Releasable nonvolatile mass-label molecules

Abstract

Releasable tag reagents for use in the detection and analysis of target molecules, particular in mass spectrometric analyses are provided. Also provided are methods of detection that employ releasable tag reagents.

What is claimed is:  
     
         1 . A method for multiplexing the detection of a target molecule comprising: 
 (a) obtaining a plurality of probes, each probe comprising a release tag compound comprising Rx, Re and M wherein: 
 Rx is a reactive group;  
 Re is a release group; and  
 M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry;  
   (b) contacting the target molecule with the plurality of probes to produce probe:target molecule complexes, wherein the target molecule is attached to the reactive group of the probe;    (c) releasing the mass labels from the probe:target molecule complexes to produce released mass labels; and    (d) determining the mass of the released mass labels by mass spectrometry, wherein each reactive group in a probe:target molecule complex is associated with a unique set of mass labels.    
     
     
         2 . The method of  claim 1 , wherein a plurality of target molecules is contacted with the plurality of probes.  
     
     
         3 . The method of  claim 1 , wherein the set of mass labels are attached to the same probe.  
     
     
         4 . The method of  claim 1 , wherein the set of mass labels are attached to different probes.  
     
     
         5 . The method of  claim 1 , wherein the target molecule is immobilized onto a solid support.  
     
     
         6 . The method of  claim 4 , wherein a plurality of target molecules are immobilized onto the solid support at spaced locations.  
     
     
         7 . The method of  claim 1 , wherein the target molecule is selected from the group consisting essentially of a polynucleotide, an antigen, a ligand, a polypeptide, a carbohydrate, and a lipid.  
     
     
         8 . The method of  claim 7 , wherein the target molecule comprises a polynucleotide.  
     
     
         9 . The method of  claim 7 , wherein the target molecule comprises a polypeptide.  
     
     
         10 . The method of  claim 1 , wherein the reactive group comprises a biomolecule capable of specific molecular recognition.  
     
     
         11 . The method of  claim 10 , wherein the reactive group comprises a polypeptide.  
     
     
         12 . The method of  claim 10 , wherein the polypeptide is selected from the group consisting essentially of an antibody, an enzyme, a receptor, a regulatory protein, a nucleic-acid-binding protein, a hormone, and a protein product of a display methods.  
     
     
         13 . The method of  claim 12 , wherein the polypeptide comprises an antibody.  
     
     
         14 . The method of  claim 12 , wherein the polypeptide comprises a product of a phage display method or a bacterial display method.  
     
     
         15 . The method of  claim 10 , wherein the reactive group comprises a polynucleotide or an oligonucleotide.  
     
     
         16 . The method of  claim 1 , wherein the unique set of mass labels comprises a mass label that indicates the presence of a specified component within the reactive group.  
     
     
         17 . The method of  claim 16 , wherein the mass label indicates the presence of the specified component at a specified location within the reactive group.  
     
     
         18 . The method of  claim 17 , wherein a reactive group comprising n specified components is associated with a unique set of mass labels having (n) members.  
     
     
         19 . The method of  claim 18 , wherein (n) is from 1 to 1000.  
     
     
         20 . The method of  claim 17 , wherein a reactive group comprising n specified components is associated with a unique set of mass labels having y members wherein n is less than y!/{x!(y−x)!}; and 
 wherein x comprises the number of mass labels used per active group.  
 
     
     
         21 . The method of  claim 17 , wherein the plurality of probes each comprise a known reactive group having a known set of mass labels.  
     
     
         22 . The method of  claim 21 , wherein the plurality of probes are prepared by combinatorial synthesis.  
     
     
         23 . The method of  claim 17 , wherein the plurality of target molecules comprise a known chemical structure.  
     
     
         24 . The method of  claim 1 , wherein the target molecule comprises an expressed gene product.  
     
     
         25 . The method of  claim 24 , wherein the expressed gene product is derived from a cloned mRNA.  
     
     
         26 . The method of  claim 1 , wherein the target molecule comprises a cloned genomic DNA.  
     
     
         27 . A method of monitoring gene expression comprising: 
 (a) obtaining a plurality of probes, each probe comprising a release tag compound comprising Rx, Re and M wherein: 
 Rx is a reactive group;  
 Re is a release group; and  
 M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry;  
   (b) obtaining a plurality of target nucleic acids with the plurality of probes to produce probe:target nucleic acid complexes;    (c) selectively releasing the mass labels from the probe:target nucleic acid complexes, wherein the complexes are in solution; and    (d) determining the mass of the released mass labels by mass spectrometry.    
     
     
         28 . The method of  claim 27 , herein the target nucleic acids comprise mRNA or first-strand cDNA.  
     
     
         29 . The method of  claim 27 , wherein the target nucleic acids comprise amplified nucleic acid products.  
     
     
         30 . The method of  claim 27 , wherein the amplification is effected by PCR, rtPCR, LCR, Qbeta Replicase, SDA, CPR, TAS, NASBA, or multiple rounds of RNA transcription or some combination thereof.  
     
     
         31 . A method of monitoring gene expression comprising: 
 (a) obtaining an mRNA pool;    (b) amplifying a subset of the mRNA pool to produce a plurality of amplified nucleic acid products;    (c) obtaining a plurality of probes, each probe comprising a release tag compound comprising Rx, Re and M wherein: 
 Rx is a reactive group;  
 Re is a release group; and  
 M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry;  
   (d) contacting the plurality of amplified nucleic acid products with the plurality of probes to produce probe:amplified nucleic acid product complexes;    (e) selectively releasing the mass label from the probe:amplified nucleic acid product complexes to produce released mass labels; and    (f) determining the mass of the released mass labels by mass spectrometry.    
     
     
         32 . The method of  claim 31 , wherein at least one probe is capable of being immobilized onto a solid support.  
     
     
         33 . The method of  claim 31 , wherein at least one amplified nucleic acid product is capable of being immobilized onto a solid support.  
     
     
         34 . A method for detecting a target molecule, said method comprising the steps of: 
 (a) contacting a target molecule with a probe comprising a release tag compound comprising Rx, Re and M, wherein: 
 Rx is a reactive group;  
 Re is a release group; and  
 M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry, to produce probe:target molecule complexes and unreacted probes;  
   (b) releasing the mass labels from the probe:target molecule complexes to produce released mass labels;    (c) selectively desorbing the released mass label from an organic matrix to produce desorbed mass labels; and    (d) determining the mass of the desorbed mass label by mass spectrometry.    
     
     
         35 . The method of  claim 34 , wherein the organic matrix comprises 2,5-dihydroxybenzoic acid, sinapinic acid, or alpha-cyano-4-hydroxycinnamic acid.  
     
     
         36 . A method for detecting the presence of a target nucleic acid molecule, said method comprising: 
 (a) obtaining a probe comprising a release tag compound comprising Rx, Re and M, wherein: 
 Rx is a reactive group;  
 Re is a release group; and  
 M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry;  
   (b) contacting the probe to a target nucleic acid molecule to produce probe:nucleic acid molecule complexes;    (c) mass modifying the probe:nucleic acid molecule complexes by attaching a nucleotide or oligonucleotide to the probe to produce mass modified mass labels;    (d) releasing the mass modified mass labels; and    (e) determining the mass of the mass-modified mass labels by mass spectrometry.    
     
     
         37 . A method for detecting specific biomolecules in an enzyme-linked affinity assay comprising: 
 (a) obtaining a substrate;    (b) contacting the target molecule with an affinity ligand-enzyme conjugate to produce an affinity ligand-enzyme conjugate:target molecule complex;    (c) contacting the affinity ligand-enzyme conjugate:target molecule complex with the substrate to produce a mass modified product; and    (d) determining the mass of the mass modified product by mass spectrometry.    
     
     
         38 . The method of  claim 37 , wherein the enzyme is a restriction endonuclease.  
     
     
         39 . The method of  claim 38 , further comprising a plurality of restriction endonuclease conjugates.  
     
     
         40 . The method of  claim 38 , wherein the substrate comprises a restriction site.  
     
     
         41 . The method of  claim 37 , wherein the affinity ligand is a biomolecule capable of specific molecular recognition.  
     
     
         42 . The method of  claim 41 , wherein the affinity ligand is a polypeptide.  
     
     
         43 . The method of  claim 42 , wherein the polypeptide is selected from the group consisting essentially of an antibody, an enzyme, a receptor, a regulatory protein, a nucleic acid-binding protein, a hormone, or a protein product of a display method.  
     
     
         44 . The method of  claim 43 , wherein the polypeptide comprises a product of a phage display method or a bacterial display method.  
     
     
         45 . The method of  claim 43 , wherein the polypeptide comprises an antibody.  
     
     
         46 . The method of  claim 41 , wherein the affinity ligand is a polynucleic acid.  
     
     
         47 . The method of  claim 37 , wherein the enzyme is a protease.  
     
     
         48 . The method of  claim 47 , wherein the substrate is a polypeptide.  
     
     
         49 . A method of determining the identity of a nucleotide at a single nucleotide polymorphism site, the method comprising the steps of: 
 (a) obtaining at least one probe comprising a release tag compound comprising Rx, Re and M, wherein: 
 Rx is a reactive group;  
 Re is a release group; and  
 M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry, with a reactive group capable of hybridizing to a target nucleic acid containing a single nucleotide polymorphism site;  
   (b) hybridizing the probe to the target nucleic acid to produce a probe:target nucleic acid complex;    (c) selectively releasing a mass label to obtain a released mass label; and    (d) determining the mass of the released mass label by mass spectrometry, wherein the identity of the nucleotide at the single nucleotide polymorphism site corresponds to the mass of the released mass label.    
     
     
         50 . The method of  claim 49 , wherein the probe is obtained by hybridizing the reactive group to the target nucleic acid to produce a reactive group:target nucleic acid complex and extending the reactive group by the addition of one or more added nucleotides.  
     
     
         51 . The method of  claim 50 , wherein at least one added nucleotide is mass-labeled.  
     
     
         52 . The method of  claim 49 , further comprising a set of probes having an invariant region and a variant region, wherein the invariant region is capable of hybridizing to a region immediately adjacent to a single nucleotide polymorphism site and wherein at least one member of the set of probes contains a variable region complementary to the nucleotide at the single nucleotide polymorphism site.  
     
     
         53 . A method of determining the identity of a plurality of nucleotides at a plurality of single nucleotide polymorphism sites, the method comprising the steps of: 
 (a) obtaining a plurality of probes each comprising a reactive group capable of hybridizing to a target nucleic acid containing a single nucleotide polymorphism site;    (b) hybridizing the probes to one or more target nucleic acids to produce one or more probe:target nucleic acid complexes;    (c) selectively releasing a plurality of mass labels to obtain released mass labels; and    (d) determining the mass of the released mass labels by mass spectrometry, wherein the identity of the nucleotide at each single nucleotide polymorphism site corresponds to the mass of a released mass label.    
     
     
         54 . The method of  claim 53 , wherein one or more probes are obtained by hybridizing a reactive group to a target nucleic acid to produce a reactive group:target nucleic acid complex and extending the reactive group by the addition of one or more added nucleotides.  
     
     
         55 . The method of  claim 54 , wherein at least one added nucleotide is mass-labeled.  
     
     
         56 . The method of  claim 53 , further comprising a set of probes having an invariant region and a variant region, wherein the invariant region is capable of hybridizing to a region immediately adjacent to a single nucleotide polymorphism site and wherein at least one member of the set of probes contains a variable region complementary to the nucleotide at the single nucleotide polymorphism site.  
     
     
         57 . The method of detecting a single-stranded nucleic acid target molecule, said method comprising the steps of: 
 (a) obtaining a double-stranded nucleic acid probe comprising a reactive group, a release group and a nonvolatile mass label, wherein the double-stranded probe further comprises a complementary strand and a mass-labeled strand, and wherein the mass-labeled strand further comprises a nonvolatile mass label and a release group;    (b) obtaining a single-stranded target molecule;    (c) contacting the single-stranded target with the double-stranded probe to produce a complementary strand:target molecule complex and a single-stranded mass-labeled strand;    (d) selectively releasing the mass label from the single-stranded mass-labeled strand; and    (e) determining the mass of the mass label by mass spectrometry.    
     
     
         58 . A method for multiplexing the detection of at least one single-stranded target molecule, said method comprising the steps of: 
 (a) obtaining a plurality of double-stranded probes, each probe comprising a release tag compound comprising 
 Rx, Re and M wherein:  
 Rx is a reactive group;  
 Re is a release group; and  
 M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry, wherein each of the double-stranded probes further comprise a complementary strand and a mass-labeled strand, and wherein the mass-labeled strand further comprises a nonvolatile mass label and a release group;  
   (b) obtaining at least one single-stranded target molecule;    (c) contacting the target molecule with the plurality of probes to produce a complementary strand:target molecule complex and a single-stranded mass-labeled strand;    (d) selectively releasing the mass label from the single-stranded mass-labeled strand;    (e) determining the mass of the mass label by mass spectrometry, wherein each reactive group of the double-stranded probe is associated with a unique set of mass labels.