Releasable nonvolatile mass-label molecules
First Claim
Patent Images
1. 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.
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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.
177 Citations
122 Claims
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1. A release tag compound comprising Rx, Re and M wherein:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 111, 112, 113, 114, 120)
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20. The compound of 19, wherein the polypeptide comprises a product of a phage display method or a bacterial display method.
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39. A release tag compound comprising Rx, Re and M, wherein:
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Rx is a reactive group comprising mass-labeled nucleotides;
Re is a release group; and
M is a mass label detectable by mass spectrometry.
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40. A release tag compound comprising Rx, Re and M, wherein:
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Rx is a reactive group comprising nucleoside phosphoramidites;
Re is a release group; and
M is a mass label detectable by mass spectrometry.
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41. A release tag compound comprising Rx, Re and M, wherein:
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Rx is a reactive group comprising a first oligonucleotide having a nucleotide or a second oligonucleotide attached thereto;
Re is a release group; and
M is a mass label detectable by mass spectrometry. - View Dependent Claims (42, 43, 44)
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45. A release tag compound comprising Rx, Re and M, wherein:
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Rx is an oligonucleotide comprising a nuclease blocking moiety;
Re is a release group; and
M is a mass label detectable by mass spectrometry. - View Dependent Claims (46)
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47. A release tag compound comprising Rx, Re and M, wherein:
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Rx is a double-stranded oligonucleotide comprising a restriction endonuclease recognition site;
Re is a release group comprising a phosphodiester linkage capable of being cleaved by a restriction endonuclease; and
M is a mass label detectable by mass spectrometry. - View Dependent Claims (48, 49, 50)
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51. A release tag compound comprising Rx, Re and M, wherein:
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Rx is a double-stranded oligonucleotide;
Re is a chemically cleavable release group; and
M is a mass label detectable by mass spectrometry;
wherein Re is located within Rx; and
wherein cleavage at the chemically cleavable release group is inhibited by the presence of a double-stranded oligonucleotide at said release group. - View Dependent Claims (52, 53)
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54. A set of release tag compounds, said set comprising one or more release tag compounds for detecting a particular target nucleic acids, each release tag compound comprising Rx, Re and M, wherein:
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Rx is an oligonucleotide comprising a variable region and an invariant region;
Re is a release group; and
M is a mass label detectable by mass spectrometry;
wherein said invariant and variable regions react with the target nucleic acid. - View Dependent Claims (55, 56, 57, 58, 59, 60, 61, 62)
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99. 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. - View Dependent Claims (100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110)
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115. 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:
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(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. - View Dependent Claims (116, 117, 118)
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119. The method of detecting a single-stranded nucleic acid target molecule, said method comprising the steps of:
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(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;
(e) determining the mass of the mass label by mass spectrometry.
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121. A method of preparing a release tag compound, the method comprising:
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(a) obtaining a target nucleic acid sequence;
(b) synthesizing a reactive group capable of hybridizing to the target nucleic acid sequence;
(c) hybridizing the reactive group to the target nucleic acid sequence; and
(d) extending the reactive group to produce a release tag compound complementary to the target nucleic acid sequence, wherein the release tag compound comprises a reactive group and release group and a mass-labeled detectable by mass spectrometry. - View Dependent Claims (122)
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Specification