cell suspensions and cultures. Employment of the methods of the invention in this way would help studies of phosphorylation where they could be used to identify heterogenous responses to a stimulus by different cells within a tissue or a localised response to a stimulus in one part of the cell. Of course the signal from each cell would be the sum of phosphorylation from many proteins .

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the pattern of phosphorylation of a heterogeneous mixture of proteins from isolated nerve endings from rat brain. 30 μg total protein was loaded per track. - 18 -

DNo Activator B+Ca/CM U+Ca/CM.+cAMP

Figure 2 shows the effect of stimulation of cultured chromaffin cells with nicotine.

BEST METHOD OF PERFORMING THE INVENTION

Synthesis of MMBA

MMBA is synthesized by the protocol described in any one of the following documents:

Rich D.H. and Gurwana S.K. (1975) J Am Chem Soc 97(6) : 1575-1579;

Turner F.A. and Gearien J.E. (1959) J Org Chem 24:1952-1955; or Brown T.M. , Carruthers W. and Pellatt M.G.

(1982) J Chem Soc (Perkins Trans 1) 483-486.

Method for Phosphoserine Immunoassay

I . SDS-PAGE 2. Western transfer to nylon 66 membrane (1 hour transfer at 300mA)

3. Wash membrane 3 x 5 minutes in TBS/T

4. Incubate membrane in 5% glutaraldehyde (in PBS) for 45 minutes at room temperature 5. Wash membrane 3x5min in ddH₂0

6. incubate membrane in 0.15M ethanolamine at room temperature (15 minutes)

7. wash 3x5min with ddH₂0

8. incubate membrane in derivatisation solution at 55°C for 45 minutes

9. wash membrane 3x5minutes in derivatisation buffer

(no MMBA)

10. wash membrane 3x5 minutes in TBS/T

II. incubate membrane in 5% dry skim milk powder in TBS/T (wt/vol) for 2 hours - 19 -

12. wash 3x5minutes in TBS/T

13. incubate membrane in rabbit antibody to MMBA [1 in

1000 dilution of whole serum in 5% dry skim milk powder in TBS/T (wt/vol) overnight] 14. wash 3x5 minutes in TBS/T

15. incubate membrane in commercial anti-rabbit Ig enzyme conjugate (alkaline phosphatase or horseradish peroxidase) according to supplier's protocol) 16. wash and visualise according to supplier's protocol

A. Buffers i) TBS/T (Tris buffered saline/tween)

0.010M N-Tris (hydroxymethyl)methylamine 0.150M sodium chloride pH to 8.0

0.05% polyoxyethylene sorbitan monolaurate (Tween 20) ii) PBS (phosphate buffered saline) 1.72mM sodium dihydrogen orthophosphate

3.21mM disodium hydrogen orthophosphate 0.154M sodium chloride pH to 7.4 iii) Ba²⁺ derivatisation buffer 24ml 85% ethanol (EtOH)

60ml dimethyl sulfoxide (DMSO) 60ml ddH₂0 pH to 12.8 with saturated Ba(OH)₂ solution iv) Na^* derivatisation buffer 24ml EtOH

60ml DMSO 60ml ddH₂0 31.5ml 5M NaOH

B. Derivatisation solutions i) Ba²⁺ derivatisation solution - 20

MMBA in Ba^2* derivatisation buffer (20mg/ml) ii) Na⁺ derivatisation solution

MMBA in Na⁺derivatisation buffer (20mg/ml)

Method for glyco-serine assay

This assay is performed as described above for the phosphoserine assay. The derivatisation under Na⁺ conditions shows both glycoserine and phosphoserine residues. To determine which residues are glycoserine, results obtained in the Ba²⁺ (phospho selective) and the Na⁺ conditions are compared and those whose labelling in Ba²" is not increased relative to the other bands are glycoserine.

Synthesis of Biotinylated Nucleophiles

X γ^ *r HO

X - any protected SH, OH or NH₂ group n = 0-5

Y = any protected SH, OH or NH₂ group

HX^ *T

A number of biotinylated nucleophiles are available via the above generic synthetic pathway. These include: amino (NH₂) based; thiol (SH) based; and alcohol (OH) based species.

The key steps in the synthesis involve the generation of suitable protected amino compounds with X = OH; SH; or NH in their protected form. Standard 21

protecting groups are used for each class of compounds. For example: OH can be protected as a SiMe₃ ether; THP ether; or MOM ether, while NH₂ can be Boc, Fmoc or phthalyl protected and SH can be protected as the disulphide or alkylated with a range of alkyl halides.

The actual coupling step Is conducted via standard peptide synthesis routes using a suitable coupling agent; e.g. BOP, DCC, EDC, HOBt, HbpyU, HOAt or HATU.

In the case of using a protected "Y" group the "X" group would be used in the de-protected form, or else no coupling reaction would take place.

The final step involves deprotection of the nucleophile using standard procedures.

Abbreviations :

Me : methyl THP: Tetrahydropyran MOM: Monomethyl Boc : tert-butoxycarbonyl Fmoc: 9-Fluorenylmethylcarbonyl

BOP: (Benzotriazolo-1-yloxy) tris (dimethylamino) phosphonium phosphate DCC: Dicyclohexylcarbodiimide

EDC: l-ethyl-3- (3-dimethyl-aminopropyl) - carbodiimide hydrochloride

HOBt: 1-hydroxybenzotriazole

HATU: O-(lH-Benzotriazol-l-yl) -N' ,N' ,N' ,N'- tetramethyluronium hexafluorophosphate HOAt : l-hydroxy-7-azabenzotriazole HbpyU: O- (Benzotriazol-1-yl) -N' ,N' ,N' ,N' -bis

( tetramethylene) uronium hexafluorophosphate Phthal : phthalamido

The skilled addressee will understand that other reagents of use in the invention can be prepared using standard chemical techniques. - 22 -

Example 1

Figure 1 shows the pattern of phosphorylation of a heterogenous mixture of proteins (30 μg total protein) from isolated nerve endings (synaptosomes) from rat brain after they were incubated in vi tro with 40 UM ATP for 30 sec at 37°C in the presence or absence of a Ca²⁺-calmodulin (CM) and/or cAMP to activate the major endogenous enzymes (protein kinases) that will phosphorylate the proteins in this mixture. After electrophoresis, transfer to Nylon 66 membrane and immobilisation of the proteins, they were derivatised with MMBA, firstly to selectively derivatise phosphoserine sites on the proteins (indicated by Ba^2*) and a control condition which detects phosphorylated and glycosylated serine residues (indicated by Na^*) . The panels on the right of the Figure show chemiluminescent detection of the adducts under these conditions. Results suggest that the technique is sensitive enough to detect less than 10 ng of protein containing phosphorylated serine residues. Figure 1 shows a large number of nerve terminal proteins detected by this technique, as expected from studies using radioactive techniques which show that synaptosomes contain many phosphoproteins . Also as expected, a brief incubation with ATP in the presence of activators of endogenous protein kinases leads to an increase in the labelling of many protein bands. When derivatisation is performed under the control conditions, (Na⁺) , the number of bands detected is much lower and the labelling of the principal bands does not change under conditions known to lead to increased phosphorylation. The graphs on the left of Figure 1 quantitate the changes in the degree of derivatisation of 4 protein bands which show different degrees of increased labelling due to the two activators. The degree of increase in labelling observed with this technique is consistent with results from radioactivity based assays for - 23 -

phosphorylation. For instance Band 2 in Figure 1 is synapsin I which is phosphorylated on separate serine residues by Ca/calmodulin and cAMP stimulated protein kinases present in synaptosomes . Band 1 is likely to be a glycoprotein.

Example 2

Figure 2 shows an example of the detection of changes in protein phosphorylation in cultured adrenal chromaffin cells. A comparison of the results with those obtained from the same cells using conventional isotopic labelling illustrates the principles of this technique. For the conventional labelling approach the cultured cells were incubated with radioactive phosphate for 1 hour in order to label intracellular pools of ATP and to achieve a basal level of phosphorylation of endogenous proteins. Subsequently the cells were treated for 10 minutes with nicotine (20μM) or control medium, the cells were harvested and the proteins analysed by SDS-PAGE and autoradiography. In these cells nicotine is known to stimulate phosphorylation of several protein bands. One of the major phosphorylation changes occurs in the band at approximately 60kDa that contains the enzyme tyrosine hydroxylase (TOH) . This is the rate limiting enzyme in the pathway for the synthesis of catecholamines and an enzyme that is particularly enriched in these cells. It is known that under these conditions TOH becomes phosphorylated at one of four serines . The band corresponding to TOH on the autoradiogram is indicated in Figure 2 ; the degree of increase in phosphate incorporation (as determined by densitometry of the autoradiogram) is shown in the bottom panel. 10⁴ chromaffin cells were loaded per track. The right side of Figure 2 shows a parallel experiment with cells that were treated in the same way except that there was no preincubation with radioactive phosphate. Instead of detecting incorporated phosphate by autoradiography the - 24 -

samples were transferred to a nylon membrane and treated with MMBA as described above. The MMBA adducts formed were detected by enzyme linked chemiluminescence (ECL) using antibody to MMBA and a secondary antibody linked to horseradish peroxidase. The results obtained using the two methods are very different. Four major points can be made :

1. Despite the different appearance of the blots the increase in phosphorylation of TOH induced by nicotine detected in the ³²P labelled cells was also detected using the MMBA technique. Furthermore densitometry showed that the increase in phosphorylation obtained using the two techniques was comparable . 2. In the MMBA derivatised sample there is a broad band (70-75kDa) above TOH that is not visible on the autoradiogram. The characteristic broad "fuzzy" appearance of this band after SDS-PAGE suggests that it contains a glycoprotein. The fact that it does not label with radioactive phosphate in the intact cell, and that its derivatisation with MMBA was unaffected by nicotine is consistent with this representing MMBA derivatisation of serine linked carbohydrates in this glycoprotein (s) . 3. In the radioactive sample there was a protein of approximately 42 kDa whose phosphorylation was increased by nicotine treatment by about the same extent as that of TOH. However, no MMBA derivatised protein band was visible in that region of the gel. We suggest that this is a protein containing phosphothreonine or phosphotyrosine side chains that are not susceptible to derivatisation with MMBA under these conditions. 4. While TOH is a major band in the autoradiogram it is not a major band in the ECL. We propose that this is due to inherent differences in the way in which the two detection mechanisms work. The isotopic - 25 -

labelling approach detects phosphate turnover and gives a strength of signal in a band that depends on the concentration of the protein, the stoichiometry of phosphorylation as well as its rate of turnover; a stably phosphorylated site would not be detected.

By contrast the MMBA technique depends only on the concentration of the protein and its stoichiometry of phosphorylation and detects both stable and labile phosphorylation sites . Therefore for some applications the MMBA and isotopic techniques may need to be used in parallel because they yield complementary information.

INDUSTRIAL APPLICABILITY The methods of the invention are of use as research tools for the identification of phosphorylated and/or glycosylated peptides, polypeptides and proteins in which serine residues are phosphorylated or glycosylated. These methods can also be applied in diagnostic settings.

Claims

- 26 -

CLAIMS 1. A method for demonstrating the presence of phosphate and/or carbohydrate bound to serine residues in proteinaceous material which method comprises : providing the proteinaceous material immobilised on a solid support; derivatising the proteinaceous material with a nucleophile under conditions which selectively replace at least some of the phosphate and/or carbohydrate groups present on serine residues in the proteinaceous material with the nucleophile to form a derivatised proteinaceous material comprising an adduct formed by the nucleophile; and detecting the derivatised proteinaceous material by recognition of the adduct formed by the nucleophile.

2. A method or demonstrating the presence of phosphate and/or carbohydrate bound to serine residues in proteinaceous material which method comprises: providing the proteinaceous material in solution; derivatising the proteinaceous material with a nucleophile under conditions which selectively replace at least some of the phosphate and/or carbohydrate groups present on serine residues in the proteinaceous material with the nucleophile to form a derivatised proteinaceous material comprising an adduct formed by the nucleophile; retrieving the derivatised proteinaceous material ; and detecting the derivatised proteinaceous material by recognition of the adduct formed by the nucleophile . - 27 -

3. A method according to claim 1 or claim 2 wherein the proteinaceous material is one or more peptides, polypeptides and/or proteins present in a crude mixture .

4. A method according to claim 1 or claim 2 wherein the proteinaceous material is one or more peptides, polypeptides and/or proteins present in purified form.

5. A method according to claim 1 or claim 2 wherein a mixture of peptide(s), polypeptide (s) and/or protein (s) is present and the method includes the separation of the peptide(s), polypeptide (s) and/or protein (s) present sufficient to permit the identification of the different components of the mixture.

6. A method according to claim 2 wherein the method is carried out in solution and the proteinaceous material consists of a single protein, peptide or polypeptide .

7. A method according to claim 1 or claim 2 wherein the nucleophile forms an adduct which does not present a component for recognition that occurs naturally in proteinaceous material .

8. A method according to claim 1 or claim 2 wherein the nucleophile is p-mercaptomethylbenzoic acid (MMBA) , or a derivative of MMBA, or a compound with a suitable nucleophilic group and detectable portion that can be detected directly or indirectly.

9. A method according to claim 8 wherein the detectable portion can be detected directly by its colour or fluorescence or radioactivity or indirectly by: binding to another molecule which other molecule can be detected either directly or indirectly by its colour or fluorescence or radioactivity; or by being coupled to; an enzyme which can produce a coloured or fluorescent product and a signal that can be amplified; or to a fluorescent chemical group; or by binding to another - 28 -

molecule which can be detected directly or indirectly.

10. A method according to claim 1 or claim 2 wherein the nucleophile is a thiol, an amine, an amide or an alkanol, in an alkaline environment.

11. A method according to claim 1 or claim 2 wherein a separate detection reagent is used to recognise the adduct.

12. A method according to claim 8 wherein the derivative of MMBA is a chemically altered MMBA in which the attributes of the molecule as a nucleophile and as a detectable molecule are retained.

13. A method according to claim 1 or claim 2 wherein the adduct formed by the nucleophile is recognised immunochemically, colorimetrically or by fluorescence or chemiluminescence.

14. A method according to claim 1 or claim 2 wherein the nucleophile is MMBA or a derivative of MMBA and the detection reagent comprises an antibody which recognises

MMBA.

15. A method according to claim 14 in which the anti-MMBA antibody is detected with a commercially available secondary antibody.

16. A method according to claim 14 wherein the secondary antibody comprises a label .

17. A method according to claim 16 wherein the label is alkaline phosphatase or horseradish peroxidase, or another reporter enzyme, together with one or more appropriate colour or light producing reagents, or a fluorescent label .

18. A method according to claim 1 or claim 2 wherein the nucleophile is a biotinylated nucleophile and the method utilises a detection reagent comprising avidin or streptavidin in labelled form.

19. A method according to claim 18 wherein the label is alkaline phosphatase or horseradish peroxidase or another reporter enzyme, together with one or more - 29 -

appropriate colour or light producing reagents, or a fluorescent label .

20. A method according to claim 17 or claim 19 wherein the colour producing reagent is: a soluble reagent giving one or more insoluble products which precipitate at the required spot on a support; or a soluble reagent giving one or more soluble products to be detected by colorimetry in an ELISA assay type application where the protein is immobilised onto a microtitre plate or tube or where the assay is done in solution.

21. A method according to claim 1 or claim 2 wherein the nucleophile is a nucleophilic group coupled to a variable sequence and length of DNA nucleotides and the detection reagent comprises complementary DNA coupled to an enzyme or fluorescent group.

22. A method according to claim 1 comprising the following steps:

(1) a sample comprising a mixture of protein (s) polypeptide (s) and/or peptide (s) is treated to separate the protein (s), polypeptide (s) and/or peptide (s) by a separation technique and the separated protein (s) , polypeptide (s) and/or peptide (s) are transferred to a support; (2) the protein(s), polypeptide (s) and/or peptide (s), are crosslinked to the support using a cross linking reagent;

(3) unreacted groups on the cross linking reagent are blocked by reaction with a suitable reagent ;

(4) protei (s) , polypeptide (s) and/or peptide (s) on the solid support are derivatised by incubation at approximately 55┬░C for 30 to 60 minutes in a solution comprising p-mercaptomethylbenzoic acid (MMBA) at a pH of at least 12.5 to form an MMBA adduct; - 30 -

(5) after removal of unreacted MMBA, the MMBA derivatised protein (s) , polypeptide (s) and/or peptide (s) are detected using an antibody prepared against the MMBA adduct using standard immunodetection methods used for western blotting and immunodetection reagents according to any one of claims 15 to 17.

23. A method according to claim 22 wherein the separation technique is SDS-PAGE or a chromatographic step.

24. A method according to claim 23 wherein the chromatographic step is HPLC .

25. A method according to claim 22 wherein the support is a sheet or strip of Nylon 66 or polyvinyl difluoride (PVDF) .

26. A method according to claim 22 wherein the support is one which survives the alkaline conditions under which the method is carried out, at 55┬░C for the length of time required; does not have a chemical substituent on it which can undergo nucleophilic attack by MMBA; and has suitable sites for attachment of the proteinaceous material .

27. A method according to claim 22 wherein purified protein (s) , polypeptide (s) or peptide (s) are applied directly to the support .

28. A method according to claim 27 wherein the transfer of protein (s), polypeptide (s) or peptide (s) to the support is performed electrophoretically, by diffusion or by spotting from a pipette or column eluate.

29. A method according to claim 22 wherein the cross linking reagent is an aldehyde.

30. A method according to claim 29 wherein the blocking is with a low molecular weight source of free amino groups .

31. A method according to claim 22 wherein the cross linking reagent is one which does not react with the support to produce a high background in the detection - 31 -

part of the method.

32. A method according to claim 30 wherein the cross linking reagent is glutaraldehyde and the low molecular weight source of free amino groups is ethanolamine.

33. A method according to claim 22 wherein the solution comprises MMBA at a concentration of at least 20mg/ml.

34. A method according to claim 22 or claim 33 wherein the solution comprising MMBA further comprises 12-17% ethanol, 34-42% DMSO made up to pH12.9 with NaOH or a Group II metal hydroxide.

35. A method according to claim 34 wherein the Group II metal hydroxide is Ba(OH)₂ .

36. A method according to claim 22 wherein the unreacted MMBA is removed by washing with solvent minus MMBA.

37. A method according to claim 2 which comprises the following steps :

1) protein(s), peptide(s) and/or polypeptide (s) are derivatised by incubation at approximately 55┬░C for 30 to 60 minutes in a solution comprising p-mercaptomethylbenzoic acid (MMBA) at a pH of at least 12.5

2) removal of unreacted excess MMBA by gel filtration;

3) after removal of the excess MMBA the MMBA derivatised protein (s) , peptide (s) and/or polypeptide (s) are detected using an antibody prepared against the MMBA adduct using standard solution based immunodetection techniques .

38. A method according to claim 37 wherein the solution comprises MMBA at a concentration of at least 20mg/ml.

39. A method according to claim 37 or claim 38 wherein the MMBA solution further comprises 12-17% ethanol and 34-42% DMSO made up to pH12. with NaOH or a Group II metal hydroxide. - 32 -

40. A method according to claim 39 wherein the Group II metal hydroxide is Ba(OH)₂.

41. A method according to claim 37 wherein removal of unreacted excess MMBA is performed instead by dialysis or selective extraction with organic or alkaline solvent (s) except when the protein (s), polypeptide (s) and/or peptide (s) are soluble under these conditions and can not still be redissolved afterwards.

42. A method according to claim 41 wherein the alkaline solvent is a sodium bicarbonate solution.

43. A method according to claim 37 wherein removal of unreacted MMBA is performed instead by filtration through a membrane to which the protein (s), peptide (s) and/ or polypeptide (s) bind and on which the subsequent detection reaction can be performed.

44. A method according to claim 14 wherein the anti- MMBA antibody is a polyclonal antibody raised in a rabbit against MMBA derivatised bovine submaxillary gland mucin or MMBA derivatised phosvitin.

45. A method according to claim 14 wherein the anti-

MMBA antibody is a monoclonal antibody raised against MMBA derivatised bovine submaxillary gland mucin or MMBA derivatised phosvitin.

46. A method according to claim 14 wherein a protein containing O-linked carbohydrate or phosphate is used to make the MMBA derivative.

47. A method according to claim 46 wherein the protein is a strong natural antigen.

48. A method according to claim 47 wherein the protein is mucin.

49. A method for differentiating between phospho- and glyco serine residues in a proteinaceous material selected from the group consisting of protein, polypeptide and peptide or mixtures thereof present in crude mixtures, and purified proteins, polypeptides or peptides, which method comprises: - 33 -

derivatising the proteinaceous material with at least one nucleophile under conditions which selectively replace at least some of the phosphate and/or carbohydrate groups present on serine residues in the proteinaceous material with the at least one nucleophile to form a derivatised proteinaceous material comprising at least one adduct formed by at least one nucleophile in separate or sequential reactions in the presence of a Group II metal hydroxide and NaOH; and detecting the derivatised proteinaceous material by recognition of the adducts formed by the nucleophiles .

50. A method according to claim 49 wherein the Group II metal hydroxide is Ba(OH)₂.

51. A method according to claim 49 wherein the method is conducted in the solid phase, and the proteinaceous material is immobilised on a solid support.

52. A method according to claim 51 comprising the method according to claim 1.

53. A method according to claim 49 performed in solution and additionally comprising retrieving the derivatised proteinaceous material prior to detection.

54. A method according to claim 49 wherein the nucleophile (s) forms an adduct (s) which does not present a component for recognition that occurs naturally in proteinaceous material .

55. A method according to claim 49 wherein one or more separate detection reagents are used to recognise the adduct (s) .

56. An anti-MMBA antibody.

57. An antibody according to claim 56 which is a polyclonal antibody.

58. An antibody according to claim 56 which is a monoclonal antibody.

59. A method for investigating phosphorylation changes in tissues from intact animals after drug or - 34 -

other treatment by comparing the phosphorylation before and after treatment using a method according to any one of claims 1, 2 or 49.

60. A method for investigating phosphorylation changes in biopsy or blood samples from a human patient or an animal as a diagnostic aid by comparing the phosphorylation in different samples using a method according to any one of claims 1, 2 or 49.

61. A method for differentiating between serine and threonine or tyrosine phosphorylation on individual proteins in a mixture wherein isotopic labelling of a protein which shows a phosphorylation site which does not label with a method of any one of claims 1, 2 or 49 indicates that the residue is threonine or tyrosine.

62. A method for the detection of serine linked carbohydrate wherein the method of any one of claims 1,2 or 49 is used in conjunction with one or more other selective probes for carbohydrates.

63. A method according to claim 62 wherein the other selective probes are lectins or antibodies directed against carbohydrate epitopes .

64. A method for differentiation between serine and threonine glycosylation on individual proteins in a mixture wherein combining a method of claim 1 or claim 2 with other selective assays which distinguish carbohydrate sites based on sugar sequences permits discrimination of serine linked from threonine linked carbohydrate .

65. A method according to claim 1 or claim 2 wherein the nucleophile is a nucleophilic group attached to a DNA molecule.

66. A method for selectively purifying peptide (s), polypeptide (s) or protein (s) from mixtures based on their content of phospho- or glyco-serine, wherein a nucleophile used to derivatise the phospho or glyco serine has an attached chemical group that can be bound to another molecule attached to a support with an - 35 -

affinity suitable for its use for purification by affinity chromatography.

67. A method according to claim 66 wherein the nucleophile is a nucleophile containing a DNA molecule consisting of a polydA sequence.

68. A method according to claim 1 or claim 2 which can additionally detect the presence of one or more of phosphosthreonine, glycothreonine or phospho yrosine wherein at least one additional reaction is performed sequentially or on a parallel sample to cause selective adduct formation and detection for one or more of phosphosthreonine, glycothreonine and phosphotyrosine .

69. A method according to claim 68 wherein the same nucleophilic group is used to detect the presence of one or more of phosphosthreonine, glycothreonine or phosphotyrosine and phospho and /or glyco serine and different detection portions or reagents are used to permit distinction of the different adducts.

70. A method according to claim 68 wherein different nucleophilic groups are used in detecting the presence of at least one of phosphosthreonine, glycothreonine and phosphotyrosine from the nucleophilic group used to detect phospho and/or glycoserine.

71. A method according to claim 49 or claim 68 wherein a silent adduct and a detectable adduct are used to distinguish the adducts formed.

72. A method according to claim 1, 2, 49, 59 60 or 68 wherein the method is carried out on a tissue section or a cell suspension or culture.

73. A method for enhancing screening of clinical samples for genetic mutations in glycoproteins comprising including a method according to any one of claims 1, 2, 49, 62 or 64 in the screening.