Mass defect labeling for the determination of oligomer sequences
First Claim
1. A method for sequencing a terminal portion of an oligomer, comprising:
- (a) contacting said oligomer with a mass defect labeling moiety to covalently attach the mass defect labeling moiety to a terminus of the oligomer and form a labeled oligomer, said mass defect labeling moiety comprising at least one element having an atomic number from 17 to 77;
(b) fragmenting said labeled oligomer using an enzymatic, chemolytic or mass spectrometric fragmentation method to produce labeled oligomer fragments;
(c) identifying a mass spectrum data corresponding to said labeled oligomer fragments; and
(d) determining the sequence of at least two terminal residues of said labeled oligomer, wherein said sequence determination step comprises discriminating between the mass of the labeled oligomer fragment and an unlabeled oligomer fragment based on the nuclear binding energy of the mass defect labeling moiety;
wherein said oligomer is selected from the group consisting of a protein, polysaccharide, nucleic acid, and lipid.
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Abstract
Mass tagging methods are provided that lead to mass spectrometer detection sensitivities and molecular discriminations that are improved over other methods. In particular the methods are useful for discriminating tagged molecules and fragments of molecules from chemical noise in the mass spectrum. These mass tagging methods are useful for oligomer sequencing, determining the relative abundances of molecules from different samples, and identifying individual molecules or chemical processing steps in combinatorial chemical libraries. The methods provided are useful for the simultaneous analysis of multiple molecules and reaction mixtures by mass spectrometric methods.
53 Citations
51 Claims
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1. A method for sequencing a terminal portion of an oligomer, comprising:
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(a) contacting said oligomer with a mass defect labeling moiety to covalently attach the mass defect labeling moiety to a terminus of the oligomer and form a labeled oligomer, said mass defect labeling moiety comprising at least one element having an atomic number from 17 to 77;
(b) fragmenting said labeled oligomer using an enzymatic, chemolytic or mass spectrometric fragmentation method to produce labeled oligomer fragments;
(c) identifying a mass spectrum data corresponding to said labeled oligomer fragments; and
(d) determining the sequence of at least two terminal residues of said labeled oligomer, wherein said sequence determination step comprises discriminating between the mass of the labeled oligomer fragment and an unlabeled oligomer fragment based on the nuclear binding energy of the mass defect labeling moiety;
wherein said oligomer is selected from the group consisting of a protein, polysaccharide, nucleic acid, and lipid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for sequencing a portion of an oligomer in an oligomer mixture, said method comprising;
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(a) contacting said oligomer mixture with a mass defect labeling moiety to covalently attach the mass defect labeling moiety to a terminus of said oligomer and form a labeled oligomer mixture, said mass defect labeling moiety comprising at least one element having an atomic number from 17 to 77;
(b) separating individual labeled oligomers in said labeled oligomer mixture;
(c) identifying a mass spectrum data corresponding to said individual labeled oligomer; and
(d) analyzing said mass spectrum data to determine the sequence of at least two terminus residues of said oligomer, wherein said analysis step comprises discriminating between the mass of the labeled oligomer and an unlabeled oligomer based on the nuclear binding energy of the mass defect labeling moiety;
wherein said oligomer is selected from the group consisting of a protein, polysaccharide, nucleic acid, and lipid. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for structure and function analysis of an oligomer having a plurality of residues, said method comprising:
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(a) contacting said oligomer with a mass defect labeling reagent to differentially label exposed residues and unexposed residues and produce a differentially labeled oligomer comprising a mass defect labeling moiety, wherein said mass defect labeling reagent comprises at least one element having an atomic number of from 17 to 77 that is other than sulfur or phosphorus;
(b) identifying a mass spectrum data corresponding to said differentially labeled oligomer; and
(c) analyzing said mass spectrum data to determine sequences of said oligomer that are exposed in the three-dimensional structure and sequences of said oligomer that are unexposed in the three-dimensional structure, wherein said analysis step comprises discriminating between the mass of the differentially labeled oligomer and an unlabeled oligomer based on the nuclear binding energy of the mass defect labeling moiety;
wherein said oligomer is selected from the group consisting of a protein, polysaccharide, nucleic acid, and lipid. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31)
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32. A method for sequencing the terminal portion of an oligomer, comprising:
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(a) contacting a first sample of said oligomer with a labeling moiety to covalently attach a label to the terminus of the oligomer and form a labeled oligomer, said labeling moiety having one element with an atomic number from 17 to 77, (b) contacting a second sample of said oligomer with a labeling moiety to covalently attach a label to a terminus of the oligomer and form a labeled oligomer, said labeling moiety having two elements with an atomic number from 17 to 77;
(c) optionally, repeating step (b) from one to three times with additional samples, wherein the labeling moieties have three, four or five elements, respectively, with an atomic number from 17 to 77;
(d) mixing the labeled oligomers from steps (a) through (c);
(e) fragmenting said labeled oligomers using an enzymatic, chemolytic or mass spectrometric fragmentation method to produce labeled oligomer fragments;
(f) identifying a mass spectrum data corresponding to said labeled oligomer fragments; and
(g) determining the sequence of at least two terminal residues of said labeled oligomer fragments, wherein said sequence determination step comprises discriminating between the mass of the labeled oligomer fragment and an unlabeled oligomer fragment based on the nuclear binding energy of the labeling moiety;
wherein said oligomer is selected from the group consisting of a protein, polysaccharide, nucleic acid, and lipid. - View Dependent Claims (33, 34, 35, 36, 37)
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38. A method for sequencing a portion of an oligomer, comprising:
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(a) fragmenting aliquots of said oligomer using one or more specific enzymatic or chemolytic fragmentation methods to produce oligomer fragments, wherein a different fragmentation method is applied to each aliquot;
(b) contacting a first aliquot of oligomer fragments with a first labeling moiety to covalently attach said first labeling moiety to a terminus of the oligomer fragments and form labeled oligomer fragments, said first labeling moiety having one element with an atomic number from 17 to 77;
(c) optionally contacting the other aliquots of oligomer fragments with other distinct labeling moieties to covalently attach said distinct labeling moieties to the termini of the oligomer fragments and form labeled oligomer fragments, said distinct labeling moiety having two or more elements with an atomic number from 17 to 77;
(d) optionally mixing the aliquots of labeled oligomer fragments;
(e) identifying a mass spectrum data corresponding to said labeled oligomer fragments; and
(f) determining the sequence of at least two residues of said labeled oligomer, wherein said sequence determining step comprises discriminating between the mass of the labeled oligomer fragments and an unlabeled oligomer fragment based on the nuclear binding energy of the labeling moiety;
wherein said oligomer is selected from the group consisting of a protein, polysaccharide, nucleic acid, and lipid. - View Dependent Claims (39, 40, 41, 42, 43, 44)
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45. A method for comparing the relative abundances of analytes from two or more samples, comprising:
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(a) contacting the analytes of the first sample with a labeling moiety to covalently attach a label to the analytes and form labeled analytes, said labeling moiety having one element with an atomic number from 17 to 77;
(b) contacting the analytes of subsequent samples with labeling moieties to covalently attach labels to the analytes in each sample, wherein the labeling moieties used for each subsequent sample contain an additional element with an atomic number from 17 to 77;
(c) mixing the aliquots of labeled analytes;
(d) identifying mass spectrum data corresponding to said labeled analytes; and
(e) analyzing said mass spectrum data to determine the relative abundances of one or more of the analytes between the samples, wherein said analysis step comprises discriminating between the mass of the labeled analytes and an unlabeled analyte based on the nuclear binding energy of the labeling moiety;
wherein said analyte is selected from the group consisting of a protein, polysaccharide, nucleic acid, and lipid. - View Dependent Claims (46, 47, 48, 49, 50, 51)
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Specification