Fluorescent Affinity Tag to Enhance Phosphoprotein Detection and Characterization

Abstract

The invention relates to target-associative tags incorporating cysteamine as the target-associative moiety. The invention further relates to a method for producing a target-associative tag by addition of cysteamine as the target-associative moiety to another molecule or entity having a property or properties useful in discriminating or selecting between members of a set, where such properties could include, for example, fluorescence, mass, affinity, reactivity, size, absorbance, magnetism, subatomic spin characteristics, or an ability to associate specifically or preferentially with certain structures. The invention further relates to a method for analyzing, identifying, or purifying phosphorylated proteins or phosphorylated protein fragments using a tag having the properties of both fluorescence and affinity.

1 - 24 . (canceled)  
     
     
         25 . A composition of matter comprising: 
 a) an isolated compound of the formula:                          b) an isolated compound comprising a cysteamine moiety directly attached to a discriminating moiety selected from fluorophores, chromophores, reactive groups, magnetic particles, gold particles, isotopic mass labels, or other discriminating moieties;    c) an isolated compound comprising a cysteamine moiety directly attached to a discriminating moiety selected from rhodamine, fluorescein, coumarin, eosin, erythrosin, lucifier yellow, malachite green, or oregon green;    d) a composition comprising a carrier and a compound comprising a cysteamine moiety directly attached to a discriminating moiety selected from fluorophores, chromophores, reactive groups, magnetic particles, gold particles, isotopic mass labels, or other discriminating moieties;    e) a composition comprising a carrier and a compound directly attached to a discriminating moiety selected from fluorophores, chromophores, reactive groups, magnetic particles, gold particles, isotopic mass labels, or other discriminating moieties, wherein said compound is                          f) a nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide attached, via a phosphate group, to a compound of the formula:                          g) a nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide attached, via a phosphate group, to a compound comprising a cysteamine moiety directly attached to a discriminating moiety selected from fluorophores, chromophores, reactive groups, magnetic particles, gold particles, isotopic mass labels, or other discriminating moieties;    h) a nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide attached, via a phosphate group, to a compound comprising a cysteamine moiety directly attached to a discriminating moiety selected from rhodamine, fluorescein, coumarin, eosin, erythrosin, lucifier yellow, malachite green, or oregon green;    i) a composition comprising a carrier and a nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide attached, via a phosphate group, to a compound comprising a cysteamine moiety directly attached to a discriminating moiety selected from fluorophores, chromophores, reactive groups, magnetic particles, gold particles, isotopic mass labels, or other discriminating moieties; or    j) a composition comprising a carrier and a nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide attached, via a phosphate group, to a compound directly attached to a discriminating moiety selected from fluorophores, chromophores, reactive groups, magnetic particles, gold particles, isotopic mass labels, or other discriminating moieties, wherein said compound is                          
     
     
         26 . A method of labeling a phosphate containing nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide comprising contacting a phosphate containing nucleotide, nucleic acid, polynucleotide, protein, peptide, or polypeptide with a tag comprising both (1) a fluorophore, chromophore, magnetic particle, gold particle, isotopic mass label, or other discriminating moiety and (2) a moiety suitable for affinity binding under conditions that allow for the incorporation of said tag into said nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide at a phosphate group.  
     
     
         27 . The method of  claim 26 , wherein said tag provides for at least one or both of the following: (1) detecting said labeled phosphate containing nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide by fluorescence; or (2) isolating said phosphate containing nucleotide, nucleic acid, polynucleotide, protein, peptide, or polypeptide by an affinity interaction.  
     
     
         28 . The method according to  claim 26 , wherein said tag has the formula:  
       
         
           
           
               
               
           
         
       
     
     
         29 . The method according to  claim 26 , wherein said moiety is cysteamine.  
     
     
         30 . The method according to  claim 26 , wherein said tag replaces at least one phosphate group in said phosphate containing nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide.  
     
     
         31 . A method of attaching a molecule to a phosphate containing (phosphorylated) nucleotide, nucleic acid, amino acid, protein, peptide, or polypeptide comprising reacting a molecule with cysteamine or with a cysteamine-containing compound to give derivatized cysteamine and reacting said derivatized cysteamine with said phosphate containing nucleotide, nucleic acid, polynucleotide, amino acid, protein, peptide, or polypeptide.  
     
     
         32 . The method of  claim 31 , wherein said molecule comprises a fluorophore.  
     
     
         33 . The method of  claim 31 , wherein said molecule comprises a moiety suitable for affinity binding.  
     
     
         34 . The method of  claim 31 , wherein said reacting of said derivatized cysteamine with said protein results in the formation of a covalent bond between the cysteaminyl sulfur atom and said protein.  
     
     
         35 . A method of mapping phosphorylation sites in proteins, peptides, or polypeptides comprising replacement of one or more phosphate group in a protein, peptide, or polypeptide with an affinity tag and mapping said phosphorylation sites and performing mass spectroscopy to map said phosphorylation sites.  
     
     
         36 . The method according to  claim 35 , wherein said affinity tag is a fluorescent affinity tag (FAT).  
     
     
         37 . The method according to  claim 36 , wherein said fluorescent affinity tag is  
       
         
           
           
               
               
           
         
       
     
     
         38 . A method of labeling a phorylated compound comprising contacting a phosphorylated compound with a compound comprising a cysteamine moiety directly attached to a discriminating moiety selected from fluorophores, chromophores, reactive groups, magnetic particles, gold particles, isotopic mass labels, or other discriminating moieties.  
     
     
         39 . The method according to  claim 38 , wherein said phosphorylated compound is a nucleotide, nucleic acid, polynucleotide, amino acid, polypeptide, peptide or protein.  
     
     
         40 . The method according to  claim 28 , further comprising the purification of said labeled compound.  
     
     
         41 . The method according to  claim 39 , further comprising the purification of said labeled compound.