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US 6,558,902 B1Granted
Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules
AssigneeSequenom, Inc.
InventorFranz Hillenkamp
Life of this patent
Estimating the term…
Abstract
Mixtures containing a biological macromolecule, such as a nucleic acid molecule or a polypeptide, and a liquid matrix, which absorbs infrared (IR) radiation, are provided. These mixtures are useful for analysis of the biological macromolecule by IR matrix assisted laser desorption/ionization (IR-MALDI) mass spectrometry. Also provided are processes for analyzing a biological macromolecule using IR-MALDI mass spectrometry. For example, processes for detecting the presence or identity of a biological macromolecule in a sample, or for sequencing a biological macromolecule are provided.
Claims226
A method for performing matrix assisted laser desorption/ionization (MALDI) of a nucleic acid molecule for analysis by mass spectrometry, comprising the steps of:
- (a) depositing a solution containing the nucleic acid and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a nucleic acid/liquid matrix solution;
- and
- (b) illuminating the substrate with infrared radiation, whereby the nucleic acid in the solution is desorbed and ionized.
A method for analyzing a nucleic acid in a sample by mass spectrometry, comprising the steps of:
- (a) depositing a mixture containing the nucleic acid and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a nucleic acid/liquid matrix mixture;
- (b) illuminating the substrate of (a) with infrared radiation from an infrared laser, so that the nucleic acid is desorbed and ionized;
- and
- (c) mass separating and detecting the ionized nucleic acid using a mass separation and analysis format.
1 more element
A method for detecting a target molecule, comprising:
- (a) obtaining a target molecule;
- (b) amplifying the target molecule to produce an amplified target molecule;
- (c) obtaining a probe comprising a reactive group, a release group and a mass label;
- (d) hybridizing the amplified target molecule to the probe to produce a probe:amplified target molecule complex;
- (e) releasing the mass label from the probe:amplified target molecule complex to obtain a released mass label;
- (f) depositing a solution containing the released mass label and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;
- (g) illuminating the substrate with infrared radiation, whereby the nucleic acid in the solution is desorbed and ionized;
- and
- (h) determining the mass of the released mass label by matrix assisted laser desorption/ionization (MALDI) mass spectrometry.
6 more elements
A method for detecting a target molecule, comprising:
- (a) obtaining a probe comprising a reactive group, a release group and a mass label;
- (b) obtaining a target molecule;
- (c) contacting the target molecule with the probe to produce a probe:target molecule complex;
- (d) releasing the mass label from the probe:target molecule complex;
- (e) depositing a solution containing the released mass label and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;
- (f) illuminating the substrate with infrared radiation, whereby the nucleic acid in the solution is desorbed and ionized;
- and
- (g) determining the mass of the released mass label by matrix assisted laser desorption/ionization (MALDI) mass spectrometry.
5 more elements
A method for multiplexing the detection of a target molecule, comprising:
- (a) obtaining a plurality of probes, each comprising a reactive group, a release group and a mass label;
- (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;
- (d) depositing a solution containing the released mass labels and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;
- (e) illuminating the substrate with infrared radiation, whereby the nucleic acid in the solution is desorbed and ionized;
- and
- (f) determining the masses of the released mass labels by matrix assisted laser desorption/ionization (MALDI) mass spectrometry, wherein each reactive group in a probe:target molecule complex is associated with a unique set of mass labels.
4 more elements
A method for multiplexing the detection of a plurality of target molecules, comprising:
- (a) obtaining a plurality of probes, each comprising a reactive group, a release group and a mass label;
- (b) contacting the plurality of target molecules with the plurality of probes to produce probe:target molecule complexes, wherein target molecules are attached to the reactive groups of the probes;
- (c) releasing the mass labels from the probe:target molecule complexes to produce released mass labels;
- (d) depositing a solution containing the released mass labels and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;
- (e) illuminating the substrate with infrared radiation, whereby the nucleic acid in the solution is desorbed and ionized;
- and
- (f) determining the masses of the released mass labels by matrix assisted laser desorption/ionization (MALDI) mass spectrometry, wherein each reactive group specific for a particular target molecule is associated with a unique mass label.
4 more elements
A method for detecting a target biological macromolecule, comprising the steps of:
- a) preparing a mixture, comprising a biological macromolecule and a liquid matrix, which absorbs infrared radiation;
- and
- b) performing IR-MALDI mass spectrometry on the mixture to identify the target biological macromolecule in the mixture, thereby detecting the target biological macromolecule.
A method for detecting the presence of a target nucleic acid sequence in a biological sample containing nucleic acid molecules, comprising the steps of:
- a) contacting the nucleic acid molecules with a detector oligonucleotide, which can hybridize to a target nucleic acid sequence present in the biological sample;
- b) preparing a mixture for IR-MALDI, comprising the product of step a) and a liquid matrix, which absorbs infrared radiation;
- c) identifying duplex nucleic acid molecules in the mixture by IR-MALDI mass spectrometry, thereby detecting presence of the target nucleic acid sequence in the biological sample.
A method for detecting the presence of a target nucleic acid sequence in a biological sample containing nucleic acid molecules, comprising the steps of:
- a) specifically digesting the nucleic acid molecules using at least one appropriate nuclease, thereby producing digested fragments;
- b) hybridizing the digested fragments with complementary capture nucleic acid sequences, which are immobilized on a solid support and can hybridize to a digested fragment of a target nucleic acid to produce immobilized fragments;
- c) preparing a mixture for IR-MALDI, comprising the immobilized fragments and a liquid matrix, which absorbs infrared radiation;
- and
- d) identifying immobilized fragments by IR-MALDI mass spectrometry, thereby detecting the presence of the target nucleic acid sequence in the biological sample.
2 more elements
A method for detecting a target nucleic acid sequence, comprising the steps of:
- a) performing at least one hybridization on a nucleic acid molecule containing the target nucleic acid sequence with a set of ligation educts and a thermostable DNA ligase;
- b) preparing a mixture for IR-MALDI, comprising the product of step a) and a liquid matrix, which absorbs infrared radiation;
- and
- c) identifying a ligation product in the mixture by IR-MALDI mass spectrometry, thereby detecting the target nucleic acid sequence.
1 more element
A method for detecting the presence of a target nucleic acid in a biological sample containing nucleic acid molecules, comprising the steps of:
- a) performing on the nucleic acid molecules, a first polymerase chain reaction using a first set of primers, which are capable of amplifying a portion of a nucleic acid molecule containing the target nucleic acid, thereby producing a first amplification product;
- b) preparing a mixture for IR-MALDI, comprising the first amplification product and a liquid matrix, which absorbs infrared radiation;
- and
- c) detecting the first amplification product in the mixture by IR-MALDI mass spectrometry, thereby detecting the presence of the target nucleic acid in the biological sample.
1 more element
A method for determining the identity of a target nucleotide, comprising the steps of:
- a) hybridizing a nucleic acid molecule containing the target nucleotide with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the target nucleotide, thereby producing a hybridized nucleic acid molecule;
- b) contacting the hybridized nucleic acid molecule with a complete set of dideoxynucleosides or 3′-deoxynucleoside triphosphates and a DNA dependent DNA polymerase, so that only the dideoxynucleosides or 3′-deoxynucleoside triphosphate that is complementary to the target nucleotide is extended onto the primer, thereby producing an extended primer;
- c) preparing a mixture for IR-MALDI, comprising the extended primer and a liquid matrix, which absorbs infrared radiation;
- and
- d) detecting the extended primer in the mixture by IR-MALDI mass spectrometry, thereby determining the identity of the target nucleotide.
2 more elements
A method for detecting the presence or absence of a mutation in a target nucleic acid sequence, comprising the steps of:
- a) hybridizing a nucleic acid molecule containing the target nucleic acid sequence with at least one primer, the primer having 3′ terminal base complementarity to the target nucleic acid sequence, thereby producing a hybridized product;
- b) contacting the hybridized product with an appropriate polymerase enzyme and sequentially with one of the four nucleoside triphosphates;
- c) preparing a mixture for IR-MALDI, comprising the product of step b) and a liquid matrix, which absorbs infrared radiation;
- and
- d) detecting the product of step b) in the mixture by IR-MALDI mass spectrometry, wherein the molecular weight of the product indicates the presence or absence of a mutation next to the 3′ end of the primer in the target nucleic acid molecule.
2 more elements
A method for detecting a mutation in a target nucleic acid, comprising the steps of:
- a) hybridizing the target nucleic acid with an oligonucleotide probe, to produce a hybridized target nucleic acid, wherein a mismatch is formed at the site of a mutation;
- b) contacting the hybridized target nucleic acid with a single strand specific endonuclease;
- c) preparing a mixture for IR-MALDI, comprising the product of step b) and a liquid matrix, which absorbs infrared radiation;
- and
- d) analyzing the mixture by IR-MALDI mass spectrometry, wherein the presence of more than one fragment of the target nucleic acid in the mixture detects a mutation the target nucleic acid.
2 more elements
A method for identifying the absence or presence of a mutation in a target nucleic acid sequence, comprising the steps of:
- a) performing at least one hybridization on a nucleic acid molecule containing the target nucleic acid sequence with a set of ligation educts and a DNA ligase;
- b) preparing a mixture for IR-MALDI, comprising the product of step a) and a liquid matrix, which absorbs infrared radiation;
- and
- c) analyzing the mixture by IR-MALDI mass spectrometry,
- wherein detecting a ligation product in the mixture identifies the absence of a mutation in the target nucleic acid sequence, and
- wherein detecting only the set of ligation educts in the mixture identifies the presence of a mutation in the target nucleic sequence.
3 more elements
A method for determining the identity of each target biological macromolecule in a plurality of target biological macromolecules, comprising the steps of:
- a) preparing a mixture for IR-MALDI, comprising a plurality of differentially mass modified target biological macromolecules and a liquid matrix, which absorbs infrared radiation;
- b) determining the molecular mass of each differentially mass modified target biological macromolecule in the plurality by IR-MALDI mass spectrometry;
- and
- c) comparing the molecular mass of each differentially mass modified target biological macromolecule in the plurality with the molecular mass of a corresponding known biological macromolecule, thereby determining the identity of each target biological macromolecule in the plurality of target biological macromolecules or fragments thereof.
1 more element
A method for determining the sequence of a target biological macromolecule, comprising the steps of:
- a) generating at least two biological macromolecule fragments from the target biological macromolecule;
- b) preparing a mixture for IR-MALDI, comprising the biological macromolecule fragments and a liquid matrix, which absorbs infrared radiation;
- and
- c) analyzing the biological macromolecule fragments in the mixture by IR-MALDI mass spectrometry, thereby determining the sequence of the target nucleic acid molecule.
1 more element
A method of determining the subunit sequence of at least one species of target biological macromolecule, i, comprising the steps of:
- a) contacting the species of target biological macromolecule with at least one agent that cleaves a bond involved in the formation of the target biological macromolecule such that each bond in involved in the formation of the target biological macromolecule is cleaved, thereby producing a nested set of deletion fragments of the species of biological macromolecule;
- b) preparing a mixture for IR-MALDI, comprising the nested set of deletion fragments and a liquid matrix, which absorbs infrared radiation;
- and
- c) determining the molecular mass of each deletion fragment in mixture by IR-MALDI mass spectrometry, thereby determining the subunit sequence of the species of target biological macromolecule.
1 more element
A method of determining the nucleotide sequence of at least one species of nucleic acid, i, comprising the steps of:
- a) synthesizing complementary nucleic acids, which are complementary to the species of nucleic acid to be sequenced, starting from an oligonucleotide primer and in the presence of chain terminating nucleoside triphosphates, thereby producing four sets of base-specifically terminated complementary polynucleotide fragments;
- b) preparing a mixture for IR-MALDI, comprising the four sets of polynucleotide fragments and a liquid matrix, which absorbs infrared radiation;
- and
- c) determining the molecular weight value of each polynucleotide fragment by IR-MALDI mass spectrometry;
- and
- d) determining the nucleotide sequence of the species of nucleic acid by aligning the molecular weight values-according to molecular weight.
3 more elements
A method for determining a sequence of a target nucleic acid, comprising the steps of:
- a) hybridizing at least one partially single stranded target nucleic acid to one or more nucleic acid probes, each probe comprising a double stranded portion, a single stranded portion, and a determinable variable sequence within the single stranded portion, thereby producing at least one hybridized target nucleic acid;
- b) preparing a mixture for IR-MALDI, comprising the hybridized target nucleic acid and a liquid matrix, which absorbs infrared radiation;
- c) determining a sequence of the hybridized target nucleic acid by IR-MALDI mass spectrometry based on the determinable variable sequence of the probe to which the target nucleic acid hybridized;
- and
- d) repeating steps a) to c) a sufficient number of times to determine a sequence of the target nucleic acid.
2 more elements
A method for analyzing biological macromolecules in a sample comprising:
- exposing the sample to infrared radiation, so that one or more of the biological macromolecules are desorbed and ionized;
- and
- analyzing the sample using matrix-assisted laser desorption/ionization mass spectrometry;
- wherein the accuracy of mass determination by matrix-assisted laser desorption/ionization is in the range of about 102 to about 5×103 ppm.
1 more element
A method for determining the presence or absence of a target biological macromolecule in a sample, comprising:
- analyzing the sample using infrared matrix-assisted laser desorption/ionization mass spectrometry and a liquid matrix;
- wherein the target biological macromolecule is detected if present in the sample.
Dependencies are inferred from the claim text.