EP1057034A1 - Detection de groupes phosphate ou hydrate de carbone sur les residus serine - Google Patents

Detection de groupes phosphate ou hydrate de carbone sur les residus serine

Info

Publication number
EP1057034A1
EP1057034A1 EP99901514A EP99901514A EP1057034A1 EP 1057034 A1 EP1057034 A1 EP 1057034A1 EP 99901514 A EP99901514 A EP 99901514A EP 99901514 A EP99901514 A EP 99901514A EP 1057034 A1 EP1057034 A1 EP 1057034A1
Authority
EP
European Patent Office
Prior art keywords
mmba
nucleophile
proteinaceous material
protein
derivatised
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99901514A
Other languages
German (de)
English (en)
Inventor
John Andrew Paul Rostas
John Edgar Thomas Corrie
Adam Mccluskey
Andrew James Delaney
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Newcastle Innovation Ltd
Original Assignee
Newcastle Innovation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Newcastle Innovation Ltd filed Critical Newcastle Innovation Ltd
Publication of EP1057034A1 publication Critical patent/EP1057034A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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 .
  • Phosphorylation and glycosylation are both ubiquitous post-translational modifications of proteins used by all pro aryotic and eukaryotic cells.
  • 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.
  • 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.
  • 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.
  • 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 -
  • 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.
  • 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 ⁇ -elimination at greatly different rates under strongly alkaline - 5 -
  • 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.
  • 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 32 P (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.
  • 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.
  • 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.
  • the nucleophile is MMBA and the detection reagent comprises an antibody which - 7 -
  • 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) .
  • the nucleophile is a biotinylated nucleophile and the detection reagent comprises avidin or streptavidin.
  • 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.
  • 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.
  • 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) .
  • 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 -
  • colour detection reagent 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:
  • 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 proteinaceous material
  • 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 ;
  • cross linking reagent which may be an aldehyde such as glutaraldehyde .
  • 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;
  • 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 -
  • MMBA p-mercaptomethylbenzoic acid
  • 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. ;
  • 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.
  • organic or alkaline solvents e.g. sodium bicarbonate solution
  • 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 ;
  • 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 -
  • 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 ⁇ -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.
  • 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
  • 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 .
  • the method is conducted in the solid phase, the proteinaceous material is immobilised on a solid support .
  • this method when conducted in the solid phase involves the steps specified above with respect to the first aspect of the invention.
  • the method 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.
  • the two derivatisation conditions can be employed sequentially.
  • 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 -
  • 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) .
  • a nucleophile such as mercaptoethanol, ethanethiol etc
  • the preferential labelling of the phospho- and gyco-serines in the two stages of the reaction can be visualised simultaneously in the same sample.
  • the invention provides an anti-MMBA antibody.
  • the anti-MMBA antibody may be a polyclonal or a monoclonal antibody.
  • 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 -
  • 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.
  • 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.
  • 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.
  • 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 -
  • 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) .
  • 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.
  • phospho- and glyco- threonine and phosphotyrosine will also be able to undergo ⁇ -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 -
  • 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 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.
  • glyco- threonine will also be able to undergo ⁇ - 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 .
  • 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.
  • 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 .
  • Figure 1 shows the pattern of phosphorylation of a heterogeneous mixture of proteins from isolated nerve endings from rat brain. 30 ⁇ g total protein was loaded per track. - 18 -
  • Figure 2 shows the effect of stimulation of cultured chromaffin cells with nicotine.
  • MMBA is synthesized by the protocol described in any one of the following documents:
  • TBS/T Tris buffered saline/tween
  • This assay is performed as described above for the phosphoserine assay.
  • the derivatisation under Na + conditions shows both glycoserine and phosphoserine residues.
  • results obtained in the Ba 2+ (phospho selective) and the Na + conditions are compared and those whose labelling in Ba 2 " is not increased relative to the other bands are glycoserine.
  • biotinylated nucleophiles are available via the above generic synthetic pathway. These include: amino (NH 2 ) based; thiol (SH) based; and alcohol (OH) based species.
  • protecting groups are used for each class of compounds.
  • OH can be protected as a SiMe 3 ether; THP ether; or MOM ether, while NH 2 can be Boc, Fmoc or phthalyl protected and SH can be protected as the disulphide or alkylated with a range of alkyl halides.
  • a suitable coupling agent e.g. BOP, DCC, EDC, HOBt, HbpyU, HOAt or HATU.
  • the final step involves deprotection of the nucleophile using standard procedures.
  • 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
  • Figure 1 shows the pattern of phosphorylation of a heterogenous mixture of proteins (30 ⁇ g 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 Ca 2+ -calmodulin (CM) and/or cAMP to activate the major endogenous enzymes (protein kinases) that will phosphorylate the proteins in this mixture.
  • CM Ca 2+ -calmodulin
  • cAMP protein kinases
  • 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.
  • 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.
  • 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 (20 ⁇ M) 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.
  • TOH tyrosine hydroxylase
  • 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.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé permettant de démontrer la présence de phosphate et/ou d'hydrates de carbone liés aux résidus sérine dans les matières protéiniques. Ce procédé consiste à: prendre des matières protéiniques immobilisées sur un support solide; à former une dérivé de la matière protéinique par l'utilisation d'un nucléophile dans des conditions propres à entraîner le remplacement sélectif d'au moins une partie des groupes phosphate et/ou hydrate de carbone présents sur les résidus sérine dans les matières protéiniques par le nucléophile, de manière à former un dérivé de matière protéinique comprenant un produit d'addition formé par le nucléophile; et détecter les matières protéiniques dérivées en identifiant le produit d'addition formé par le nucléophile.
EP99901514A 1998-01-21 1999-01-21 Detection de groupes phosphate ou hydrate de carbone sur les residus serine Withdrawn EP1057034A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AUPP1480A AUPP148098A0 (en) 1998-01-21 1998-01-21 Assay
AUPP148098 1998-01-21
PCT/AU1999/000043 WO1999038012A1 (fr) 1998-01-21 1999-01-21 Detection de groupes phosphate ou hydrate de carbone sur les residus serine

Publications (1)

Publication Number Publication Date
EP1057034A1 true EP1057034A1 (fr) 2000-12-06

Family

ID=3805757

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99901514A Withdrawn EP1057034A1 (fr) 1998-01-21 1999-01-21 Detection de groupes phosphate ou hydrate de carbone sur les residus serine

Country Status (3)

Country Link
EP (1) EP1057034A1 (fr)
AU (1) AUPP148098A0 (fr)
WO (1) WO1999038012A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001267485A1 (en) * 2000-05-26 2001-12-11 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Method for the analysis of picomole amounts of carbohydrates
NZ539734A (en) 2002-10-09 2011-07-29 Dmi Biosciences Inc Diagnosis and monitoring of inflammation, ischemia and appendicitis

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686310A (en) * 1995-06-06 1997-11-11 University Of Virginia Patent Foundation Method for determination of the amount of either phosphotyrosine or phosphoserine in a protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9938012A1 *

Also Published As

Publication number Publication date
WO1999038012A1 (fr) 1999-07-29
AUPP148098A0 (en) 1998-02-19

Similar Documents

Publication Publication Date Title
US5585277A (en) Screening method for identifying ligands for target proteins
EP1456632B1 (fr) Procedes permettant d'isoler et de marquer des molecules d'echantillon
US20090023144A1 (en) Method and its kit for quantitatively detecting specific analyte with single capturing agent
JP6756611B2 (ja) 中和抗体を検出するための競合リガンド結合アッセイ
US20080020374A1 (en) Reagents, Kits and Methods for Immunodetection of Epitopes on Molecules
AU2002303760A1 (en) Methods for isolating and labeling sample molecules
KR20200051699A (ko) 프로테오믹스를 위한 멀티플렉스화된 비드 어레이
US7794947B2 (en) Affinity capture of peptides by microarray and related methods
JP2517187B2 (ja) アナライト変異体分析
WO2001088543A2 (fr) Methodes et compositions destinees a etre utilisees dans le diagnostic et la caracterisation de maladies immunitaires chroniques
EP3264085A1 (fr) Procédé de dosage immunologique et réactif de dosage utilisé dans le procédé
JP4515099B2 (ja) Ｌａｓｐ−１免疫反応性の決定によって炎症性疾患および感染を診断するための方法
EP1101114B1 (fr) Methode d'analyse de proteines
KR20040012854A (ko) 미생물의 동정 방법
EP1589341A2 (fr) Méthode pour l'isolation des peptides pour les identifier et analyser dans des mixtures complexes
FI100276B (fi) Menetelmä analyyttien ei-kilpailevaa määritystä varten
US5225322A (en) Immunoassays for detection of viral subtypes and other antigens
EP1057034A1 (fr) Detection de groupes phosphate ou hydrate de carbone sur les residus serine
US11199539B2 (en) Methods, compositions and devices for improving the sensitivity of assays
US8153367B2 (en) Amplified array analysis system
JP2003502665A (ja) 検出可能な競合リガンドを使用した、親和性リガンドのスクリーニングに適したキャピラリ電気泳動法
WO2002061114A9 (fr) Substrats peptidiques solubles dans l'eau, fluorescents, mobiles par electrophorese pour des reactions enzymatiques et leurs procedes d'utilisation dans des essais de criblage extremement productifs
US20100003698A1 (en) Kit of Materials for Decreasing Interference in Results of Immunochemical Methods
KR101432714B1 (ko) 벤조안트라센 오염 검출을 위한 마커 및 키트
US20050239078A1 (en) Sequence tag microarray and method for detection of multiple proteins through DNA methods

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000821

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): DE FR GB

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 20020328