[go: up one dir, main page]

WO1996018899A1 - Procede de detection d'un acide amine phosphoryle dans une proteine intacte - Google Patents

Procede de detection d'un acide amine phosphoryle dans une proteine intacte Download PDF

Info

Publication number
WO1996018899A1
WO1996018899A1 PCT/US1995/016421 US9516421W WO9618899A1 WO 1996018899 A1 WO1996018899 A1 WO 1996018899A1 US 9516421 W US9516421 W US 9516421W WO 9618899 A1 WO9618899 A1 WO 9618899A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
amino acid
group
nucleophile
intact protein
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.)
Ceased
Application number
PCT/US1995/016421
Other languages
English (en)
Inventor
Malcolm Whitman
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.)
Harvard University
Original Assignee
Harvard University
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 Harvard University filed Critical Harvard University
Publication of WO1996018899A1 publication Critical patent/WO1996018899A1/fr
Anticipated expiration legal-status Critical
Ceased 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This invention relates to methods and compositions for detecting phosphorylated serine and threonine amino acid residues in intact proteins.
  • the methods involve dephosphorylating the phosphorylated amino acid and covalently coupling a detectable tag to the dephosphorylated amino acid in the intact protein.
  • metabolic labeling with inorganic 32 [P] typically involve using millicurie quantities of radioisotope for the detection of phosphorylated amino acids in non-abundant proteins and cannot easily be adapted for studying protein phosphorylation in tissues derived from whole animals.
  • metabolic labeling techniques together with the risk of exposure to high levels of radioactivity and the concomitant generation of large amounts of radioactive waste, have precluded the widespread acceptance of metabolic labeling with 32 [P] for studying protein phosphorylation in vivo.
  • the present invention overcomes these problems by providing methods and compositions for detecting phosphoserine and/or phosphothreonine residues in an intact protein.
  • the methods of the invention can be practiced using radiolabel or non-radiolabel detection systems that are well known to those of skill in the art of protein labeling. Thus, in contrast to the prior art, the methods do not require metabolic labeling in vivo and do not require large quantities of radiolabel for detection of the phosphorylated amino acids.
  • kits for determining the presence of a phosphoserine or phosphothreonine residue in an intact protein are based upon the recognition that two reactions, a beta- elimination reaction and an addition reaction, can be used in combination to detect phosphorylated amino acids in an intact protein.
  • the methods and compositions of the invention are useful for detecting phosphoserine and phosphothreonine residues in a variety of protein samples, including crude protein extracts and purified protein preparations containing, for example, immunoprecipitated proteins. Accordingly, the invention provides a useful alternative to metabolic labeling for studying the changes in regulatory serine and/or threonine phosphorylations in vivo.
  • phosphorylated amino acid refers to a phosphoserine or phosphothreonine residue in an intact protein.
  • the method involves: (1) subjecting the protein to conditions for effecting a beta-elimination reaction in the intact protein to convert the phosphorylated amino acid to a dehydroamino acid including a double bond containing carbon atoms that are susceptible to attack by an addition reaction; (2) contacting the protein of step (1) with a reactant under conditions for adding the reactant to the carbon atom of the double bond of the dehydroamino acid to form an amino acid addition product in the intact protein; and (3) detecting the amino acid addition product in the intact protein, wherein the presence of the amino acid addition product is indicative of the presence of the phosphorylated amino acid in the intact protein.
  • cysteine, cystine and/or lysine residues which may interfere with the sensitivity and/or accuracy of the detection methods disclosed herein. Accordingly, it is recommended that cysteine, cystine and/or lysine residues be modified prior to conducting the beta-elimination to avoid their participation in side reactions which could interfere with the determination of phosphoserine and phosphothreonine residues in the intact protein. Methods for modifying cysteine, cystine and lysine residues are described in more detail below.
  • the conditions for effecting a beta-elimination reaction involve exposing the protein to a dilute basic aqueous solution having a Ph between about 10 and 14 for between about 10 minutes and twelve hours.
  • the beta-elimination reaction will proceed faster at a higher pH. Accordingly, optimization of the precise incubation conditions for effecting the beta-elimination reaction, while at the same time minimizing protein degradation due to exposure to high Ph, can be accomplished using no more than routine experimentation.
  • a beta-elimination catalyst e.g., a group II metal ion
  • be added to facilitate the rate of reaction for the beta-elimination reaction can be added to facilitate the rate of reaction for the beta-elimination reaction.
  • beta-elimination catalyst refers to a substance which increases the kinetic rate of the beta-elimination reaction.
  • the beta-elimination reaction results in conversion of the phosphorylated amino acids to the corresponding dehydroamino acids.
  • the dehydroamino acid includes a double bond containing carbon atoms that are susceptible to attack by an addition reactant in an addition reaction.
  • an addition reactant refers to a molecule that is capable of forming a covalent bond with a carbon atom of the dehydroamino acid in an addition reaction.
  • the invention is not limited in scope to a particular mechanism, it is believed that the addition reactions disclosed herein primarily proceed via nucleophilic addition.
  • the dehydroamino acid double bond is subject to a nucleophilic addition reaction to form a nucleophilic addition product.
  • nucleophile The "reactant" in a nucleophilic addition reaction is referred to herein as a nucleophile and the nucleophilic addition product is referred to herein as a nucleophilic amino acid addition product.
  • the invention embraces two types of nucleophile: (1) a monofunctional nucleophile which contains a detectable tag for determining the presence of the phosphorylated amino acid in the intact protein in a "one-step” reaction and (2) a bifunctional nucleophile which does not contain a detection reagent and which therefore must be further reacted with a detection reagent to determine the presence of the phosphorylated protein in a "two-step” reaction.
  • a monofunctional nucleophile is a nucleophile which contains a single functional group for covalent attachment to an amino acid residue in an intact protein and further includes a detectable tag for detecting the presence of the phosphorylated amino acid in the intact protein.
  • a bifunctional nucleophile is a nucleophile which contains two functional groups for forming two distinct covalent bonds, i.e., the bifunctional nucleophile contains a first functional group for reacting with the carbon atom of the double bond of the dehydroamino acid and a second functional group for reacting with the detection reagent.
  • detectable tag refers to a molecule that can be directly detected (i.e., the molecule itself can be detected) or indirectly detected (i.e., the molecule is detected by a further reaction).
  • exemplary detectable tags include radioactive tags (e.g., 35 [S], 32 [P], l4 [C]), chromophores, fluorophores, luminescent tags, biotin, haptens and enzymes. In general, such detection procedures are performed using routine procedures well known to those of ordinary skill in the art.
  • a bifunctional nucleophile is used for the addition reaction involves: (1) subjecting the intact protein to conditions for effecting a beta- elimination reaction to convert the phosphorylated amino acid to a dehydroamino acid, (2) contacting the protein of step (1) with the bifunctional nucleophile under conditions for adding the nucleophile to the carbon atom of the double bond to form a nucleophilic addition product in the intact protein, (3) contacting the protein of step (2) with the detection reagent under conditions for the detection reagent to covalently bond to the second functional group of the bifunctional nucleophile, and (4) detecting the nucleophilic addition product in the intact protein as described above.
  • the bifunctional nucleophile contains a second functional group that is an amine group or a thiol group and the detection reagent is an amino group or thiol group labeling reagent, respectively.
  • the detection reagent is an amino group or thiol group labeling reagent, respectively.
  • Exemplary amino group and/or thiol group labeling reagents are well known to those of skill in the art of protein labeling.
  • the phosphorylated amino acids can be detected by oxidizing the double bond of the dehydroamino acid (the product of the above-described beta-elimination reaction) with KMn0 4 to yield a cis diol which then can be oxidized with periodate to a highly reactive aldehyde.
  • the resultant aldehyde is reacted with, for example, biotin-hydrazide to yield a biotin- labeled amino acid in the intact protein at the location of the dehydroamino acid.
  • kits for detecting phosphoserine and/or phosphothreonine in an intact protein include a nucleophile (monofunctional or bifunctional) which can add to the carbon atoms of the double bond of a dehydroamino acid and instructions for using the nucleophile to perform the method of the invention.
  • Kits which include the bifunctional nucleophile further include a detection reagent which can form a covalent complex with one functional group of the bifunctional nucleophile.
  • the kits further include protein controls for evaluating the sensitivity and accuracy of the method for detecting the presence of a phosphorylated amino acid in an unknown protein.
  • the detection of phosphoserine and phosphothreonine in accordance with the methods of the invention is useful for characterizing the molecular events leading to and resulting from the phosphorylation of serine and/or threonine residues, in a manner analogous to studies performed using antibodies to phosphotyrosine to characterize the molecular events leading to and resulting from the phosphorylation of tyrosine residues in vivo.
  • agents and the physiological targets and/or conditions to which they are directed include the immunosuppressive cyclosporin (which targets calcineurin, a serine/threonine protein kinase).
  • immunosuppressive rapamycin which reportedly inhibits p70 So kinase, a serine/threonine kinase
  • beta-adrenergic blockers which prevent adrenergic activation of cAMP- dependent protein kinases.
  • the methods and compositions of the invention are useful for evaluating the efficacy of putative pharmacological agents to specifically block a particular targeted metabolic pathway in vivo. Further, because changes in protein phosphorylation have been correlated to a broad range of normal physiological functions (e.g., glycogen metabolism, cholesterol metabolism, neurotransmitter function) and disease pathogenesis (e.g., leukemia), the methods and compositions of the invention also have utility in evaluating disease pathologies and assessing clinical prognosis.
  • normal physiological functions e.g., glycogen metabolism, cholesterol metabolism, neurotransmitter function
  • disease pathogenesis e.g., leukemia
  • the receptors for insulin and certain growth factors are membrane-bound tyrosine protein kinases for which the kinase enzymatic activity reportedly is essential for proper receptor function in vivo. See, e.g., Harrison's Principles of Internal Medicine, 12th ed., J. Wilson, et al. editors, McGraw-Hill, Inc., New York, NY (1991 ). Such results are consistent with more recent reports that the phosphorylation state of certain protein amino acid residues (e.g., tyrosines) correlates to neoplastic disease states (e.g., leukemia).
  • neoplastic disease states e.g., leukemia
  • the methods and compositions of the invention also have utility in improving routine protein characterization methods, e.g., peptide mapping and amino acid analysis, with respect to phosphoserine and/or phosphothreonine detection.
  • routine protein characterization methods e.g., peptide mapping and amino acid analysis
  • the methods and compositions disclosed herein can be used to increase the sensitivity and accuracy of phosphoserine and/or phosphothreonine detection, e.g., by providing a detection method that is more sensitive than the detection methods that are currently available for detecting these phosphorylated amino acids and/or by providing a method that can be adapted to generate a variety of detectable signals depending upon the selection of the particular detectable tag for labeling (e.g., a fluorophore, a chemiluminescent tag).
  • a fluorophore e.g., a fluorophore, a chemiluminescent tag
  • Figure 1 shows the results obtained following dephosphorylation/biotinylation of BSA and casein as described in Example 1.
  • Purified bovine serum albumin (B) an unphosphorylated protein
  • casein (C) a serine/threonine phosphoprotein
  • Figure 1 A shows the streptavidin detection results of approximately 500 ng BSA (B) or 250 ng casein (C) after dephosphorylation/ DTP/ biotin- labeling.
  • Figure IB shows the Coomassie staining results of the starting material that was used in the detection procedure, showing the relative amounts of total BSA and casein protein.
  • the instant invention embraces methods for detecting phosphorylated amino acids in an intact protein and related compositions. More specifically, methods for identifying phosphoserine and/or phosphothreonine amino acid residues in an intact protein are provided. The invention is useful for detecting these phosphorylated amino acids in virtually any intact protein without regard to the primary, secondary and/or tertiary protein structure in the vicinity of the phosphorylated amino acids.
  • phosphorylated amino acid refers to a phosphoserine or a phosphothreonine residue.
  • the phosphoesters of the primary and secondary aliphatic alcohols in phosphoserine and phosphothreonine are base-labile.
  • the aromatic phosphoester in phosphotyrosine is acid-labile and base-stable. Accordingly, subjecting the phosphotyrosine residues to basic aqueous solution in accordance with the method of the invention (discussed in detail below) does not effect a beta-elimination reaction, an essential step in practicing the method of the invention.
  • the methods of the invention specifically detect phosphoserine and phosphothreonine in an intact protein without interference from phosphotyrosine.
  • the methods of the invention involve: (1) subjecting the protein to conditions for effecting a beta-elimination reaction in the intact protein to convert the phosphorylated amino acid to a dehydroamino acid containing a double bond containing carbon atoms that are susceptible to attack by an addition reaction; (2) contacting the protein of step (1) with a reactant under conditions for adding the reactant to the carbon atom of the double bond of the dehydroamino acid to form an amino acid addition product in the intact protein; and (3) detecting the amino acid addition product in the intact protein, wherein the presence of the amino acid addition product is indicative of the presence of the phosphorylated amino acid in the intact protein.
  • the proteins that are analyzed according to the methods of the invention contain at least one cysteine and/or cystine residue. Accordingly, to avoid participation of the cysteine/cystine residues in side reactions (discussed below), typically the protein is subjected to conditions (prior to exposing the protein to conditions for effecting the beta-elimination reaction) to convert the cysteine(s)/cystine(s) to a product (referred to herein as a "cysteine product") which cannot undergo a beta-elimination reaction or otherwise interfere with the detection reactions disclosed herein.
  • the intact protein can be exposed to an oxidizing reagent (e.g., performic acid) to oxidize the cysteine(s)/cystine(s) to cysteic acid residue(s) or modified by alkylating the sulfhydryl group of the cysteine(s)/cystine(s) prior to performing the beta-elimination reaction.
  • an oxidizing reagent e.g., performic acid
  • conditions for effecting a beta-elimination reaction are well known to those of ordinary skill in the art.
  • relatively mild conditions for effecting a beta-elimination reaction are selected to prevent peptide bond hydrolysis. Accordingly, subjecting the protein to the selected beta-elimination conditions results in an intact protein in which phosphoserine and phosphothreonine are dephosphorylated to their respective dehydroamino acids.
  • the conditions for effecting a beta- elimination reaction involve placing the intact protein in a dilute basic aqueous solution (i.e., a solution having a pH between 10 and 14, inclusive) for between about 10 minutes and 12 hours. More preferably, the dilute basic aqueous solution has a pH between 1 1 and 13 and the intact protein is placed in basic solution for between about 0.5 and 6 hours.
  • the basic aqueous solution is a sodium hydroxide solution having a sodium hydroxide concentration ranging between 0.05 and 0.5 normal, more preferably, the basic solution is 0.10 N NaOH and the protein is placed in this basic solution for between 0.5 and 6.0 hours.
  • the protein is placed in the basic solution for between 0.5 and 3.0 hours.
  • aqueous solutions which can be used to practice the invention include sodium borate, potassium borate, potassium hydroxide, calcium hydroxide, barium hydroxide and 3-[cyclohexylamino]- 1 -propane-sulfonic acid (CAPS) buffers.
  • CAPS cyclohexylamino]- 1 -propane-sulfonic acid
  • a dilute basic buffer refers to any basic solution which can maintain a pH in the range of about 10 to 14 and further, which is not reactive with the dehydroamino acid that is formed by the beta-elimination reaction.
  • the basic aqueous solution further includes a catalytic amount of a beta-elimination catalyst.
  • a "beta-elimination catalyst” refers to a substance which increases the kinetic rate of the beta-elimination reaction.
  • a “catalytic amount” of the beta-elimination reaction catalyst is an amount which increases the kinetic rate of the reaction to an extent that is statistically significant.
  • Exemplary beta- elimination reaction catalysts include the group II metal ions. See, e.g., M. Byford, Biochem. J. 280:261-265 (1991).
  • the basic aqueous solution contains between 0.01 and 1.0 moles per liter of the group II metal ion beta-elimination reaction catalyst.
  • the beta-elimination catalyst is SrCl 2 or Ba(OH)-, which is present in the basic aqueous solution at a concentration ranging between 0.10 and 0.20 moles per liter.
  • the basic aqueous solution is 0.10 N NaOH containing 0.10 M SrCl 2 or Ba(OH) 2 .
  • other basic aqueous solutions can be substituted for the sodium hydroxide solution and other catalysts can be substituted for SrCU or Ba(OH) 2 without departing from the essence of the invention.
  • the beta-elimination reaction results in conversion of the phosphorylated amino acids to the corresponding dehydroamino acids, i.e., phosphoserine is converted to dehydroalanine and phosphothreonine is converted to 2-aminodehydroxybutyric acid.
  • the dehydroamino acid includes a double bond containing carbon atoms that are susceptible to attack by an addition reactant in an addition reaction.
  • an addition reactant refers to a molecule that is capable of forming a covalent bond with a carbon atom of the dehydroamino acid in an addition reaction.
  • nucleophilic addition J.
  • a nucleophilic addition is favored because of the electron- withdrawing effect of the carbonyl group present in the adjacent amide (peptide) bond which enhances nucleophilic addition and inhibits electrophilic addition (under mild basic conditions) by lowering the electron density of the dehydroamino acid double bond, i.e., the dehydroamino acid acts as a Michael substrate which is particularly susceptible to nucleophilic attack.
  • nucleophile in a nucleophilic addition reaction
  • nucleophilic addition product is referred to herein as a nucleophilic amino acid addition product.
  • nucleophile and “electrophile” have their common meanings. See, e.g., J. March, J., ibid.
  • electrophile such as a carbon atom of a carbon-carbon double bond
  • nucleophile such as an amine, a thiol or an alcohol
  • the nucleophilic addition reaction is performed using a molar excess of the nucleophile in order to drive the addition reaction to completion.
  • the molar ratio of nucleophile to intact protein is at least about 10 : 1, more preferably, the molar ratio of nucleophile to protein is between 10 5 :1 and lO 14 :! .
  • the invention embraces two types of nucleophile: (1) a monofunctional nucleophile which contains a detectable tag for determining the presence of the phosphorylated amino acid in the intact protein in a "one-step” reaction and (2) a bifunctional nucleophile which does not contain a detection reagent and which therefore must be further reacted with a detection reagent to determine the presence of the phosphorylated protein in a "two-step” reaction.
  • a monofunctional nucleophile is a nucleophile which contains a single functional group for covalent attachment to an amino acid residue in an intact protein.
  • a bifunctional nucleophile is a nucleophile which contains two functional groups for forming two distinct covalent bonds, i.e., the bifunctional nucleophile contains a first functional group for reacting with the carbon atom of the double bond of the dehydroamino acid and a second functional group for reacting with the detection reagent.
  • the nucleophile contains a first functional group ("X", a nucleophilic group such as a thiol or an amine) for reacting with the carbon atom of the double bond of the dehydroamino acid and a detectable tag ("D") for determining the presence of the phosphorylated amino acid in the intact protein.
  • the first functional group and the detection reagent are separated from one another by between zero to ten carbon atoms.
  • the number of carbon atoms in the chain separating the first functional group and the detection reagent is selected so that the nucleophile is soluble at a sufficient concentration to achieve the purposes of the reaction. Solubility of the chain in aqueous solution can be increased by increasing the proportion of hydroxyl groups present on the carbon chain.
  • the monofunctional nucleophile has the formula,
  • n 0 to 10, inclusive; Rl is H or OH; R2 is H or OH and wherein Rl and R2 can be the same or different from one another.
  • Rl and R2 optionally can embrace alkyl groups containing between one and ten carbon atoms, provided that the presence of the alkyl group (1) does not reduce the solubility of the nucleophile to a degree that would interfere with the efficiency or sensitivity of the reaction, (2) does not inhibit formation of a covalent bond between the first functional group and the dephosphorylated amino acid and (3) does not interfere with detection of the detectable tag.
  • detectable tag refers to a molecule that can be directly detected (i.e., the molecule itself can be detected) or indirectly detected (i.e., the molecule is detected by a further reaction).
  • Exemplary detectable tags include radioactive tags (e.g., 35 [S], 32 [P], l4 [C]), chromophores (e.g., Texas Red dye), fluorophores (e.g., fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC)), luminescent tags (e.g., aminobutylethylisoluminol), biotin, haptens (e.g., fluorescein, dinitrophenol) and enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta galactosidase).
  • radioactive tags e.g., 35 [S], 32 [P], l4 [C]
  • chromophores e.g., Texas Red dye
  • fluorophores e.g., fluorescein isothiocyanate (FITC), tetramethylrhodamine isothio
  • Exemplary procedures for detecting a biotin-, hapten- or enzyme-labeled dephosphorylated amino acid in an intact protein are discussed below. In general, such detection procedures are performed using routine procedures well known to those of skill in the art.
  • contacting the protein with the (detection reagent-containing) nucleophile under conditions for adding the nucleophile to the double bond of the dehydroamino acid yields a biotin-labeled intact protein, i.e., an intact protein in which the biotin moiety is covalently coupled to the dehydroamino acid.
  • detecting the nucleophilic addition amino acid product in the intact protein involves detecting the biotin moiety in the intact protein.
  • a biotin/streptavidin detection system involves coupling biotin to a molecule in a process referred to as biotinylation to form a biotinylated molecule (e.g., H 2 N-CH 2 -CH 2 -Biotin) and coupling an indicator molecule such as an enzyme or a fluorochrome to streptavidin or avidin for detecting the presence of the biotinylated molecule.
  • biotinylated molecule e.g., H 2 N-CH 2 -CH 2 -Biotin
  • an indicator molecule such as an enzyme or a fluorochrome
  • streptavidin or avidin for detecting the presence of the biotinylated molecule.
  • assays e.g.. ELISA, immunoblotting, immunohistochemical staining and fluorescent cell sorting
  • biotin/avidin labeling system immunoassay kits and reagents are commercially available (e.g., ECL kits and reagents available from Amersham Corp., Arlington Heights, IL; Sigma Chem. Co., St. Louis, MO) and can be used without undue experimentation to detect the biotin-labeled dehydroamino acid in the intact protein.
  • the phosphorylated amino acids in the intact protein are detected as described in Example 1.
  • This detection method employs a commercially available chemiluminescence kit (an ECL system, Amersham) containing a streptavidin- horseradish peroxidase (HRP) conjugate as the detection reagent in a chemiluminescence assay.
  • the enzyme e.g., peroxidase
  • An exemplary ECL assay is provided in Example 1. Additional ECL protocols and references describing the commercially available chemiluminescence assay are provided in the Amersham 1994 catalog. Other non- biotin ECL detection protocols are discussed in more detail below.
  • avidin conjugates that are useful for detecting a biotin-labeled amino acid include avidin-alkaline phosphatase, avidin-beta galactosidase, avidin-fluorescein isothiocyanate (FITC), avidin-peroxidase, avidin-tetramethylrhodamine isothiocyanate (TRITC), avidin-gold (i.e., avidin adsorbed to colloidal gold), streptavidin-FITC, streptavidin-beta- galactosidase, streptavidin-gold, streptavidin-peroxidase, and streptavidin-Texas Red. See, e.g., Sigma Chemical Co. Catalog, ibid.
  • an enhanced chemiluminescent reaction for detection of phosphorylated amino acids in an intact protein also can be performed using a hapten-based detection system.
  • the reactant e.g., a monofunctional nucleophile
  • contains a hapten e.g., fluorescein
  • the addition reaction e.g., nucleophilic addition
  • yields a hapten-labeled amino acid addition product e.g., the dehydroamino acid is labeled with the hapten.
  • Detection of the hapten-labeled addition product in the intact protein is performed by reacting the intact protein with an antibody conjugate.
  • the antibody conjugate contains an antibody which specifically recognizes the hapten and an enzyme which catalyzes the above-described chemiluminescent reaction (e.g.. luminol is the substrate).
  • the antibody conjugate contains an anti-fluorescein antibody covalently coupled to horse radish peroxidase.
  • the reagents for performing the hapten-based detection of the dehydroamino acids in the intact protein are commercially available from Amersham Corp., Arlington Heights, IL.
  • an 35 [S]-labeled nucleophile e.g., H 2 N-CH 2 -CH 2 - 35 [S]H 2 , H 2 S-CH 2 -CH 2 - 35 [S]H 2
  • an 35 [S]-labeled nucleophile e.g., H 2 N-CH 2 -CH 2 - 35 [S]H 2 , H 2 S-CH 2 -CH 2 - 35 [S]H 2
  • the dehydroamino acid-containing protein can be reacted with 35 [S]-sulfite to directly introduce a radioactive tag to the intact protein at the location of the dehydroamino acid double bond.
  • detectable tags as well as the selection of monofunctional nucleophiles that contain the detectable tags, which can be used in accordance with the methods of the invention can be made using routine experimentation, for example, by substitution a putative detectable tag for the detectable tag of Example 1 and determining whether the putative detectable tag provides a sensitivity and specificity that is at least comparable to that of a detectable tag which is known to be useful for detecting phosphorylated amino acids in an intact protein.
  • a bifunctional nucleophile can be used to practice the methods of the invention in accordance with a "two-step" reaction, i.e., the bifunctional nucleophile serves as a linker to covalently attach a detection reagent containing a detectable tag to the dehydroamino acid in the intact protein.
  • the nucleophile contains a first functional group ("X", a nucleophilic group such as a thiol or an amine) for reacting with the carbon atom of the double bond of the dehydroamino acid and a second functional group (“Y”) for reacting with a detection reagent.
  • the first and the second functional groups are separated from one another by between zero to ten carbon atoms, i.e., the bifunctional nucleophile has the formula,
  • n 0 to 10, inclusive; Rl is H or OH; R2 is H or OH and wherein Rl and R2 can be the same or different from one another.
  • the same limitations apply to the carbon chain between the X and Y groups and of the Rl and R2 groups as discussed above in regard to the monofunctional nucleophile.
  • nucleophiles that can be used in accordance with the "two-step" embodiment include H 2 N-CH 2 -CH 2 -NH 2 (1,2- diaminoethane), H,N-CH 2 -CH 2 -CH 2 -NH 2 (1,3- diaminopropane), diaminopropanol, H 2 S-CH 2 -CH 2 -SH 2 (dithioethane) and H 2 S-CH 2 - CH 2 -CH 2 -SH 2 (1,3-propane dithiol; also referred to herein as dithiopropane, DTP), dithiothreitol and dithioerythritol.
  • DTP dithiopropane
  • Alternative bifunctional nucleophiles can be selected from putative bifunctional nucleophiles in an analogous manner to that described above for the selection of monofunctional nucleophiles and detectable tags, namely, by substituting a putative bifunctional nucleophile for a bifunctional nucleophile that is known to be useful for detecting a phosphorylated amino acid in an intact protein and determining whether the putative bifunctional nucleophile provides a sensitivity and specificity that is at least comparable to that obtained using the reference bifunctional nucleophile.
  • Reaction of the intact protein with the bifunctional nucleophile results in the introduction into the protein of a new functional group ("Y") to which a detection reagent can be covalently coupled. Accordingly, it is desirable to remove as much as possible (e.g., by evaporation, extraction) any uncoupled bifunctional nucleophile from the reaction mixture prior to contacting the nucleophile-modified protein with the detection reagent.
  • Y new functional group
  • Practicing the invention in which a bifunctional nucleophile is used for the addition reaction involves: (1) subjecting the intact protein to conditions for effecting a beta-elimination reaction to convert the phosphorylated amino acid to a dehydroamino acid, (2) contacting the protein of step (1) with the bifunctional nucleophile under conditions for adding the nucleophile to a carbon atom of the double bond to form a nucleophilic addition product in the intact protein. (3) contacting the protein of step (2) with the detection reagent under conditions for the detection reagent to covalently bond to the second functional group of the bifunctional nucleophile, and (4) detecting the nucleophilic addition product in the intact protein as described above.
  • bifunctional nucleophile contains a second functional group that is an amine group and the detection reagent is an amino group labeling reagent.
  • exemplary amino group labeling reagents are well known to those of skill in the art of protein labeling and include biotin-succinimidyl ester, the Bolton and Hunter reagent and its derivatives (e.g., N-succinimidyl 3-(4-hydroxy, 5-[ ,25 -I]-iodophenyl) propionate and the corresponding di-iodo derivative), as well as [ 35 S]-sulfur labeling reagents which label free amino groups in the intact protein (e.g., [ 35 S] sulfur labeling reagent product no.
  • Exemplary thiol labeling reagents include N-ethyl[2,3- l4 C]-maleimide, biotin-maleimide, [ 14 C]-methyl iodide, [ 14 C]- succinic anhydride, iodoacetylbiotin and N-hydroxysuccinimido-biotin (NHS-biotin).
  • the foregoing labeling reagents are known to label free amine or thiol groups in proteins. Accordingly, to prevent unwanted side reactions between the labeling reagents and the N- terminal or lysine side chain amine groups, it is essential that the intact protein be treated to block free amine groups (e.g., by alkylating the free N-terminal amine group and lysine side chain amine groups) prior to contacting the protein with the bifunctional nucleophile.
  • proteins which contain cysteine or cystine residues should be subjected to conditions to convert the cysteine thiol groups to a form (referred to herein as a "cysteine product") which cannot undergo a beta- elimination reaction and thus, which cannot be labeled with the above-noted thiol labeling reagents.
  • cysteine product a form which cannot undergo a beta- elimination reaction and thus, which cannot be labeled with the above-noted thiol labeling reagents.
  • this is accomplished by exposing the intact protein to an oxidizing agent under conditions to oxidize cysteine to cysteic acid or by alkylating the sulfhydryl groups of the cysteine residues using routine procedures.
  • phosphorylated amino acids are detected by oxidizing the double bond of the derivatized dehydroamino acid with KMnO 4 to yield a cis diol which then can be oxidized with periodate to a highly reactive aldehyde.
  • the resultant aldehyde is allowed to react with biotin-hydrazide to yield a biotin-labeled amino acid in the intact protein at the location of the dehydroamino acid.
  • kits for detecting the presence of a phosphoserine and/or phosphothreonine in an intact protein contain an amount of nucleophile sufficient for effecting the above-described beta-elimination, addition, and detection reactions and instructions for using the nucleophile in accordance with the methods of the invention.
  • kits further include a negative (e.g., bovine serum albumin, "BSA") or positive (e.g., casein) protein control for evaluating the sensitivity and accuracy of the beta-elimination, nucleophile and/or detection reagents for detecting the presence of phosphoserine and/or phosphothreonine in an unknown protein sample.
  • BSA bovine serum albumin
  • casein casein
  • ECL enhanced chemiluminescence kit
  • Amersham Amersham (Arlington Heights, IL).
  • Casein kinase, protein kinase A, and pp60 src were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
  • Radiochemicals were purchased from Amersham (Arlington Heights. IL). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO).
  • cysteines and cystines are removed by oxidation (part 1 , below) or reduction/alkylation (part 2, below) to a form (referred to herein as a "cysteine product") which cannot undergo a beta-elimination reaction and/or which cannot be subject to attack in an addition reaction.
  • the protein to be subjected to the detection procedure can be present in an unpurified extract, isolated by means of immunoprecipitation, chromatography or other standard methods well known in the art, or prepared by standard in vitro methods well known in the art (Molecular Cloning: a Laborator y Manual. Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
  • Performic acid is the preferred oxidation reagent to efficiently oxidize cysteine or cystine residues in intact proteins.
  • Performic acid is prepared by combining 1 part 30% H 2 O 2 and 9 parts 70% formic acid and allowing the mixture to incubate for about one hour at room temperature.
  • Oxidation of cysteines and cystines is accomplished as follows. One part performic acid is added to 20 parts protein solution (about 1 mg/ml) and incubated for two hours on ice. One hundred parts cold (4°C) 10 mM ammonium acetate solution and 0.2 parts beta-mercaptoethanol are added to the reaction mixture; the mixture is lyophilized to dryness using, for example, a Speed- Vac rotoevaporator (Savant Instruments, Inc., Farmingdale, NY). The modified protein is dissolved in 100 microliters 10 mM ammonium acetate and relyophilized to completely remove formic acid. Failure to completely remove formic acid may result in a pH which is less than the optimum pH for effecting the beta-elimination reaction and/or the addition reaction, thereby reducing the efficiencies of these reactions.
  • the oxidation reaction time and performic acid concentration are optimized by assessing the degree of cysteine/cystine oxidation and the amount of sample degradation as a function of reaction time, temperature and performic acid concentration.
  • Assessment of cysteine/cystine oxidation and protein degradation is made by comparing the banding patterns of performic acid treated and untreated (control) protein samples on SDS-PAGE.
  • BSA which contains cysteine residues but which does not contain phosphoserine or phosphothreonine residues, can be used as a control protein to assess the extent of cysteine oxidation to cysteic acid.
  • Elimination of cysteines and cystines can be accomplished by reduction and alkylation reactions using NEM and DTT. This alternative procedure degrades the protein sample to a lesser extent than the performic acid procedure. However, this reaction should be optimized to ensure complete removal of cysteines and cystines in the intact protein. Conditions for performing the reduction/alkylation of intact proteins are well known to those of ordinary skill in the art. An exemplary protocol for the reduction/alkylation removal of cysteine/cystine residues is presented below.
  • the protein of interest was isolated by a standard immunoprecipitation using Protein A sepharose beads to which was coupled an antibody that was specifically reactive to the protein of interest.
  • the protein was eluted from the sepharose beads by boiling 3 minutes in 1% SDS. 25 mM HEPES pH 8.0. Thereafter, the eluted protein was incubated in 1% SDS, 2 mM DTT, 0.5 mM EDTA in 25 mM HEPES pH 8.0 for 30 minutes at 45 °C to denature the protein and reduce the cystine residues. 10 mM N-ethyl maleimide was added to the denatured protein and this mixture was incubated for an additional 60 minutes to alkylate the cysteine residues. Dephosphorylation was performed using DTP/NaOH/SrCl 2 procedure described herein.
  • Beta-elimination reaction Alkaline dephosphorylation of phosphoserine and phosphothreonine residues is accomplished by beta-elimination of the phosphate moieties from these amino acid residues.
  • a 10% stock solution of DTP in DMSO is freshly prepared.
  • a protein sample e.g., the lyophilized oxidized protein of Part A above
  • a protein sample is dissolved in a volume of 0.1% SDS and the following reactants (concentration is the final concentration) are added to the protein solution: 0.1N NaOH, 0. IN SrCl 2 and 1 % DTP.
  • the mixture is incubated at 44 °C for about 30 minutes to yield an intact protein containing the dehydroamino acids corresponding to the former phosphoserine and phosphothreonine residues.
  • the resulting beta-dehydroalanine residues in the intact protein are subjected to an addition reaction (e.g., a nucleophilic addition reaction) to introduce a detectable tag into the former phosphoserine or phosphothreonine residues.
  • an addition reaction e.g., a nucleophilic addition reaction
  • the first step of this "two-step" reaction is performed by adding an equal volume of 3% DTP in 1.0M CAPS buffer, pH 1 1.0 to the above- described protein solution reaction mixture and incubating this reaction mixture for about 3 hours at 44 °C. Thereafter, the reaction mixture is neutralized to pH 7-8 with glacial acetic acid and EDTA is added to a concentration of 0.2M to chelate the SrCl 2 .
  • the reaction mixture is extracted three times with an equal volume of diethyl ether, saving and pooling the lower aqueous layer after each extraction. Complete removal of the nucleophile enhances the efficiency of subsequent labeling reactions.
  • a commercially available chemiluminescence kit (ECL, Amersham) that employs streptavidin conjugated horseradish peroxidase as a luminol substrate converting enzyme is used to visualize the biotin-labeled protein.
  • Casein contains between five and ten phosphate groups per polypeptide chain. Reportedly, these phosphate groups are present as phosphoserine residues or phosphothreonine residues and are not present as phosphotyrosine residues.
  • phosphate groups are present as phosphoserine residues or phosphothreonine residues and are not present as phosphotyrosine residues.
  • Protein controls include serine- (or threonine-) containing proteins which are either phosphorylated using an appropriate kinase and ATP using manufacturer's recommended procedure, mock phosphorylated using the appropriate kinase without ATP, or not treated.
  • BSA was used as a negative control. BSA does not contain phosphoserine residues but does contain seventeen cystine residues. Accordingly, BSA also served as a control for assessing completion of the performic acid oxidation reaction.
  • the lyophilized sample was dissolved in 100 ul 0.1% SDS; 20 ul 1.0N NaOH, 20 ul 1.ON SrCl 2 , 20 ul 10% DTP in DMSO and 40 ul water were added to initiate the beta-elimination (dephosphorylation) reaction and the reaction was allowed to proceed for 30 minutes at 44oC.
  • the nucleophilic addition of DTP to the dehydroamino acids i.e., dephosphorylated serine and threonine residues
  • Glacial acetic acid was added to neutralize the pH of the mixture, followed by 500 ul of 0.4M EDTA to chelate the Sr 2* ions.
  • the mixture was extracted thrice with 1.0 ml of diethyl ether.
  • the gel was transferred to nitrocellulose (S&S, Keene, N.H.) using an electroblotting device (Bio-Rad, Richmond, CA) according to standard practice.
  • the Western blot was processed according to the manufacturer's instructions for chemiluminescent detection of proteins using an ECL kit (Amersham, Arlington Heights, IL). 250 nanograms of casein were easily detectable with a ten second exposure of the Western blot to X-ray film.
  • Example 2 Sensitivity and Specificity of the Labeling and Detection Procedure
  • a titration experiment is described herein to determine the sensitivity of the above- described methods for detecting phosphorylated serines and threonines in intact proteins. Casein is used as a positive control.
  • Other serine-and/or threonine-containing proteins can be phosphorylated on serines and threonines using the appropriate protein kinase according to the manufacturer's recommendations. For example, ovalbumin is phosphorylated on serines and threonines using protein kinase A according to the manufacturer's instructions.
  • pp60 src kinase is autophosphorylated on tyrosine residues according to the manufacturer's instructions.
  • Each of the phosphorylated proteins is mixed with its corresponding unphosphorylated protein to constitute 100%, 50%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or 0% of 1.0 ug total protein for use in the beta-elimination (dephosphorylation) and addition/detection reactions.
  • Fifty micrograms of phosphorylated pp60 src (a negative control which contains phosphorylated tyrosine residues) is mixed with unphosphorylated bovine serum albumin added as a carrier protein.
  • the nucleophile dithiothreitol (DTT) is substituted for DTP in the method of Example 1. Because DTT is more soluble in aqueous solution than DTP, higher effective concentrations of DTT can be attained in the reaction mixture. In the beta-elimination (dephosphorylation) and addition reactions, DTT is added to the reaction as a 1-10% solution in water, preferably as a 3% solution in water. Phosphorylated casein is detectable with similar sensitivity as when labeled using DTP.
  • Example 4 Nucleophilic Attack with 1.3-Diaminopropane
  • Primary amine-containing nucleophiles also can be used in accordance with the methods of the invention to detect phosphoserine and/or phosphothreonine residues in an intact protein.
  • the detection reagent which is reactive with one of the primary amine groups of the nucleophile
  • the protein sample should be treated to block free amine groups (e.g., by alkylation) prior to performing the method of Example 1.
  • the protein samples be subjected to conditions to block lysine, cysteine, and cystine amino acid side chains to prevent their respective amine and sulfhydryl groups from participating in the beta-elimination, addition and labeling reactions of the invention.
  • this is accomplished by alkylating the cysteine/cystine sulfhydryl groups and/or the lysine primary amine according to standard procedures.
  • modification of cysteine/cystine and/or lysine residues prior to conducting the beta-elimination is recommended to preclude potential side reactions which could interfere with the determination of phosphoserine and phosphothreonine residues in the intact protein.
  • DAP 1,3-diaminopropane
  • DTP 1,3-diaminopropane
  • DAP is substituted for DTP in the method of Example 1.
  • DAP is added to the reaction mixture as a 1-10% solution in DMSO, preferably a 3% solution in DMSO.
  • the results of the labeling procedure indicate that DAP can be used as a bifunctional nucleophile for the detection of phosphorylated serine and threonine residues in an intact protein.
  • Example 5 Nucleophilic Attack Following Oxidation of Beta-elimination Products An alternative addition reaction for introducing a detectable tag to a dehydroamino acid is described herein. After beta-elimination under alkaline conditions as described in Example 1 , a solution of KMnO 4 of sufficient molar strength to oxidize a beta-dehydroalanine double bond to a cis-diol configuration is added to the beta-elimination reaction mixture. Periodic acid is added in a sufficient molar amount to oxidize the cis-diol to an aldehyde compound.
  • the resulting aldehyde is a reactive species that is subject to nucleophilic attack by, for example, a monofunctional nucleophile that contains a detectable tag.
  • biotin-hydrazide is added to the periodic acid- treated protein in a molar amount sufficient to stoichiometrically label the reactive aldehyde species in a Wolff-Kishner reaction in which the biotin moiety is covalently attached to a carbon atom of the dehydroamino acid in the intact protein.
  • a standard Western blot/ECL detection protocol is performed to detect the biotin moiety in the intact protein at a level of sensitivity comparable to that observed in Example 2.
  • Example 6 Radioactive Labeling with 32 P-Phosphoric Acid
  • An in vitro method of radiolabeling serines and threonines phosphorylated in vivo is described in this example.
  • One of the difficulties of in vivo 3 P radiolabeling of proteins to determine the phosphorylation state is the relatively large amounts of radioactive materials required. Since the reaction is highly inefficient in vivo, an large excess of radioactive material is used, resulting in contamination of experimental apparatus and creation of large quantities of hazardous waste.
  • the method described in Example 1 is used to in vitro label the phosphorylated serine and threonine residues.
  • cysteines/cystines are removed (e.g., oxidized or subjected to reduction/alkylation) and phosphorylated serines and threonines converted to dehydroamino acids by alkaline beta-elimination.
  • the resulting double bond is attacked by the addition of a sufficient molar quantity of 32 P-phosphoric acid to the beta-elimination reaction mixture.
  • the addition of the radioactive phosphorus moiety labels the site of in vivo or in vitro phosphorylation in the intact protein. The presence of radiolabel is determined by standard methods well known in the art, such as SDS-PAGE followed by autoradiography or phosphorimager detection, or scintillation counting.
  • Example 7 Radioactive Labeling with 35 S-Sodium Sulfite
  • An in vitro method of radiolabeling serines and threonines phosphorylated in vivo is described in this example.
  • One of the difficulties of in vivo 35 S radiolabeling of proteins is that it is difficult to determine the phosphorylation state of the protein. Since 35 S radiolabeling requires uptake of 35 S labeled methionine and/or cysteine, it is highly inefficient in vivo and large excess of radioactive material must be used, resulting in contamination of experimental apparatus and creation of large quantities of hazardous waste. Furthermore, the reaction is not specific for phosphorylated proteins. Using the method described in Example 1, one can effect in vitro labeling of phosphorylated serine and threonine residues.
  • cysteines/cystines are removed (e.g., oxidized or subjected to reduction/alkylation) and phosphorylated serines and threonines converted to dehydroamino acids by alkaline beta-elimination.
  • the resulting double bond is attacked by the addition of a sufficient molar quantity of 35 S-sodium sulfite to the reaction.
  • the addition of the radioactive sulfite moiety labels the site of in vivo or in vitro phosphorylation in the intact protein. The presence of radiolabel is determined by standard methods well known in the art, such as SDS-PAGE followed by autoradiography or phosphorimager detection, or scintillation counting.

Landscapes

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

Abstract

Procédés et compositions permettant de déterminer la présence d'un acide aminé phosphorylé dans une protéine intacte. Les procédés de cette invention consistent à déphosphoryler l'acide aminé phosphorylé pour former un acide déshydroaminé puis à coupler de manière covalente une étiquette détectable sur l'acide aminé déphosphorylé présent dans la protéine intacte. Ces procédés et compositions sont utiles pour déterminer la présence de phosphosérine et de phosphothréonine dans des protéines intactes.
PCT/US1995/016421 1994-12-16 1995-12-15 Procede de detection d'un acide amine phosphoryle dans une proteine intacte Ceased WO1996018899A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35749894A 1994-12-16 1994-12-16
US08/357,498 1994-12-16

Publications (1)

Publication Number Publication Date
WO1996018899A1 true WO1996018899A1 (fr) 1996-06-20

Family

ID=23405875

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/016421 Ceased WO1996018899A1 (fr) 1994-12-16 1995-12-15 Procede de detection d'un acide amine phosphoryle dans une proteine intacte

Country Status (1)

Country Link
WO (1) WO1996018899A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100560127B1 (ko) 2004-09-04 2006-03-13 한국기초과학지원연구원 인산화 단백질 분석용 겔-내 표지화 및 겔-내 단리 방법및 이를 이용한 단백질의 인산화 위치 동정 방법
WO2022103990A1 (fr) * 2020-11-12 2022-05-19 Phosfish Llc Procédés de modification de résidus de tyrosine phosphorylés ou sulfatés de polypeptides

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001771A1 (fr) * 1992-07-14 1994-01-20 Patchornik, Zipora Reactifs standard universels, procede de preparation et utilisation de ces reactifs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001771A1 (fr) * 1992-07-14 1994-01-20 Patchornik, Zipora Reactifs standard universels, procede de preparation et utilisation de ces reactifs

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, Vol. 243, No. 2, issued December 1985, R. FUJISAWA et al., "In Vivo Cleavage of Dentin Phosphophoryn Following Beta Elimination of Its Phosphoserine Residues", pages 619-623. *
BIOCHEMICAL JOURNAL, Vol. 275, issued 1991, J.W. POLLI et al., "Preparation, Characterization and Biological Properties of Biotinylated Derivatives of Calmodulin", pages 733-743. *
BIOCHEMICAL JOURNAL, Vol. 280, issued 1991, M.F. BYFORD, "Rapid and Selective Modification of Phosphoserine Residues Catalysed by Ba2+ Ions for Their Detection During Peptide Microsequencing", pages 261-265. *
BIOCHEMISTRY, Vol. 11, No. 10, issued 1972, D.L. SIMPSON et al., "Beta Elimination and Sulfite Addition as a Means of Localization and Identification of Substituted Seryl and Threonyl Residues in Proteins and Proteoglycans", pages 1849-1856. *
BIOCHEMISTRY, Vol. 27, issued 1988, L. VARTICOVSKI et al., "Location of Sites in Human Lipocortin I That are Phosphorylated by Protein Tyrosine Kinases and Protein Kinases A and C", pages 3682-3690. *
FEBS LETTERS, Vol. 215, No. 1, issued May 1987, C.F.B. HOLMS, "A New Method for the Selective Isolation of Phosphoserine-Containing Peptides", pages 21-24. *
FEBS LETTERS, Vol. 231, No. 2, issued April 1988, T.G. HASTINGS et al., "Beta-Elimination of Phosphate and Subsequent Addition of Pyridoxamine as a Method for Identifying and Sequencing Peptides Containing Phosphoseryl Residues", pages 431-436. *
R.L. SHRINER et al., "The Systematic Identification of Organic Compounds", Published 1980, by JOHN WILEY & SONS, INC. (N.Y.), pages 192-195. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100560127B1 (ko) 2004-09-04 2006-03-13 한국기초과학지원연구원 인산화 단백질 분석용 겔-내 표지화 및 겔-내 단리 방법및 이를 이용한 단백질의 인산화 위치 동정 방법
WO2022103990A1 (fr) * 2020-11-12 2022-05-19 Phosfish Llc Procédés de modification de résidus de tyrosine phosphorylés ou sulfatés de polypeptides

Similar Documents

Publication Publication Date Title
Mendoza et al. Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry
King et al. The Mr 78,000 intermediate chain of Chlamydomonas outer arm dynein interacts with alpha-tubulin in situ
US5763198A (en) Screening assays for compounds
Bayer et al. 3-(N-Maleimido-propionyl) biocytin: a versatile thiol-specific biotinylating reagent
JP2870704B2 (ja) ホモシステインアッセイ
EP0132948B1 (fr) Méthode biochimique de détection et kit pour son utilisation
US5132226A (en) Method for the determination of maleimide groups
EP1261621B1 (fr) Bioanalyses realisees au cours de la phosphorylation
Suwal et al. Phosphorylation-dependent kinase–substrate cross-linking
CA2661995C (fr) Conjugues de proteines antigeniques et procede de preparation associe
JP2005510234A (ja) ポリペプチドの定量
Barth et al. Evaluation of NHS-Acetate and DEPC labelling for determination of solvent accessible amino acid residues in protein complexes
US7090993B2 (en) Method of assaying pyrrole-containing biological compounds
US5948630A (en) Method and composition for reducing the effects of endogenous alkaline phosphatase
WO2008022355A2 (fr) Procédés et réactifs de détection de groupes phosphomonoester
WO1996018899A1 (fr) Procede de detection d'un acide amine phosphoryle dans une proteine intacte
WO2008033992A2 (fr) Essai de criblage à haut débit de modulateurs d'enzymes modifiant les histones
JPS62502633A (ja) 体液の酵素検定
Klingenberg et al. Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 2.[5-(Dimethylamino)-1-naphthoyl] adenine nucleotides as probes for the transition between c and m states of the ADP/ATP carrier
Al-Bassam et al. Double-antibody enzyme immunoassay for nortriptyline.
US6159699A (en) Enzyme linked chemiluminescent assay
JP4455688B2 (ja) 分析物遊離段階を可能にするアッセイ用表面
Sauer et al. A comparison of alkaline phosphatase, β-galactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtitre plate enzymeimmunoassay
JP4437003B2 (ja) Ｅｖｈ１ドメインまたはｅｖｈ１ドメインを有するタンパク質とｅｖｈ１結合ドメインまたはｅｖｈ１結合ドメインを有するタンパク質との相互作用を調節する化合物をスクリーニングする方法およびその相互作用を検出する方法
JPH07198721A (ja) 免疫測定用緩衝液

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA