Methods for isolating and labeling sample molecules

Abstract

The invention provides methods for labeling a molecule by contacting a sample molecule with a solid support coupled to a chemical group comprising a cleavable functional group, one or more functional groups, and a reactive group for the sample molecule, under conditions allowing the sample molecule to covalently bind to the reactive group; and cleaving the cleavable functional group, thereby releasing the sample molecule comprising the one or more functional groups, which can be a tag. The invention also provides a solid support covalently coupled to a chemical group comprising a cleavable functional group, a mass spectrometry tag and a reactive group for covalently attaching a sample molecule, wherein the cleavable functional group, the tag and the reactive group are positioned relative to each other to allow transfer of the tag to the sample molecule upon cleavage of the cleavable functional group.

What is claimed is:  
     
         1 . A method for labeling a molecule, comprising the steps of: 
 (a) contacting a sample molecule with a solid support coupled to a chemical group comprising a cleavable functional group, one or more functional groups, and a reactive group for said sample molecule, under conditions allowing said sample molecule to covalently bind to said reactive group; and    (b) cleaving said cleavable functional group, thereby releasing said sample molecule comprising said one or more functional groups.    
     
     
         2 . The method of  claim 1 , wherein said sample molecule is selected from the group consisting of a polypeptide, a nucleic acid, a lipid, a second messenger, and a metabolite.  
     
     
         3 . The method of  claim 1 , wherein said sample molecule is a polypeptide.  
     
     
         4 . The method of  claim 3 , wherein said polypeptide has a modification selected from the group consisting of phosphorylation, glycosylation, ubiquitination, acetylation, prenylation, palmitylation, myristylation, sulfation, and hydroxylation.  
     
     
         5 . The method of  claim 4 , wherein said polypeptide is a phosphopolypeptide.  
     
     
         6 . The method of  claim 1 , wherein said solid support is a glass bead.  
     
     
         7 . The method of  claim 1 , wherein said cleavable functional group is a chemical linker cleavable by light, an acid, a base or an enzyme.  
     
     
         8 . The method of  claim 1 , wherein one of said functional groups is a tag.  
     
     
         9 . The method of  claim 8 , wherein said tag is a mass spectrometry tag.  
     
     
         10 . The method of  claim 8 , wherein said tag is selected from the group consisting of a stable isotope tag, an isotope distribution tag, and a charged amino acid.  
     
     
         11 . The method of  claim 10 , wherein said tag is a stable isotope coded amino acid.  
     
     
         12 . The method of  claim 11 , wherein said tag is a deuterated or non-deuterated amino acid.  
     
     
         13 . The method of  claim 8 , wherein said tag is a gas-phase basic group or a hydrophobic group.  
     
     
         14 . The method of  claim 13 , wherein said gas-phase basic group is pyridyl.  
     
     
         15 . The method of  claim 8 , wherein said tag is selected from a fluorophore, chromophore, and spin label.  
     
     
         16 . The method of  claim 8 , wherein one of said functional groups comprises an element having a characteristic isotope distribution.  
     
     
         17 . The method of  claim 16 , wherein said element is chlorine or bromine.  
     
     
         18 . The method of  claim 3 , wherein said reactive group of said chemical group is selected from the group consisting of a succinimide ester group and an iodoacetyl group.  
     
     
         19 . The method of  claim 3 , wherein a primary amine group of said polypeptide is modified by treatment with N-succinimidyl S-acetylthioacctate, hydroxylamine, and tris(2-carboxyethyl)phosphine.  
     
     
         20 . The method of  claim 4 , wherein said polypeptide is isolated using an antibody has specific binding activity to said modification of said polypeptide.  
     
     
         21 . The method of  claim 1 , wherein the method steps are performed by an automated process.  
     
     
         22 . The method of  claim 1 , wherein at least 50 percent of said sample molecule contacted with said solid support is released.  
     
     
         23 . A method for analyzing a sample molecule, comprising the steps of: 
 (a) contacting a sample molecule with a solid support coupled to a chemical group comprising a cleavable functional group, one or more functional groups, and a reactive group for said sample molecule, under conditions allowing said sample molecule to covalently bind to said reactive group;    (b) cleaving said sample molecule from said solid support, wherein one or more specific functional groups are transferred to the released sample molecule; and    (c) analyzing said released sample molecule.    
     
     
         24 . The method of  claim 23 , wherein the released sample molecule is analyzed by mass spectrometry.  
     
     
         25 . The method of  claim 23 , wherein a plurality of a class of molecules expressed by a cell or tissue is analyzed.  
     
     
         26 . The method of  claim 23 , wherein said sample molecule is selected from the group consisting of a polypeptide, a nucleic acid, a lipid, a second messenger, and a metabolite.  
     
     
         27 . The method of  claim 26 , wherein said sample molecule is a polypeptide.  
     
     
         28 . The method of  claim 27 , wherein said polypeptide has a modification selected from the group consisting of phosphorylation, glycosylation, ubiquitination, acetylation, palmitylation, prenylation, sulfation, hydroxylation, and myristylation.  
     
     
         29 . The method of  claim 28 , wherein said polypeptide is a phosphopolypeptide.  
     
     
         30 . The method of  claim 23 , wherein said solid support is a glass bead.  
     
     
         31 . The method of  claim 23 , wherein said cleavable functional group is a chemical linker cleavable by light, an acid, a base or an enzyme.  
     
     
         32 . The method of  claim 23 , wherein one of said functional groups is a tag.  
     
     
         33 . The method of  claim 32 , wherein said tag is a mass spectrometry tag.  
     
     
         34 . The method of  claim 32 , wherein said tag is selected from the group consisting of a stable isotope tag, an isotope distribution tag, and a charged amino acid.  
     
     
         35 . The method of  claim 34 , wherein said tag is a stable isotope coded amino acid.  
     
     
         36 . The method of  claim 35 , wherein said tag is a deuterated or non-deuterated amino acid.  
     
     
         37 . The method of  claim 32 , wherein said tag is a gas-phase basic group or hydrophobic group.  
     
     
         38 . The method of  claim 37 , wherein said gas-phase basic group is pyridyl.  
     
     
         39 . The method of  claim 32 , wherein said tag is selected from a fluorophore, chromophore, and spin label.  
     
     
         40 . The method of  claim 32 , wherein one of said functional groups comprises an element having a characteristic isotope distribution.  
     
     
         41 . The method of  claim 40 , wherein the elements are chlorine or bromine.  
     
     
         42 . The method of  claim 23 , wherein said reactive group of said chemical group is selected from the group consisting of a succinimide ester group and an iodoacetyl group.  
     
     
         43 . The method of  claim 27 , wherein a primary amine group of said polypeptide is modified by treatment with N-succinimidyl S-acetylthioacctate, hydroxylamine, and tris(2-carboxyethyl)phosphine.  
     
     
         44 . The method of  claim 28 , wherein said polypeptide is isolated using an antibody having specific binding activity to said modification of the polypeptide.  
     
     
         45 . The method of  claim 23 , wherein the method steps are performed by an automated process.  
     
     
         46 . The method of  claim 23 , wherein at least 50 percent of said sample molecule contacted with said solid support is released.  
     
     
         47 . The method of  claim 23 , wherein molecules from two or more samples are comparatively analyzed.  
     
     
         48 . The method of  claim 47 , wherein said two or more samples are differentially labeled.  
     
     
         49 . The method of  claim 48 , wherein said samples are differentially labeled with a mass spectrometry tag.  
     
     
         50 . The method of  claim 48 , wherein said samples are differentially labeled with a stable isotope tag, an isotope distribution tag, or a charged amino acid.  
     
     
         51 . The method of  claim 48 , wherein said samples are differentially labeled with a fluorophore, chromophore, or spin label.  
     
     
         52 . A method for labeling a molecule, comprising the steps of: 
 (a) contacting a sample molecule with a solid support coupled to a chemical group comprising a cleavable functional group, one or more functional groups, and a reactive group for said sample molecule, under conditions allowing said sample molecule to covalently bind to said reactive group;    (b) modifying said sample molecule bound to said solid support; and    (c) cleaving said cleavable functional group, thereby releasing said modified sample molecule comprising said one or more functional groups.    
     
     
         53 . The method of  claim 52 , wherein said modifying step is a chemical or enzymatic modification.  
     
     
         54 . The method of  claim 52 , wherein said sample molecule is selected from the group consisting of a polypeptide, a nucleic acid, a lipid, a second messenger, and a metabolite.  
     
     
         55 . The method of  claim 52 , wherein said sample molecule is a polypeptide.  
     
     
         56 . The method of  claim 55 , wherein said polypeptide has a modification selected from the group consisting of phosphorylation, glycosylation, ubiquitination, acetylation, prenylation, palmitylation, myristylation, sulfation, and hydroxylation.  
     
     
         57 . The method of  claim 56 , wherein said polypeptide is a phosphopolypeptide.  
     
     
         58 . The method of  claim 57 , wherein said modifying step modifies a phosphate group on said phosphopolypeptide.  
     
     
         59 . The method of  claim 52 , wherein said solid support is a glass bead.  
     
     
         60 . The method of  claim 52;  wherein said cleavable functional group is a chemical linker cleavable by light, an acid, a base or an enzyme.  
     
     
         61 . The method of  claim 52 , wherein one of said functional groups is a tag.  
     
     
         62 . The method of  claim 61 , wherein said tag is a mass spectrometry tag.  
     
     
         63 . The method of  claim 61 , wherein said tag is selected from the group consisting of a stable isotope tag, an isotope distribution tag, and a charged amino acid.  
     
     
         64 . The method of  claim 63 , wherein said tag is a stable isotope coded amino acid.  
     
     
         65 . The method of  claim 64 , wherein said tag is a deuterated or non-deuterated amino acid.  
     
     
         66 . The method of  claim 61 , wherein said tag is a gas-phase basic group or a hydrophobic group.  
     
     
         67 . The method of  claim 66 , wherein said gas-phase basic group is pyridyl.  
     
     
         68 . The method of  claim 61 , wherein said tag is selected from a fluorophore, chromophore, and spin label.  
     
     
         69 . The method of  claim 61 , wherein one of said functional groups comprises an element having a characteristic isotope distribution.  
     
     
         70 . The method of  claim 69 , wherein said element is chlorine or bromine.  
     
     
         71 . The method of  claim 55 , wherein said reactive group of said chemical group is selected from the group consisting of a succinimide ester group and an iodoacetyl group.  
     
     
         72 . The method of  claim 55 , wherein a primary amine group of said polypeptide is modified by treatment with N-succinimidyl S-acetylthioacctate, hydroxylamine, and tris(2-carboxyethyl)phosphine.  
     
     
         73 . The method of  claim 56 , wherein said polypeptide is isolated using an antibody has specific binding activity to said modification of said polypeptide.  
     
     
         74 . The method of  claim 52 , wherein the method steps are performed by an automated process.  
     
     
         75 . The method of  claim 52 , wherein at least 50 percent of said sample molecule contacted with said solid support is released.  
     
     
         76 . A composition comprising a solid support coupled to a chemical group comprising a cleavable functional group, a tag and a reactive group covalently linked to a sample molecule, wherein said cleavable functional group, said tag and said reactive group are positioned relative to each other to allow transfer of said tag to said sample molecule upon cleavage of said cleavable functional group.  
     
     
         77 . The composition of  claim 76 , wherein said sample molecule is selected from the group consisting of a polypeptide, a nucleic acid, a lipid, a second messenger, and a metabolite.  
     
     
         78 . The composition of  claim 77 , wherein said sample molecule is a polypeptide.  
     
     
         79 . The composition of  claim 78 , wherein said polypeptide has a modification selected from the group consisting of phosphorylation, glycosylation, ubiquitination, acetylation, palmitylation, prenylation, sulfation, hydroxylation, and myristylation.  
     
     
         80 . The composition of  claim 79 , wherein said polypeptide is a phosphopolypeptide.  
     
     
         81 . The composition of  claim 76 , wherein the solid support is a glass bead.  
     
     
         82 . The composition of  claim 76 , wherein said cleavable functional group is a chemical linker cleavable by light, an acid, a base or an enzyme.  
     
     
         83 . The composition of  claim 76 , wherein said tag is a mass spectrometry tag.  
     
     
         84 . The composition of  claim 76 , wherein said tag is selected from the group consisting of a stable isotope tag, an isotope distribution tag, and a charged amino acid.  
     
     
         85 . The composition of  claim 84 , wherein said tag is a stable isotope coded amino acid.  
     
     
         86 . The composition of  claim 85 , wherein said tag is a deuterated or non-deuterated amino acid.  
     
     
         87 . The composition of  claim 76 , wherein said tag is a gas-phase basic group or a hydrophobic group.  
     
     
         88 . The composition of  claim 87 , wherein the gas-phase basic group is pyridyl.  
     
     
         89 . The method of  claim 76 , wherein said tag is selected from a fluorophore, chromophore, and spin label.  
     
     
         90 . The composition of  claim 76 , wherein said tag comprises an element having a characteristic isotope distribution.  
     
     
         91 . The composition of  claim 90 , wherein said element is chlorine or bromine.  
     
     
         92 . The composition of  claim 76 , wherein said covalently linked reactive group is derived from a succinimide ester group or an iodoacetyl group.  
     
     
         93 . The composition of  claim 78 , wherein a primary amine group of said polypeptide is modified by treatment with N-succinimidyl S-acetylthioacctate, hydroxylamine, and tris(2-carboxyethyl)phosphine.  
     
     
         94 . A composition comprising a solid support covalently coupled to a chemical group comprising a cleavable functional group, a mass spectrometry tag and a reactive group for covalently attaching a sample molecule, wherein said cleavable functional group, said tag and said reactive group are positioned relative to each other to allow transfer of said tag to said sample molecule upon cleavage of said cleavable functional group.  
     
     
         95 . The composition of  claim 94 , wherein the solid support is a glass bead.  
     
     
         96 . The composition of  claim 94 , wherein said cleavable functional group is a chemical linker cleavable by light, an acid, a base or an enzyme.  
     
     
         97 . The composition of  claim 94 , wherein said tag is selected from the group consisting of a stable isotope tag, an isotope distribution tag, and a charged amino acid.  
     
     
         98 . The composition of  claim 97 , wherein said tag is a stable isotope coded amino acid.  
     
     
         99 . The composition of  claim 98 , wherein said tag is a deuterated or non-deuterated amino acid.  
     
     
         100 . The composition of  claim 94 , wherein said tag is a gas-phase basic group or a hydrophobic group.  
     
     
         101 . The composition of  claim 100 , wherein said gas-phase basic group is pyridyl.  
     
     
         102 . The method of  claim 94 , wherein said tag is selected from a fluorophore, chromophore, and spin label.  
     
     
         103 . The composition of  claim 94 , wherein said tag comprises an element having a characteristic isotope distribution.  
     
     
         104 . The composition of  claim 103 , wherein said element is chlorine or bromine.  
     
     
         105 . The composition of  claim 94 , wherein said reactive group of said chemical group is selected from the group consisting of a succinimide ester group and an iodoacetyl group.