Time-of-flight mass spectrometry analysis of biomolecules
First Claim
Patent Images
1. A time-of-flight mass spectrometer comprising:
- a) a sample holder for providing a source of ions from a liquid or solid sample;
b) an ionizer for ionizing the source of ions to form sample ions;
c) means for controllably generating a preselected non-periodic non-zero electric field which imposes a retarding force on the sample ions; and
d) means for generating a different electric field at a time subsequent to ionizing the source of ions and generating the preselected electric field to extract the ions.
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Abstract
A time-of-flight mass spectrometer for measuring the mass-to-charge ratio of a sample molecule is described. The spectrometer provides independent control of the electric field experienced by the sample before and during ion extraction. Methods of mass spectrometry utilizing the principles of the invention reduce matrix background, induce fast fragmentation, and control the transfer of energy prior to ion extraction.
277 Citations
109 Claims
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1. A time-of-flight mass spectrometer comprising:
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a) a sample holder for providing a source of ions from a liquid or solid sample; b) an ionizer for ionizing the source of ions to form sample ions; c) means for controllably generating a preselected non-periodic non-zero electric field which imposes a retarding force on the sample ions; and d) means for generating a different electric field at a time subsequent to ionizing the source of ions and generating the preselected electric field to extract the ions. - View Dependent Claims (2)
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3. A time-of-flight mass spectrometer for measuring the mass-to-charge ratio of ions generated from a sample comprising:
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a) a sample holder; b) a sample ionizer for generating a pulse of sample ions from a sample disposed on the holder; c) a first element spaced apart from the sample holder; and d) a power source electrically coupled to the first element and the holder for i) applying a variable potential to each of the first element and the holder to establish a non-zero retarding electric field wherein the first element and holder potentials are variable independently before ion extraction, and ii) applying a second variable potential for ion extraction to each of the first element and the holder, wherein the second variable potential is applied subsequent to establishing the retarding electric field and the first element and holder potentials are variable indpendently. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A time-of-flight mass spectrometer for measuring the mass-to-charge ratio of ions generated from a sample comprising:
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a) a sample holder; b) a laser which generates a pulse of energy for irradiating and thereby ionizing a sample disposed on the holder; c) a first element spaced apart from the holder; d) a second element spaced apart from the first element; and e) a power source responsive to the pulse of energy and electrically coupled to the first element, second element, and the holder for applying a potential to each of the first element, second element, and holder wherein i) the potential between the first element and holder defines a first electric field and the potential between the second element and the first element defines a second electric field, ii) the potentials on the first element and the holder are independently variable before ion extraction, and iii) the potentials on the first element, the second element, and the holder form a non-zero retarding electric field, and initiate ion extraction at a predetermined time subsequent to generation of the pulse of energy and subsequent to formation of the retarding electric field. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method of determining the mass-to-charge ratio of ions generated from molecules in a sample by time-of-flight mass spectrometry comprising:
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a) applying a first potential to a sample holder; b) applying a second potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a non-zero first electric field between the sample holder and the first element; c) ionizing a sample proximately disposed to the holder to form sample ions; and d) varying at least one of the first or second potentials at a predetermined time subsequent to steps a) through c) to defined a second different electric field between the sample holder and the first element which extracts ions for a time-of-flight measurement. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42)
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43. A method of improving mass resolution in time-of-flight mass spectrometry by compensating for an initial velocity distribution of ions to at least second order comprising:
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a) applying a potential to a sample holder; b) applying a potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a non-zero first electric field between the sample holder and the first element operative spatially to separate ions by their mass prior to ion extraction; c) ionizing a sample proximately disposed to the holder to form sample ions; d) applying a second potential to either the sample holder or the first element at a predetermined time subsequent to steps a) through e) which, together with the potential on the first element, defines a second electric field between the sample holder and the first element, and which extracts the ions from the first element after the predetermined time; and e) energizing an ion reflector spaced apart from the first element, the first and second electric fields and the predetermined time are chosen such that a flight time of extracted ions of like mass-to-charge ratio from the reflector to a detector will be independent to second order of the initial velocity. - View Dependent Claims (44, 45, 46, 47, 48)
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49. A method of improving resolution in laser desorption/ionization time-of-flight mass spectrometry by reducing the number of high energy collisions during ion extraction comprising:
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a) applying a potential to a sample holder; b) applying a potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a non-zero first electric field between the sample holder and the first element; c) ionizing a sample proximately disposed to the holder to form a cloud of ions with a laser which generates a pulse of energy; and d) applying a second potential to either the sample holder or to the sample at a predetermined time subsequent to steps a) through c) which; i) together with the potential on the first element, defines a second electric field between the sample holder and the first element; and ii) extracts the ions after the predetermined time, wherein the predetermined time is long enough to allow the cloud of ions to expand enough to substantially eliminate the addition of excessive collisional energy to the ions during ion extraction. - View Dependent Claims (50, 51, 52, 53, 54, 55)
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56. A method of reducing matrix ion signal in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry comprising:
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a) incorporating a matrix molecule into a sample; b) applying a first potential to a sample holder; c) applying a potential to a first element spaced apart from the sample holder to create a first electric field between the sample holder and the first element, wherein the potential on the first element is more positive than the potential on the sample holder for measuring positive ions and is more negative than the potential on the sample holder for measuring negative ions; d) irradiating a sample proximately disposed to the holder with a laser producing a pulse of energy which is absorbed by the matrix molecule for facilitating desorption and ionization of the sample and the matrix, wherein the first electric field spatially separates the sample ions from the matrix ions by their mass-to-change ratio and the lighter matrix ions are directed back to the sample where they are neutralized on the sample surface; and e) applying a second potential to either the sample holder or the first element at a predetermined time subsequent to the pulse of energy so that the second potential creates a second electric field between the sample holder and the first element to extract the ions. - View Dependent Claims (57, 58, 59, 60)
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61. A method of reducing background chemical ionization noise in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by ion extraction comprising:
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a) incorporating a matrix compound into a sample comprising one or more kinds of molecules to be analyzed so that the matrix substance facilitates desorption and ionization of the one or more molecules; b) applying a potential to a sample holder; c) applying a potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a first non-zero electric field between the sample holder and the first element; d) ionizing a sample proximately disposed to the holder with a laser which generates a pulse of energy which is absorbed by the matrix molecules; and e) applying a second potential to the sample holder or to the first element at a predetermined time subsequent to steps a) through d) which, i) together with the potential on the first element, defines a second electric field between the sample holder and the first element and ii) which extracts the ions, wherein the predetermined time is long enough to allow substantially all fast fragmentation processes to complete. - View Dependent Claims (62, 63, 64, 65, 66, 67)
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68. A method of improving resolution in long-pulse laser desorption/ionization time-of-flight mass spectrometry comprising:
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a) applying a first potential to a sample holder; b) applying a second potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a first electric field between the sample holder and the first element; c) ionizing a sample proximately disposed to the holder to form ions with a laser which generates a pulse of energy with a long time duration; and d) varying at least one of the first or second potentials at a predetermined time subsequent to steps a) through c) to define a second different electric field between the sample holder and the first element which extracts ions for a time-of-flight measurement. - View Dependent Claims (69, 70, 71, 72, 73, 74, 75)
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76. A method for increasing the yield of sequence defining fragment ions of biomolecules arising from fast fragmentation, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry comprising:
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a) incorporating a matrix molecule into a sample comprising one or more biomolecules to be analyzed, to facilitate desorption and ionization of the molecule; b) applying a potential to a sample holder proximately disposed to the sample; c) applying a potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a first non-zero electric field between the sample holder and the first element; d) ionizing and fragmenting the molecules with a laser which generates a pulse of energy which is absorbed by the matrix; and e) applying a second potential to either the sample holder or the first element at a predetermined time subsequent to steps a) through d) which, i) together with the potential on the first element, defines a second electric field between the sample holder and the first element and ii) which extracts the ions after the predetermined time, wherein the predetermined time is long enough to allow substantially all the fast fragmentation processes to complete. - View Dependent Claims (77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 89, 90)
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86. The method of 85 wherein the additive absorbs at the wavelength of the laser pulse but it is not effective as a matrix in itself.
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87. The method of 85 wherein the additive does not absorb at the wavelength of the laser and it is not effective as a matrix in itself.
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91. A method of sequencing DNA by mass spectrometry comprising the steps of:
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a) applying a first potential to a sample holder comprising fragments of a piece of DNA of unknown sequence; b) applying a second potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a non-zero first electric field between the sample holder and the first element; c) ionizing a sample proximately disposed to the holder to form sample ions; d) varying at least one of the first or second potentials at a predetermined time subsequent to steps a) through c) to define a second different electric field between the sample holder and the first element which extracts ions for a time-of-flight measurement; and e) obtaining mass-to charge ratios of the ions generated and using the ratios to obtain the sequence of the piece of DNA. - View Dependent Claims (92, 93, 94)
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95. A method of improving resolution in laser desorption/ionization time-of-flight mass spectrometry for nucleic acids by reducing collisions and ion charge exchange during ion extraction comprising:
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a) applying a potential to a sample holder comprising a nucleic acid; b) applying a potential to a first clement spaced apart from the sample holder which, together with the potential on the sample holder, defines a non-zero first electric field between the sample holder and the first element; c) ionizing a sample proximately disposed to the holder to form a cloud of ions with a laser which generates a pulse of energy; and d) applying a second potential to the sample holder at a predetermined time subsequent to steps a) through c) which; i) together with the potential on the first element, defines a second electric field between the sample holder and the first element; and ii) extracts the ions after the predetermined time, wherein the predetermined time is long enough to allow the cloud of ions to expand enough to substantially eliminate the addition of collisional energy and charge transfer from the ions during ion extraction. - View Dependent Claims (96, 97, 98, 99)
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100. A method of reducing matrix noise in matrix-assisted laser desorption/ionization time-of-flight mass spectrometer comprising:
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a) incorporating a matrix molecule into a sample comprising a nucleic acid; b) applying a first potential to a sample holder; c) applying a potential to a first element spaced apart from the sample holder to create a first electric field between the sample holder and the first element, wherein the potential on the first element is more positive than the potential on the sample holder for measuring positive ions and is more negative than the potential on the sample holder for measuring negative ions; d) irradiating a sample proximately disposed to the holder with a laser producing a pulse of energy having an energy substantially corresponding to an absorption energy of the matrix molecule for facilitating desorption and ionization of the sample and the matrix, wherein the first electric field spatially separates the sample ions from the matrix ions by their mass; and e) applying a second potential to either the sample holder or the first element at a predetermined time subsequent to the pulse of energy so that the second potential creates a second electric field between the sample holder and the first element to extract the ions.
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101. A method of reducing background chemical ionization noise in matrix-assisted laser desorption ionization time-of-flight mass spectrometry of nucleic acids by inducing fragmentation prior to ion extraction comprising:
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a) incorporating a matrix molecule into a sample comprising one or more nucleic acid molecules to be analyzed so that the matrix substance facilitates desorption and ionization of the one or more molecules; b) applying a potential to a sample holder; c) applying a potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a non-zero first electric field between the sample holder and the first element; d) ionization and fragmenting a sample proximately disposed to the holder with a laser which generates a pulse of energy substantially corresponding to an absorption energy of the matrix molecule; and e) applying a second potential to the sample holder at a predetermined time subsequent to steps a) through d) which, i) together with the potential on the first element, defines a second electric field between the sample holder and the first element and ii) which extracts the ions, wherein the predetermined time is long enough to allow substantially all fast fragmentation to complete.
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102. A method of determining the mass-to-charge ratio of ions generated from molecules in a sample by time-of-flight mass spectrometry comprising:
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a) applying a first potential to a sample holder; b) applying a second potential to a first element spaced apart from the sample holder which, together with the potential on the sample holder, defines a first electric field between the sample holder and the first element, wherein the first electric field is retarding so that ions are accelerated toward the sample holder with an approximately optimum magnitude, E1 given by
space="preserve" listing-type="equation">E.sub.1 =5mv.sub.0 /Δ
t,where m is a smallest mass of interest in Daltons, v0 is a most probable initial velocity in meters/second, and Δ
t is a delay time, in nanoseconds, between ionization and extraction;c) ionizing a sample proximately disposed to the holder to form sample ions; and d) varying at least one of the first or second potentials at a predetermined time subsequent to step c to define a second different electric field between the sample holder and the first element which extracts ions for a time-of-flight measurement. - View Dependent Claims (103, 104, 105, 106, 107, 108)
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109. A method of obtaining accurate molecular weights by matrix assisted laser desorption/ionization time-of-flight mass spectrometry by delaying ion extraction long enough for a plume of ions to dissipate such that substantially no energy loss is due to collisions the method comprising:
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a) applying a potential to a sample holder; b) applying a potential to a first element spaced apart from the sample holder, wherein the potential on the first element with respect to the sample holder is more positive when measuring positive ions and more negative for measuring negative ions to spatially separate ions by their mass prior to ion extraction; c) ionizing a sample proximately disposed to the holder to form a cloud of ions with a laser which generates a pulse of energy; and d) applying a second potential to either the sample holder or to the sample at a predetermined time subsequent to steps a) through c) which; i) together with the potential on the first element, defines a second electric field between the sample holder and the first element; and ii) extracts the ions after the predetermined time, wherein the predetermined time is long enough to allow the cloud of ions to expand enough to substantially eliminate the addition of excessive collisional energy to the ions during ion extraction.
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Specification