U.S. patent application number 10/308889 was filed with the patent office on 2004-04-29 for time-of-flight mass spectrometry analysis of biomolecules.
This patent application is currently assigned to PerSeptive Biosystems, Inc.. Invention is credited to Juhasz, Peter, Vestal, Marvin L..
Application Number | 20040079878 10/308889 |
Document ID | / |
Family ID | 23772982 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040079878 |
Kind Code |
A1 |
Vestal, Marvin L. ; et
al. |
April 29, 2004 |
Time-of-flight mass spectrometry analysis of biomolecules
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 this invention
reduce matrix background, induce fast fragmentation, and control
the transfer of energy prior to ion extraction.
Inventors: |
Vestal, Marvin L.;
(Framingham, MA) ; Juhasz, Peter; (Watertown,
MA) |
Correspondence
Address: |
Bowditch & Dewey, LLP
161 Worcester Road
P.O . Box 9320
Framingham
MA
01701-9320
US
|
Assignee: |
PerSeptive Biosystems, Inc.
Framingham
MA
|
Family ID: |
23772982 |
Appl. No.: |
10/308889 |
Filed: |
December 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10308889 |
Dec 3, 2002 |
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09086861 |
May 29, 1998 |
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6541765 |
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09086861 |
May 29, 1998 |
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08730822 |
Oct 17, 1996 |
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5760393 |
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08730822 |
Oct 17, 1996 |
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08488127 |
Jun 7, 1995 |
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5627369 |
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08488127 |
Jun 7, 1995 |
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08446544 |
May 19, 1995 |
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5625184 |
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Current U.S.
Class: |
250/287 ;
250/282; 250/288 |
Current CPC
Class: |
H01J 49/403 20130101;
H01J 49/164 20130101 |
Class at
Publication: |
250/287 ;
250/288; 250/282 |
International
Class: |
H01J 049/40 |
Claims
What is claimed is:
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 force on the
sample ions; and d) means for generating a different electric field
to extract the ions.
2. The mass spectrometer of claim 1 wherein the ionizer is a laser
which generates a pulse of energy with a duration substantially
greater than a time corresponding to a required mass
resolution.
3. A time-of-flight mass spectrometer for measuring the
mass-to-charge ratio of ions generated from a sample comprising: 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 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 first element and holder potentials are
variable independently.
4. The mass spectrometer of claim 3 comprising a means for
controlling the power source to establish a retarding electric
field before ion extraction.
5. The mass spectrometer of claim 3 further comprising means for
controlling the power source to set the potential of the first
element with respect to the potential of the holder more positive
when measuring positive ions and more negative for measuring
negative ions prior to ion extraction.
6. The mass spectrometer of claim 3 further comprising a second
element, spaced apart from the first element, for producing an
electric field for accelerating sample ions.
7. The mass spectrometer of claim 3 or 6 further comprising an ion
reflector spaced apart from the first element.
8. The mass spectrometer of claim 3 wherein the ionizer is a pulsed
light source.
9. The mass spectrometer of claim 3 wherein the ionizer is a laser
which generates a pulse of energy.
10. The mass spectrometer of claim 9 further comprising a sample
electrically coupled to the holder, the sample comprising one or
more molecules to be analyzed and a matrix substance which absorbs
radiation at a wavelength substantially corresponding to the pulse
of energy, the matrix facilitating desorption and ionization of
molecules.
11. The mass spectrometer of claim 10 wherein the sample comprises
at least one compound of biological interest selected from the
group consisting of DNA, RNA, polynucleotides and synthetic
variants thereof.
12. The mass spectrometer of claim 10 wherein the sample comprises
at least one biomolecule selected from the group consisting of
peptides, proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
13. The mass spectrometer of claim 3 wherein the first element
comprises a grid.
14. The mass spectrometer of claim 3 wherein the first element
comprises an electrostatic lens.
15. A time-of-flight mass spectrometer for measuring the
mass-to-charge ratio of ions generated from a sample comprising: 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
initiates ion extraction at a predetermined time subsequent to
generation of the pulse of energy.
16. The mass spectrometer of claim 15 further comprising means for
controlling the power source to set the potential of the first
element with respect to the potential of the holder more positive
when measuring positive ions and more negative for measuring
negative ions prior to ion extraction.
17. The mass spectrometer of claim 15 wherein the power source
further comprises a fast high voltage switch comprising: a) a first
high voltage input; b) a second high voltage input; c) a high
voltage output connectable to the first or second inputs; and d) a
trigger input for operating the switch wherein the output is
switched from the first input to the second input for a
predetermined time when a trigger signal is applied to the trigger
input.
18. The mass spectrometer of claim 17 wherein the first and second
high voltage inputs are electrically connected to at least a 3 kV
power supply and the switch has a turn-on rise time under 200
ns.
19. The mass spectrometer of claim 17 wherein the power source is
at least 1 kV and the switch has a turn-on rise time under 1
microsecond.
20. The mass spectrometer of claim 17 further comprising a delay
generator responsive to the pulse of energy with an output
operatively connected to the trigger input of the switch which
generates a trigger signal to operate the switch in coordination
with the pulse of energy.
21. The mass spectrometer of claim 20 wherein the laser comprises a
means for controlling the delay generator.
22. The mass spectrometer of claim 17 further comprising a delay
generator which initiates the pulse of energy and a signal to the
trigger input of the switch.
23. The mass spectrometer of claim 15 further comprising an ion
reflector spaced apart from the first element.
24. The mass spectrometer of claim 15 further comprising a sample
electrically coupled to the holder, the sample comprising one or
more molecules to be analyzed and a matrix substance which absorbs
radiation at a wavelength substantially corresponding to the pulse
of energy, the matrix facilitating desorption and ionization of
molecules.
25. The mass spectrometer of claim 24 wherein the sample comprises
at least one compound of biological interest selected from the
group consisting of DNA, RNA, polynucleotides and synthetic
variants thereof.
26. The mass spectrometer of claim 24 wherein the sample comprises
at least one biomolecule selected from the group consisting of
peptides, proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
27. The mass spectrometer of claim 15 wherein the first and second
elements comprise grids.
28. The mass spectrometer of claim 15 wherein at least one of the
first or second elements comprises an electrostatic lens.
29. The mass spectrometer of claim 15 further comprising a circuit
for comparing the voltage between the holder and the first
element.
30. The mass spectrometer of claim 15 further comprising an ion
detector for detecting ions generated by the laser and accelerated
by the second element.
31. The mass spectrometer of claim 30 further comprising a guide
wire to attract the ions to the detector.
32. The mass spectrometer of claim 15 wherein the second element is
connected to ground potential.
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: 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 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.
34. The method of claim 33 comprising independently varying the
potential on the first element from the potential on the sample
holder.
35. The method of claim 33 comprising independently varying the
potential on the first element from the potential on the sample
holder to establish a retarding electric field to spatially
separate ions by mass-to-charge ratio.
36. The method of claim 33 wherein the potential of the first
element with respect to the potential on the sample holder is more
positive for measuring positive ions and is more negative for
measuring negative ions to spatially separate ions by their mass
prior to ion extraction.
37. The method of claim 33 wherein the sample is ionized by a laser
producing a pulse of energy.
38. The method of claim 37 wherein the sample comprises a matrix
substance which absorbs radiation at a wavelength substantially
corresponding to the pulse of energy, the matrix facilitating
desorption and ionization of molecules.
39. The method of claim 33 further comprising the step of applying
a potential to a second element spaced apart from the first element
which, together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
40. The method of claim 33 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
41. The method of claim 33 wherein the sample comprises at least
one biomolecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
42. The method of claim 33 wherein the first electric field is
equal to zero.
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: 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 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 the ionization
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.
44. The method of claim 43 wherein the potential on the first
element with respect to the potential of the sample holder is more
positive for measuring positive ions and more negative for
measuring negative ions prior to ion extraction.
45. The method of claim 43 further comprising the step of applying
a potential to a second element spaced between the first element
and the reflector which creates an electric field between the first
and second elements to accelerate the ions.
46. The method of claim 43 wherein the first electric field is
zero.
47. The method of claim 43 wherein the sample is ionized by a laser
producing a pulse of energy.
48. The method of claim 43 wherein the sample comprises a matrix
substance which absorbs radiation at a wavelength substantially
corresponding to the pulse of energy, the matrix facilitating
desorption and ionization of molecules.
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: 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 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 the ionization 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.
50. The method of claim 49 wherein the predetermined time is
greater than the time in which the mean free path of ions in the
cloud becomes greater than the distance between the holder and the
first element.
51. The method of claim 49 wherein the potential on the first
element with respect to the sample holder is more positive for
measuring positive ions and more negative for measuring negative
ions to spatially separates ions by their mass prior to ion
extraction.
52. The method of claim 49 wherein the sample comprises a matrix
substance which absorbs radiation at a wavelength substantially
corresponding to the pulse of energy, the matrix facilitating
desorption and ionization of molecules.
53. The method of claim 49 further comprising the step of applying
a potential to a second element spaced apart from the first element
which, together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
54. The method of claim 49 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
55. The method of claim 49 wherein the sample comprises at least
one biomolecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
56. A method of reducing matrix ion signal in matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry comprising:
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.
57. The method of claim 56 further comprising the step of applying
a potential to a second element spaced apart from the first element
which creates an electric field between the first and second
elements to accelerate the ions.
58. The method of claim 56 wherein the potential on the first
element is about 0.1-5.0% greater than the potential on the sample
holder for measuring positive ions and about 0.1-5.0% lower than
the potential on the sample holder for measuring negative ions.
59. The method of claim 56 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
60. The method of claim 56 wherein the sample comprises at least
one bio-molecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
61. A method of reducing background chemical ionization noise in
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry by ion extraction comprising: 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 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 the
ionization 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.
62. The method of claim 61 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.
63. The method of claim 61 further comprising the step of applying
a potential to a second element spaced apart from the first element
which, together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
64. The method of claim 61 wherein the predetermined time is
greater than the time it takes for substantially all of the ions to
fragment.
65. The method of claim 61 wherein the predetermined time is
greater than 50 ns.
66. The method of claim 61 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
67. The method of claim 61 wherein the sample comprises at least
one bio-molecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
68. A method of improving resolution in long-pulse laser
desorption/ionization time-of-flight mass spectrometry comprising:
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 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.
69. The method of claim 68 wherein the time duration of the pulse
of energy is greater than 50 ns.
70. The method of claim 68 wherein the predetermined time is
greater than the duration of the pulse of energy.
71. The method of claim 68 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 separates ions by their mass prior to ion
extraction.
72. The method of claim 68 wherein the sample comprises a matrix
substance which absorbs radiation at a wavelength substantially
corresponding to the pulse of energy, the matrix facilitating
desorption and ionization of molecules.
73. The method of claim 68 further comprising the step of applying
a potential to a second element spaced apart from the first element
which, together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
74. The method of claim 68 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
75. The method of claim 68 wherein the sample comprises at least
one bio-molecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates glycoclyugates, and glycoproteins.
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: 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 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
the ionization 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.
77. The method of claim 76 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 separates ions by their mass prior to ion
extraction.
78. The method of claim 76 further comprising the step of applying
a potential to a second element spaced apart from the first element
which, together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
79. The method of claim 76 further comprising detecting the mass of
the sequence specific fragments generated.
80. The method of claim 79 comprising identification of the
sequence of at least one biomolecule in the sample.
81. The method of claim 76 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
82. The method of claim 76 wherein the sample comprises at least
one bio-molecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
83. The method of claim 76 comprising increasing the yield of
fragments generated by increasing the energy transfer to the
biomolecule during ionization.
84. The method of claim 83 wherein the energy transfer is increased
by selecting a laser wavelength at which the biomolecule
absorbs.
85. The method of claim 83 wherein the energy transfer is increased
by incorporating an additive to the matrix.
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.
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.
88. The method of claim 76 wherein the matrix is selected to
specifically promote fragmentation of biomolecules.
89. The method of claim 88 wherein the biomolecule is an
oligonucleotide and the matrix comprises at least one of
2,5-dihydroxybenzoic acid and picolinic acid.
90. The method of claim 76 wherein the biomolecule is an
polynucleotide.
91. A method of sequencing DNA by mass spectrometry comprising the
steps of: 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 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
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; and e) obtaining mass-to charge ratios
of the ions generated and using the ratios to obtain the sequence
of the piece of DNA.
92. The method of claim 91 wherein the DNA in the sample is
fragmented to produce sets of DNA fragments, each having a common
origin and terminating at a particular base along the DNA
sequence.
93. The method as defined in claim 92, wherein the sample comprises
different sets of DNA fragments mixed with a matrix substance
absorbing at a wavelength substantially corresponding to the
quantum energy of the pulse which facilitates desorption and
ionization of the sample.
94. The method of claim 91, wherein the step (e) of obtaining the
sequence of the piece of DNA comprises: a) determining the absolute
mass difference between the detected molecular weight of a peak of
one of the sets of DNA fragments compared to a peak of another of
the sets of DNA fragments.
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: a) applying a potential to a sample holder comprising a
nucleic acid; 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 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 the ionization 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.
96. The method of claim 95 wherein the predetermined time is
greater than the time in which the mean free path of ions in the
cloud approximately equals the distance between the holder and the
first element.
97. The method of claim 95 wherein the predetermined time is
greater than the time it takes for substantially all of fast
fragmentation to complete.
98. The method of claim 95 wherein the sample comprises a matrix
substance absorbing at a wavelength substantially corresponding to
the quantum energy of the pulse to facilitate desorption and
ionization of the sample.
99. The method of claim 95 further comprising the step of applying
a potential to a second element spaced apart from the first element
which, together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
100. A method of reducing matrix noise in matrix-assisted laser
desorption/ionization time-of-flight mass spectrometer comprising:
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.
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: 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 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 the ionization 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.
102. 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: a) applying a potential to a sample holder; b) applying
a potential to a first element spaced apart from the sample holder
which is substantially equal to the potential on the sample holder;
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 the ionization
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.
103. The method of claim 102 further comprising the step of
measuring the time of flight to a detector and calculating the
mass-to-charge ratio from the time of flight measurement.
104. The method of claim 102 wherein the sample comprises a matrix
substance which absorbs radiation at a wavelength substantially
corresponding to the pulse of energy, the matrix facilitating
desorption and ionization of molecules.
105. The method of claim 102 further comprising the step of
applying a potential to a second element spaced apart from the
first element which, together with the potential on the first
element, defines an electric field between the first and second
elements for accelerating the ions.
106. The method of claim 102 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 separates ions by their mass prior to ion
extraction.
107. The method of claim 102 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
108. The method of claim 102 wherein the sample comprises at least
one biomolecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
109. A method of determining the mass-to-charge ratio of ions
generated from molecules in a sample by time-of-flight mass
spectrometry comprising: 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, E.sub.1 given
byE.sub.1=5mv.sub.0/.DELTA.t, where m is a smallest mass of
interest in Daltons, v.sub.0 is a most probable initial velocity in
meters/second, and .DELTA.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.
110. The method of claim 109 comprising independently varying the
potential on the first element from the potential on the sample
holder.
111. The method of claim 109 wherein the sample is ionized by a
laser producing a pulse of energy.
112. The method of claim 109 wherein the sample comprises a matrix
substance which absorbs radiation at a wavelength substantially
corresponding to the pulse of energy, the matrix facilitating
desorption and ionization of molecules.
113. The method of claim 109 further comprising the step of
applying a potential to a second element spaced apart from the
first element which, together with the potential on the first
element, defines an electric field between the first and second
elements for accelerating the ions.
114. The method of claim 109 wherein the sample comprises at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof.
115. The method of claim 109 wherein the sample comprises at least
one biomolecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and glycoproteins.
Description
FIELD OF THE INVENTION
[0001] The invention relates generally to the field of mass
spectrometry. In particular, the invention relates to a pulsed ion
source for time-of-flight mass spectrometry and to methods of
operating a mass spectrometer.
BACKGROUND OF THE INVENTION
[0002] Mass spectrometry is an analytical technique for accurate
determination of molecular weights, the identification of chemical
structures, the determination of the composition of mixtures, and
qualitative elemental analysis. In operation, a mass spectrometer
generates ions of sample molecules under investigation, separates
the ions according to their mass-to-charge ratio, and measures the
relative abundance of each ion.
[0003] Time-of-flight (TOF) mass spectrometers separate ions
according to their mass-to-charge ratio by measuring the time it
takes generated ions to travel to a detector. TOF mass
spectrometers are advantageous because they are relatively simple,
inexpensive instruments with virtually unlimited mass-to-charge
ratio range. TOF mass spectrometers have potentially higher
sensitivity than scanning instruments because they can record all
the ions generated from each ionization event. TOF mass
spectrometers are particularly useful for measuring the
mass-to-charge ratio of large organic molecules where conventional
magnetic field mass spectrometers lack sensitivity. The prior art
technology of TOF mass spectrometers is shown, for example, in U.S.
Pat. Nos. 5,045,694 and 5,160,840 specifically incorporated by
reference herein.
[0004] TOF mass spectrometers include an ionization source for
generating ions of sample material under investigation. The
ionization source contains one or more electrodes or electrostatic
lenses for accelerating and properly directing the ion beam. In the
simplest case the electrodes are grids. A detector is positioned a
predetermined distance from the final grid for detecting ions as a
function of time. Generally, a drift region exists between the
final grid and the detector. The drift region allows the ions to
travel in free flight a predetermined distance before they impact
the detector.
[0005] The flight time of an ion accelerated by a given electric
potential is proportional to its mass-to-charge ratio. Thus the
time-of-flight of an ion is a function of its mass-to-charge ratio,
and is approximately proportional to the square root of the
mass-to-charge ratio. Assuming the presence of only singly charged
ions, the lightest group of ions reaches the detector first and are
followed by groups of successively heavier mass groups.
[0006] In practice, however, ions of equal mass and charge do not
arrive at the detector at exactly the same time. This occurs
primarily because of the initial temporal, spatial, and kinetic
energy distributions of generated ions. These initial distributions
lead to broadening of the mass spectral peaks. The broadened
spectral peaks limits the resolving power of TOF spectrometers.
[0007] The initial temporal distribution results from the
uncertainty in the time of ion formation. The time of ion formation
may be made more certain by utilizing pulsed ionization techniques
such as plasma desorption and laser desorption. These techniques
generate ions during a very short period of time.
[0008] An initial spatial distribution results from ions not being
generated in a well-defined plane perpendicular to the flight axis.
Ions produced from gas phase samples have the largest initial
spatial distributions. Desorption techniques such as plasma
desorption or laser desorption ions result in the smallest initial
spatial distributions because ions originate from well defined
areas on the sample surface and the initial spatial uncertainty of
ion formation is negligible. The initial energy distribution
results from the uncertainty in the energy of the ions during
formation. A variety of techniques have been employed to improve
mass resolution by compensating for the initial kinetic energy
distribution of the ions. Two widely used techniques use an ion
reflector (also called ion mirror or reflectron) and pulsed ion
extraction.
[0009] Pulsed ionization such as plasma desorption (PD) ionization
and laser desorption (LD) ionization generate ions with minimal
uncertainty in space and time, but relatively broad initial energy
distributions. Conventional LD typically employs sufficiently short
pulses (frequently less than 10 nanoseconds) to minimize temporal
uncertainty. However, in some cases, ion generations may continue
for some time after the laser pulse terminates causing loss of
resolution due to temporal uncertainty. Also, in some cases, the
laser pulse generating the ions is much longer than the desired
width of mass spectral peaks (for example, several IR lasers). The
longer pulse length can seriously limit mass resolution. The
performance of LD may be substantially improved by the addition of
a small organic matrix molecule to the sample material, that is
highly absorbing, at the wavelength of the laser. The matrix
facilitates desorption and ionization of the sample.
Matrix-assisted laser desorption/ionization (MALDI) is particularly
advantageous in biological applications since it facilitates
desorption and ionization of large biomolecules in excess of
100,000 Da molecular mass while keeping them intact.
[0010] In MALDI, samples are usually deposited on a smooth metal
surface and desorbed into the gas phase as the result of a pulsed
laser beam impinging on the surface of the sample. Thus, ions are
produced in a short time interval, corresponding approximately to
the duration of the laser pulse, and in a very small spatial region
corresponding to that portion of the solid matrix and sample which
absorbs sufficient energy from the laser to be vaporized. This
would very nearly be the ideal source of ions for time-of-flight
(TOF) mass spectrometry if the initial ion velocities were also
small. Unfortunately, this is not the case. Rapid ablation of the
matrix by the laser produces a supersonic jet of matrix molecules
containing matrix and sample ions. In the absence of an electrical
field, all of the molecular and ionic species in the jet reach
nearly uniform velocity distributions as the result of frequent
collisions which occur within the jet.
[0011] The ion ejection process in MALDI has been studied by
several research groups. R. C. Beavis, B. T. Chait, Chem. Phys.
Lett., 181, 1991, 479. J. Zhou, W. Ens, K. G. Standing, A.
Verentchilkov, Rapid Commun. Mass Spectrom., 6, 1992, 671-678. In
the absence of an electrical field, the initial velocity
distributions for peptide and protein ions produced by MALDI are
very nearly independent of mass of the analyte and laser intensity.
The average velocity is about 550 m/sec with most of the velocity
distibution between 200 and 1200 m/sec. The velocity distribution
for matrix ions is essentially identical to that for the peptides
and proteins near threshold irradiance, but shifts dramatically
toward higher velocities at higher irradiance. The total ion
intensity increases rapidly with increasing laser irradiance,
ranging from about 104 ions per shot near threshold to more than
10.sup.8 at higher irradiance. In the presence of an electrical
field, the ions show an energy deficit due to collisions between
ions and neutrals. This energy deficit increases with both laser
intensity and electrical field strength and is higher for higher
mass analyte ions than it is for matrix ions.
[0012] The observation that the initial velocity distribution of
the ions produced by MALDI is nearly independent of mass implies
that the width of the initial kinetic energy distribution is
approximately proportional to the square root of the mass as well
as the energy deficit arising from collisions with neutral
particles in the accelerating field. Thus the mass resolution, at
high mass, in conventional MALDI decreases with the increasing
mass-to-charge ratio of the ions. Use of high acceleration
potential (25-30 kV) increases the resolution at high mass in
direct proportion to the increase in accelerating potential.
[0013] The adverse effect of the initial kinetic energy
distribution can be partly eliminated by pulsed ion extraction.
Pulsed or delayed ion extraction is a technique whereby a time
delay is introduced between the formation of the ions and the
application of the accelerating field. During the time lag, the
ions move to new positions according to their initial velocities.
By properly choosing the delay time and the electric fields in the
acceleration region, the time of flight of the ions can be adjusted
so as to render the flight time independent of the initial velocity
to the first order.
[0014] Considerable improvements in mass resolution were achieved
by utilizing pulsed ion extraction in a MALDI ion source.
Researchers reported improved resolution as well as fast
fragmentation of small proteins in J. J Lennon and R. S. Brown,
Proceedings of the 42nd ASMS Conference on Mass Spectrometry and
Allied Topics, May 29-Jun. 3, 1994, Chicago, Ill., p 501 Also,
researchers reported significant resolution enhancement when
measuring smaller synthetic polymers on a compact MALDI instrument
with pulsed ion extraction in Breuker et al., 13th International
Mass Spectrometry Conference, Aug. 29-Sep. 3, 1994. Breuker et al.,
13th International Mass Spectrometry Conference, Aug. 29-Sep. 3,
1994, Budapest, Hungary. In addition, researchers reported
considerably improved mass resolution on small proteins with a
pulsed ion extraction MALDI source in Reilly et al. Rapid Commun.,
Mass Spectrometry, 8, 1994, 865-868. S. M. Colby, T. B. King, J. P.
Reilly, Rapid Commun. Mass Spectrom., 8, 1994, 865-868.
[0015] Ion reflectors (also called ion mirrors and reflectrons) are
also used to compensate for the effects of the initial kinetic
energy distribution. An ion reflector is positioned at the end of
the free-flight region. An ion reflector consists of one or more
homogeneous, retarding, electrostatic fields. As the ions penetrate
the reflector, with respect to the electrostatic fields, they are
decelerated until the velocity component in the direction of the
field becomes zero. Then, the ions reverse direction and are
accelerated back through the reflector. The ions exit the reflector
with energies identical to their incoming energy but with
velocities in the opposite direction. Ions with larger energies
penetrate the reflector more deeply and consequently will remain in
the ion reflector for a longer time. In a properly designed
reflector, the potentials are selected to modify the flight paths
of the ions such that ions of like mass and charge arrive at the
detector at the same time regardless of their initial energy.
[0016] The performance of a mass spectrometer is only partially
defined by the mass resolution. Other important attributes are mass
accuracy, sensitivity, signal-to-noise ratio, and dynamic range.
The relative importance of the various factors defining overall
performance depends on the type of sample and the purpose of the
analysis, but generally several parameters must be specified and
simultaneously optimized to obtain satisfactory performance for a
particular application.
[0017] Unfortunately, utilizing the prior art techniques, the
performance of TOF mass spectrometers is inadequate for analysis of
many important classes of compounds. These inadequacies are
particularly apparent with MALDI. There are several mechanisms that
may limit the performance of TOF mass spectrometry in addition to
the loss of mass resolution associated with the initial kinetic
energy distribution. An excess of generated matrix ions may cause
saturation of the detector. Due to a long recovery time of many
detectors, saturation seriously inhibits the true reproduction of
the temporal profile of the incoming ion current which constitutes
essentially the TOF spectrum.
[0018] Fragmentation processes have been observed to proceed at
three different time scales in MALDI TOF, E. Nordhoff, et al.,
J.Mass Spectrom., 30 1995, 99-112. Extremely fast fragmentation can
take place essentially during the time of the ionization event.
This process is referred to as prompt fragmentation. and the
fragment ions will give a correlated ion signal in a continuous ion
extraction MALDI TOF measurement, that is, fragment ions behave
exactly as if they were present in the sample. Fragmentation can
also take place at a somewhat lower rate during the acceleration
stage (typically with less than one usec characteristic time). This
kind of fragmentation is referred to as fast fragmentation. High
energy collisions (more energetic than thermal collisions) between
ions and neutrals can also contribute to fast fragmentation. These
collisions are particularly frequent in the early stage of ion
acceleration when the ablated material forms a dense plume.
Fragment ions from the fast fragmentation processes, as opposed to
prompt fragments, contribute to uncorrelated noise (chemical noise)
since they will be accelerated to a wide range of kinetic energies
unlike the original sample ions which are accelerated to one
well-defined kinetic energy.
[0019] Fragmentation of sample ions may also occur in the
free-flight region which occurs on a longer time scale comparable
with the flight time of the ions. This may or may not be desirable
depending on the particular type of data that is required from the
time-of-flight mass spectrometer. Generally, fragmentation
decreases the intensity of the signal due to the intact molecular
ions. In mixture analysis, these fragment ions can produce
significant chemical noise which interferes with detection of the
signals of interest. Also, fragmentation within a reflector further
reduces the intensity of the signal of interest and further
increases the interfering background signal.
[0020] When fragmentation occurs in a drift region, except for the
very small relative velocity of the separating fragments, both the
ion and neutral fragment continue to move with nearly the same
velocity as the intact ions and arrive at the end of the field-free
region at essentially the same time, whether or not fragmentation
has occurred. Thus in a simple TOF analyzer, without reflector,
neither the resolution nor the sensitivity is seriously degraded by
fragmentation after acceleration.
[0021] On the other hand, in the reflecting analyzer the situation
is quite different. Fragment ions have essentially the same
velocity as the intact ions, but having lost the mass of the
neutral fragment, have proportionally lower energy. Thus the
fragment ions penetrate a shorter distance into the reflecting
field and arrive earlier at the detector than do the corresponding
intact ions. By suitable adjustment of the mirror potentials these
fragment ions may be focused to produce a high quality post-source
decay (PSD) spectrum which can be used to determine molecular
structure.
[0022] It is therefore a principal object of this invention to
improve the performance of time-of-flight mass spectrometers,
particularly in regard to applications involving production of ions
from surfaces, by improving resolution, increasing mass accuracy,
increasing signal intensity, and reducing background noise. It is
another object to reduce the matrix ion signal in MALDI
time-of-flight mass spectrometers. Another objective is to provide
TOF mass spectrometers suitable for fast sequencing of biopolymers
such as nucleic acids, peptides, proteins, and polynucleotides by
the analysis of chemically or enzymatically generated ladder
mixtures. Still another objective is to utilize fast fragmentation
processes for obtaining structural information on biomolecules such
as oligonucleotides, carbohydrates, and glycoconjugates. Yet,
another objective is to control the extent of fast fragmentation by
selecting the most appropriate experimental conditions in a pulsed
ion extraction TOF mass spectrometer.
SUMMARY OF THE INVENTION
[0023] The invention features a time-of-flight (TOF) mass
spectrometer for measuring the mass-to-charge ratio of ions
generated from a sample. The mass spectrometer includes a sample
holder for providing a source of ions from a liquid or solid sample
and an ionizer for ionizing the source of ions to form sample ions.
The mass spectrometer also includes a means for controllably
generating a preselected non-periodic non-zero electric field which
imposes a force on the sample ions prior to extracting the ions and
a means for generating a different electric field to extract the
ions. The ionizer may be a laser which generates a pulse of
energy.
[0024] Alternatively, the mass spectrometer includes a sample
holder, a means for ionizing a sample disposed on the holder to
generate sample ions, and a first element spaced apart from the
sample holder. The mass spectrometer may include a drift tube and a
detector. The ionizer may be a laser which generates a pulse of
energy for irradiating and thereby ionizing a sample disposed on
the holder. The first element may be a grid or an electrostatic
lens. A power source is electrically coupled to the first element
and the holder. The source generates a variable potential to each
of the first element and the holder wherein the first element and
holder potentials are independently variable. The potential on the
first element together with the potential on the holder defines an
electric field between the holder and the first element. The mass
spectrometer may also include a circuit for comparing the voltage
between the holder and the first element.
[0025] The mass spectrometer may include a second element for
producing an electric field spaced apart from the first element for
accelerating sample ions. The second element is connectable to an
electrical potential independent of the potential on the holder and
the first element. The second element may be connected to ground or
may be connected to the power supply. The second element may be a
grid or an electrostatic lens. The potential on the second element
together with the potential on the first element defines an
electric field between the first and second elements. The mass
spectrometer may also include an ion reflector spaced apart from
the first element which compensates for energy distribution of the
ions after acceleration.
[0026] The mass spectrometer may include a power supply, a fast
high voltage switch comprising a first high voltage input, a second
high voltage input, a high voltage output connectable to the first
or second inputs; and a trigger input for operating the switch. The
output is switched from the first input to the second input for a
predetermined time when a trigger signal is applied to the trigger
input. The first and second high voltage inputs are electrically
connected to at least a 1 kV power supply and the switch has a
turn-on rise time less than 1 .mu.s.
[0027] The mass spectrometer may include a delay generator
responsive to the laser output pulse of energy with an output
operatively connected to the trigger input of the switch which
generates a trigger signal to operate the fast high voltage switch
in coordination with the pulse of energy. The laser may initiate
timing control by means of a photodetector responsive to the laser
pulse, or the laser itself may include a circuit which generates an
electrical signal synchronized with the pulse of energy (for
example, a Pockels cell driver). Alternately, the delay generator
may initiate both the pulse of energy and the trigger input.
[0028] The mass spectrometer must include an ion detector for
detecting ions generated by the ionizer. The mass spectrometer may
also include a guide wire to limit the cross sectional area of the
ion beam so that a small area detector can be used. The mass
spectrometer may include a computer interface and computer for
controlling the power sources and the delay generator, and a
computer algorithm for calculating the optimum potentials and time
delay for a particular application.
[0029] The present invention also features a method of determining
the mass-to-charge ratio of molecules in a sample by time-of-flight
mass spectrometry. The method includes applying a first potential
to a sample holder. A second potential is applied 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. The potential on
the first element is independently variable from the potential on
the sample holder.
[0030] A sample proximately disposed to the holder is ionized to
generate sample ions. The method may include ionizing the sample
with a laser or a light source producing a pulse of energy. At
least one of the first or second potentials are varied at a
predetermined time subsequent to the ionization event to define a
second electric field between the sample holder and the first
element which extracts the ions for a time-off-light measurement.
The optimum time delay between the ionization pulse and application
of the second electrical field (the extraction field) depends on a
number of factors, including the distance between the sample
surface and the first element, the magnitude of the second
electrical field, the mass-to-charge ratio of sample ions for which
optimum resolution is required, and the initial kinetic energy of
the ion. The method may also include a computer algorithm for
calculating the optimum values of the time delay and electric
fields, and use of a computer and computer interface to
automatically adjust the outputs of the power sources and the delay
generator.
[0031] The method may include independently varying the potential
on the first element from the potential on the sample holder. The
potential on the first element may be independently varied from the
potential on the sample holder to establish a retarding electric
field to spatially separate ions by mass-to-charge ratio prior to
ion extraction.
[0032] The method may include the step of applying a potential to a
second element spaced apart from the first element which, together
with the potential on the first element, defines an electric field
between the first and second elements for accelerating the ions.
The method may also include analyzing a sample comprising at least
one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one compound of biological interest selected
from the group consisting of peptides, proteins, PNA,
carbohydrates, and glycoproteins. The sample may include a matrix
substance absorbing at the wavelength of the laser pulse to
facilitate desorption and ionization of the one or more
molecules.
[0033] Utilizing this method improves the resolution of
time-of-flight mass spectrometers by reducing the effect of the
initial temporal and energy distributions on the time-of-flight of
the sample ions. The method may also include the step of energizing
an ion reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0034] The present invention also features 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. A potential is applied to a sample holder
comprising one or more molecules to be analyzed. A potential is
applied 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. A sample proximately disposed to the holder is ionized
with a laser, which generates a pulse of energy to form a cloud of
ions.
[0035] A second potential is applied at either the sample holder or
the first element at a predetermined time subsequent to ionization
which, together with the potential on the sample holder or first
element, defines a second electric field between the sample and the
first element. The second electric field extracts the ions after
the predetermined time. The predetermined time is long enough to
allow the cloud of ions and neutrals to expand enough to
substantially reduce the number of high energy collisions when the
extracting field is activated. The predetermined time may be
greater than the time it takes the mean free path of the ions in
the plume to become greater than the size of the accelerating
region.
[0036] The method may also include the step of applying a potential
to a second element spaced apart from the first element which,
together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
[0037] Parameters such as the magnitude and direction of the first
and second electric fields, and the time delay between the
ionization pulse and application of the second electric field are
chosen so that the delay time is long enough to allow the plume of
neutrals and ions produced in response to application of the laser
pulse to expand into the vacuum sufficiently so that further
collisions between ions and neutrals are unlikely. Parameters are
also chosen to insure that sample ions of a selected mass are
detected with optimum mass resolution. The parameters may be
determined manually or by use of a computer, computer interface,
and computer algorithm.
[0038] The method may also include analyzing a sample comprising at
least one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one bio-molecule selected from the group
consisting of peptides, proteins, PNA, carbohydrates,
glycoconjugates and glycoproteins. The sample may include a matrix
substance absorbing at the wavelength of the laser pulse to
facilitate desorption and ionization of the one or more
compounds.
[0039] The method may also include the step of energizing an ion
reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0040] The present invention also features a method of reducing the
matrix ion signal in matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry. The method includes incorporating
a matrix molecule into a sample. A first potential is applied to
the sample holder. A potential is applied to a first element spaced
apart from the sample holder to create a first electric field
between the sample holder and the first element. A sample
proximately disposed to the holder is irradiated with a laser which
produces a pulse of energy. The matrix absorbs the energy and
facilitates desorption and ionization of the sample and the matrix.
The first electric field is retarding and thus accelerates ions
toward the sample surface.
[0041] A second potential is applied to the sample holder at a
predetermined time, subsequent to the pulse of energy, which
creates a second electric field between the sample holder and the
first element to accelerate ions away from the sample surface. The
first electric field is chosen to retard the ions generated from
the sample. This field decelerates and directs the ions back toward
the sample surface.
[0042] The method may include the step of applying a potential to a
second element spaced apart from the first element which creates an
electric field between the first and second elements to accelerate
the ions Parameters such as the magnitude and direction of the
first and second electric fields and the time delay between the
ionization pulse and the application of the second electric field
are chosen so that matrix ions having a mass less than a selected
mass are suppressed while sample ions having a mass greater than a
selected mass are detected with optimum mass resolution. The
parameters may be determined manually or by use of a computer,
computer interface, and computer algorithm.
[0043] The method may include analyzing a sample comprising at
least one biological molecule selected from the group consisting of
DNA, RNA, polynucleotides and synthetic variants thereof or at
least one biological molecule selected from the group consisting of
peptides, proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
[0044] The method may also include the step of energizing an ion
reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0045] The present invention also features a method of reducing
background chemical noise in matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry by allowing
time for fast fragmentation processes to complete prior to ion
extraction. A matrix molecule is incorporated into a sample
comprising one or more molecules to be analyzed so that the matrix
substance facilitates intact desorption and ionization of the one
or more molecules. A potential is applied to the sample holder. A
potential is applied 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.
[0046] A sample proximately disposed to the holder is ionized with
a laser which generates a pulse of energy which is absorbed by the
matrix molecule. A second potential is applied to the sample holder
at a predetermined time subsequent to the ionization which,
together with the potential on the first element, defines a second
electric field between the sample and the first element to extracts
the ions. The predetermined time is long enough to substantially
allow all fast fragmentation processes to complete.
[0047] The method may include the step of applying a potential to a
second element spaced apart from the first element which, together
with the potential on the first element, defines an electric field
between the first and second elements for accelerating the
ions.
[0048] Parameters such as the magnitude and direction of the first
and second electric fields, and the time delay between the
ionization pulse and application of the second electric field are
chosen so that the time delay is long enough to allow fast
fragmentation processes to complete. The parameters are also chosen
so that the selected mass is detected with optimum mass resolution.
The parameters may be determined manually or by use of a computer,
computer interface, and computer algorithm.
[0049] The method may include analyzing a sample comprising at
least one bio molecule selected from the group consisting of DNA,
RNA, polynucleotides and synthetic variants thereof or at least one
bio molecule selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and
glycoproteins.
[0050] The method may also include the step of energizing an ion
reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0051] The present invention also features a method of improving
resolution in long-pulse laser desorption/ionization time-of-flight
mass spectrometry. A first potential is applied to a sample holder.
A second potential is applied 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. A sample proximately disposed to the holder
is ionized with a long pulse length laser. The time duration of the
pulse of energy may be greater than 50 ns.
[0052] The potential on the first element with respect to the
sample holder may be more positive for measuring positive ions and
more negative for measuring negative ions to reduce the spatial and
velocity spreads of ions prior to ion extraction. At least one of
the first or second potentials is varied at a predetermined time
subsequent ionization to define a second different electric field
between the sample holder and the first element which extracts ions
for a time-of-flight measurement. The predetermined time may be
greater than the duration of the laser pulse.
[0053] The method may include the step of applying a potential to a
second element spaced apart from the first element which, together
with the potential on the first element, defines an electric field
between the first and second elements for accelerating the
ions.
[0054] The sample may comprise a matrix substance absorbing at the
wavelength of the laser pulse to facilitate desorption and
ionization of sample molecules. The sample may also comprise at
least one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one compound of biological interest selected
from the group consisting of peptides, proteins, PNA,
carbohydrates, glycoconjugates and glycoproteins.
[0055] The present invention also features a method of generating
sequence defining fragment ions of biomolecules using
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry. The method includes incorporating a matrix molecule
into a sample comprising one or more molecules to be analyzed, to
facilitate desorption, ionization, and excitation of the molecule.
A potential is applied to the sample. A potential is applied to a
first element spaced apart from the sample which, together with the
potential on the sample, defines a first electric field between the
sample and the first element.
[0056] The molecules are ionized and fragmented with a laser which
generates a pulse of energy substantially corresponding to an
absorption energy of the matrix. A second potential is applied to
the sample at a predetermined time subsequent to the ionization
which, together with the potential on the first element, defines a
second electric field between the sample and the first element. The
second electric field extracts the ions after the predetermined
time.
[0057] The method may include the step of applying a potential to a
second element spaced apart from the first element which, together
with the potential on the first element, defines an electric field
between the first and second elements for accelerating the
ions.
[0058] Parameters such as the magnitude and direction of the first
and second electric fields, and the time delay between the
ionization pulse and application of the second electric field are
chosen so that the time delay is long enough to allow fast
fragmentation processes to complete. These parameters are also
chosen to detect the selected mass with optimum mass resolution.
The parameters may be determined manually or by use of a computer,
computer interface, and computer algorithm.
[0059] The method may include the step of detecting the
mass-to-charge ratio of the sequence specific fragments generated
and the step of identifying a sequence of at least one kind of
biomolecule in the sample wherein the biomolecule is selected from
the group consisting of DNA, RNA, polynucleotides and synthetic
variants thereof or at least one compound of biological interest
selected from the group consisting of peptides, proteins, PNA,
carbohydrates, glycoconjugates and glycoproteins.
[0060] The method may also include the step of increasing the yield
of fragments generated by increasing the energy transfer to the
biomolecule during ionization. The energy transfer may be increased
by selecting a laser wavelength at which the biomolecule absorbs
Yield of fragment ions may be increased by incorporating an
additive in the matrix. The additive may or may not absorb at the
wavelength of the laser but it is not effective as a matrix in
itself. The additive may facilitate the transfer of energy from the
matrix to the sample.
[0061] The matrix may be selected to specifically promote
fragmentation of biomolecules The biomolecule may be an
oligonucleotide and the matrix may comprise at least one of
2,5-dihydroxybenzoic acid and picolinic acid. The biomolecule may
be a polynucleotide.
[0062] The method may also include the step of energizing an ion
reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0063] The present invention also features a novel form of sample
holder for the claimed mass spectrometer as fully described and
claimed in U.S. application Ser. No. ______ (attorney docket No.
SYP-115, filed concurrently herewith) specifically incorporated
herein by reference. Briefly, the sample holder comprises spatially
separate areas adapted to hold differing concentration ratios of
polymer sample and hydrolyzing agent. After a suitable incubation
period during which the hydrolyzing agent hydrolyzes inter monomer
bonds in the polymer sample in each area, a plurality, typically
all, of the areas containing the species are ionized, typically
serially, in the mass spectrometer, and data representative of the
mass-to-charge ratios of the species in the areas are obtained.
[0064] In another embodiments the invention provides a method for
obtaining sequence information about a polymer comprising a
plurality of monomers of known mass as fully described and claimed
in U.S. application Ser. No. ______ (attorney docket No. SYP-114,
filed concurrently herewith) specifically incorporated herein by
reference. One skilled in the art first provides a set of
fragments, created by the hydrolysis of the polymer, each set
differing by one or more monomers. The difference between the
mass-to-charge ratio of at least one pair of fragments is
determined. One then asserts a mean mass-to-charge ratio which
corresponds to the known mass-to-charge ratio of one or more
different monomers. The asserted mean is compared with the measured
mean to determine if the two values are statistically different
with a desired confidence level. If there is a statistical
difference, then the asserted mean difference is not assignable to
the actual measured difference. In some embodiments, additional
measurements of the difference between a pair of fragments are
taken, to increase the accuracy of the measured mean difference.
The steps of the method are repeated until one has asserted all
desired .mu.s for a single difference between one pair of
fragments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] The foregoing and other objects, features and advantages of
the invention will become apparent from the following more
particular description of preferred embodiments of the invention,
as illustrated in the accompanying drawings. The drawings are not
necessarily to scale, emphasis instead being placed on illustrating
the principles of the present invention.
[0066] FIG. 1 is a schematic diagram of prior art pulsed ion
two-stage acceleration laser desorption/ionization time-of-flight
mass spectrometer.
[0067] FIG. 2 is a schematic diagram of a laser
desorption/ionization time-of-flight mass spectrometer
incorporating certain principles of this invention.
[0068] FIG. 3 is one embodiment of a laser desorption/ionization
time-of-flight mass spectrometer incorporating principles of this
invention.
[0069] FIGS. 4a-b illustrates improvements of mass resolution in
oligonucleotides with a MALDI TOF mass spectrometer incorporating
principles of this invention FIG. 4a is a spectrum of a DNA 22mer
sample recorded by a conventional MALDI TOF mass spectrometer. FIG.
4b is a spectrum of a DNA 22mer sample recorded with a MALDI TOF
mass spectrometer incorporating the principles of this
invention.
[0070] FIG. 5 is a schematic diagram of a laser/desorption
time-of-flight mass spectrometer embodying the invention which
includes a single stage ion reflector.
[0071] FIGS. 6a-b illustrates resolution in excess of 7,000 mass
resolution for a RNA 12mer sample at m/z 3839 and about 5,500 mass
resolution for a RNA 16mer sample at m/z 5154 recorded with a MALDI
TOF mass spectrometer of the type illustrated in FIG. 5.
[0072] FIGS. 7a-c illustrates a reduction and elimination of matrix
signal with a MALDI TOF mass spectrometer incorporating the
principles of the present invention.
[0073] FIGS. 8a-c illustrates induced fragmentation for structural
characterization of oligonucleotides in MALDI TOF mass
spectrometry, including the nomenclature of fragment ion types.
[0074] FIGS. 9a-c illustrates the ability to analyze very complex
oligonucleotide mixtures with a MALDI TOF mass spectrometer
incorporating principles of this invention.
DETAILED DESCRIPTION
[0075] FIG. 1 is a schematic diagram of a prior art pulsed ion
two-stage acceleration laser desorption/ionization time-of-flight
mass spectrometer. A high voltage power supply 11 generates a
variable high voltage at an output 13. A second high voltage power
supply 10 generates a variable high voltage at an output 12 which
is referenced to the output 13 of high voltage power supply 11. The
power supply outputs 12 and 13 are electrically coupled to inputs
14 and 15 of a pulse generator 16. A control circuit 18 for
generating a trigger signal to control the output of the pulse
generator 16 is electrically or optically connected to the trigger
input 20 of the pulse generator 16. The pulse generator 16 passes
the high voltage output of the power supply 11 to a pulse generator
output 22 when the trigger input is inactive. The pulse generator
generates a high voltage pulse whose amplitude is determined by the
high voltage output of high voltage power supply 10 at the pulse
generator output 22 for a predetermined time when the trigger input
is active.
[0076] The pulse generator output 22 is electrically coupled to a
holder 24. A sample under investigation 26 is deposited on a smooth
surface 28 of the holder 24. The holder 24 is an electrically
conductive body on which the sample 26 is typically located. A
laser 30 for irradiating the sample 26 with a pulse of energy is
positioned with an output 32 directed at the sample 26. Molecules
in the sample 26 are ionized and desorbed into the gas phase as the
result of a pulsed laser beam impinging on the surface of the
sample 26. A matrix material highly absorbing at the wavelength of
the laser 30 may be added to the sample in order to facilitate
desorption and ionization of the sample 26. Other means for causing
sample material to be ionized such as plasma desorption, particle
bombardment, etc. also may be used.
[0077] The power supply output 13 is also coupled to a first
element 34 spaced apart from the holder 24. The first element 34
may be a grid or an electrostatic lens. The potential on the holder
24 and on the first element 34 defines an electric field between
the holder 24 and the first element 34 A second element 36 spaced
apart from the first element 34 is electrically connected to a
potential which may be ground. The second element may also be a
grid or an electrostatic lens. A detector 38 spaced apart from the
second element 36 detects ionized sample material as a function of
time.
[0078] In operation, the trigger input 20 is inactive before and
during the time when the laser 30 irradiates the sample 26 with a
pulse of energy. The potential on the holder 24 and on the first
element 34 are both equal to the power supply potential. At a
predetermined time subsequent to the laser pulse, the trigger input
20 becomes active and the pulse generator 16 produces a high
voltage pulse of a predetermined amplitude on the holder. During
the pulse, the potential on the holder 24 exceeds the potential on
the first element 34 in either a positive or a negative direction
depending whether positive or negative ions are under
investigation. The electric field between the holder 24 and the
first element 34 becomes non-zero and the ions are accelerated
towards the second element 36 and the detector 38.
[0079] Thus, with the prior art pulsed ion LD TOF mass
spectrometer, sample ions are generated in a region in which the
same potential is applied to both the holder 24 and the first
element 34 prior to ion extraction. Ions are extracted from the
field free region with the application of a pulse of a
predetermined amplitude at a predetermined time delay subsequent to
the initial ion formation. Initial kinetic energy effects may be
reduced by properly choosing the predetermined pulse amplitude and
time delay.
[0080] FIG. 2 is a schematic diagram of a laser
desorption/ionization time-of-flight mass spectrometer
incorporating principles of this invention. A first high voltage
power supply 50 generates a first variable high voltage at a first
output 52. A second high voltage power supply 54 generates a second
variable high voltage at a second output 56. The first and second
power supplies may be independent, manually controlled or
programmable power supplies or may be a single multi-output
programmable power supply.
[0081] The first and second power supply outputs are electrically
connected to a first input 58 and second input 60 of a fast high
voltage switch 62. An output 64 of the switch is connectable
between the first 58 and second 60 switch inputs. A control circuit
66 for generating a control signal to operate the switch is
electrically connected to a trigger input 68 of the switch. The
output of the switch 64 is electrically coupled to a holder 70
[0082] The holder 70 is an electrically conductive body on which
the sample is located. A sample 72 under investigation is disposed
on a smooth surface 74 of the holder 70. An insulating layer (not
shown) could be interposed between the sample and holder. In an
alternative embodiment, the sample is orthogonally located with
respect to an electric field generated by the holder.
[0083] A laser 76 for irradiating the sample 72 with a pulse of
energy is positioned with an output 78 directed at the sample 72.
The sample 72 is ionized and desorbed into the gas phase as the
result of a pulsed laser beam 80 impinging on the surface of the
sample 72. A matrix material highly absorbing at the wavelength of
the laser 76 may be added to the sample 72 in order to facilitate
desorption and ionization of the sample 72. Other means for causing
sample material to be ionized and desorbed such as plasma
desorption, particle bombardment, etc. also may be used.
[0084] A third power supply 82 is electrically connected to a first
element 84 spaced apart from the holder 70 and generates a third
high voltage. The first element 84 may be a grid or an
electrostatic lens. The potential on the holder 70 and on the first
element 84 defines an electric field between the holder 70 and the
first element 84. A second element 86 spaced apart from the first
element 84 is electrically connected to a potential which may be
ground. The second element 86 may also be a grid or an
electrostatic lens. A detector 88 spaced apart from the second
element 88 detects ionized sample material as a function of
time.
[0085] In operation, the trigger input 68 is inactive before and
during the time when the laser 76 irradiates the sample 72 with a
pulse of energy. The potential on the holder 70 is equal to the
first high voltage generated by the first high voltage power supply
50. The potential on the first element is equal to the third high
voltage generated by the third high voltage power supply 82. If the
first high voltage is different from the third high voltage, there
will be a non-zero static electric field between the holder 70 and
the first element 84.
[0086] At a predetermined time subsequent to the laser pulse, the
control circuit 66 causes the trigger input 68 to become active.
The switch 62 rapidly disconnects the first high voltage power
supply 50 from the holder 70 and rapidly connects the second high
voltage power supply 54 to the holder 70 for a predetermined time.
The potential on the holder 70 rapidly changes from the first high
voltage to the second high voltage. The second high voltage exceeds
the first and third high voltages in either a positive or a
negative direction, depending whether positive or negative ions are
under investigation. Because of the higher potential on the holder
70, an electric field between the holder 70 and the first element
84 is established which extracts and accelerates the ions towards
the second element 86 and the detector 88.
[0087] Thus, with a laser desorption/ionization time-of-flight mass
spectrometer incorporating principles of this invention, there may
be a non-zero non-periodic electric field in the region between the
holder 70 and the first element 84 prior to ion extraction that may
be varied. The mass spectrometer of this invention, therefore,
allows control over the electric field experienced by generated
ions both before and during ion extraction.
[0088] FIG. 3 depicts one embodiment of a laser desorption
time-of-flight mass spectrometer incorporating the principles of
this invention. This embodiment utilizes three independent power
supplies and a fast high voltage switch to independently control
the potential on a sample holder and a first element before and
during ion extraction.
[0089] A first power supply 100 is electrically connected to a
first input 102 of a fast high voltage switch 104. The switch could
be an HTS 300-02 manufactured by Behlke and available from Eurotek,
Inc., Morganville, N.J. with a turn-on delay of approximately 150
ns, a risetime of approximately 20 ns, and an on-time of
approximately 10 microseconds. A second power supply 106 is
electrically connected to a second input 108 of the switch 104. An
output 110 of the switch 104 is connectable to either the first 102
or second 108 inputs but is normally connected to the first input
102 absent a trigger signal. A trigger input 112 causes the switch
104 to disconnect the first power supply 100 from the switch output
110 and to connect the second power supply 106 to the switch output
110 for a predetermined time. The output of the switch 110 is
electrically connected to a sample holder 114. A sample 116 under
investigation is deposited on a smooth metal surface 118 of the
holder such that it is electrically coupled to the holder 114. A
matrix material highly absorbing at the wavelength of a laser 120
used for ionization may be added to the sample 116 in order to
facilitate desorption and ionization of the sample 116.
[0090] A laser 120 for irradiating the sample with a pulse of
energy is positioned with an output 122 directed at the sample 116.
The laser pulse is detected by a photodector 124 for generating an
electrical signal synchronously timed to the pulse of energy. A
delay generator 126 has an input 128 responsive to the
synchronously timed signal and an output 130 electrically connected
to the trigger input of the switch 112. The delay generator 120
produces a trigger signal delayed by a predetermined time with
respect to the synchronously timed signal. Thus in coordination
with the pulse of energy, the switch 104 will disconnect the first
power supply 100 from the switch output 110 and connect the second
power supply 106 to the switch output 110 for a predetermined
time.
[0091] A third power supply 130 which generates a third high
voltage is electrically connected to a first element 132 spaced
apart from the holder 114. The first element 132 may be a grid or
an electrostatic lens. The potential on the holder 114 and on the
first element 132 defines an electric field between the holder 114
and the first element 132. A second element 134 spaced apart from
the first element is electrically connected to a potential which
may be ground. The second element may also be a grid or an
electrostatic lens. A detector 136 such as a channel plate detector
spaced apart from the second element 134 detects ionized sample
material as a function of time. Note that it is the relative
potential and not a particular potential of the holder 114 with
respect to the first and second elements that is important to the
operation of the mass spectrometer.
[0092] A comparing circuit 138 measures and compares the voltage on
the first 100 and third 130 power supplies and indicates the
difference between the first and third voltages The voltage
difference represents the electric field strength between the
holder 114 and the first element 132 prior to ion extraction.
[0093] In operation, before the laser 120 irradiates the sample
116, the holder 114 is electrically connected to the first high
voltage power supply 100 through the switch and the third high
voltage power supply 130 is electrically connected to the first
element 132 Thus before an ionization event, a first electric field
is established between the holder 114 and the first element 132
This electric field is indicated by the comparing circuit 138 and
is adjustable by varying the first and third high voltages.
[0094] To initiate the mass-to-charge measurement, the laser 120
irradiates the sample 116 with a pulse of energy. The laser 120
generates an electrical signal synchronously timed to the pulse of
energy. The delay generator 126 is responsive to the signal. At a
predetermined time subsequent to the signal, the delay generator
126 produces a trigger signal. The fast high voltage switch 104 is
responsive to the trigger signal and causes the switch 104 to
rapidly disconnect the first power supply 100 and rapidly connect
the second power supply 106 to the switch output 110 for a
predetermined time During the predetermined time, the potential on
the holder 114 or the first element 132 changes in magnitude
creating an electric field that causes the ions to be accelerated
towards the second element 134 and the detector 136.
[0095] The present invention also features a method of determining
the mass-to-charge ratio of molecules in a sample by utilizing a
laser desorption/ionization time-of-flight mass spectrometer which
incorporates the principles of this invention. The method includes
applying a first potential to a sample holder having a sample
proximately disposed to the sample holder which comprises one or
more molecules to be analyzed. A second potential is applied 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. The
potential on the first element is independently variable from the
potential on the sample holder. The sample is ionized to generate
sample ions The method may include ionizing the sample with a laser
or a light source producing a pulse of energy. At least one of the
first or second potentials are varied at a predetermined time
subsequent to an ionization event to define a second electric field
between the sample holder and the first element which extracts the
ions for a time-of-flight measurement.
[0096] The optimum time delay between the ionization pulse and
application of the second electrical field depends on a number of
parameters including the distance between the sample surface and
the first element, the magnitude of the second electrical field,
the mass-to-charge ratio of sample ion for which optimal resolution
is required, and the initial kinetic energy of the ion. If the
first electric field is small compared to the second, the time
delay which minimizes the variation in the total flight time with
initial velocity is approximately given by
.DELTA.=144.5d.sub.a(m/V.sub.a).sup.1/2[1/w+(V.sub.o/V.sub.a).sup.1/2]
(1)
[0097] where the time is in nanoseconds, the distance, d.sub.a,
between the sample and the first elements is in millimeters, the
mass, m, in Daltons, the potential difference, V.sub.a, is in
volts, and the initial kinetic energy of the ions of mass, m, is
V.sub.o, in electron volts. The dimensionless parameter, w, depends
upon the geometry of the TOF analyzer. The geometrical parameters
of the TOF analyzer must be chosen so that w is greater than unity.
For the case in which the time of flight analyzer consists only of
the sample plate, a first element, a field-free drift space, and a
detector, the value of w is given by
w=d/d.sub.a-1 (2)
[0098] where d is the length of the field free region between the
first element and the detector.
[0099] This method improves the resolution of time-of-flight mass
spectrometers by reducing the effect of the initial temporal and
energy distributions on the time-of-flight of the sample ions. The
method may include the step of applying a potential to a second
element spaced apart from the first element which, together with
the potential on the first element, defines an electric field
between the first and second elements for accelerating the
ions.
[0100] In this case, the time delay is also given by equation one
(1), but the geometric parameter w is given by
w=(.chi./1+.chi.).sup.3/2[(d/2d.sub.a)-(d.sub.o/d.sub.a)(1+x)]+.chi.(d.sub-
.o/d.sub.a)-1 (3)
[0101] where .chi.=V.sub.a/V, with V being the potential difference
between the first and second element, and d.sub.o is the distance
between the first and second element. For cases in which the
initial temporal distribution of sample ions is relatively broad,
for example, as the result of using a relatively long laser pulse,
it is necessary that the time delay be longer than the total
ionization time. For a given mass this can be accomplished by
reducing the value of V.sub.a. Thus for given initial temporal and
energy distributions for an ion of a particular mass-to-charge
ratio, and for a given TOF analyzer geometry, the magnitude of the
second electric field and the time difference between application
of the laser pulse to the sample and application of the second
electric field can be determined for optimum mass resolution.
[0102] The first electric field is retarding and thus accelerates
ions toward the sample surface. The magnitude of this field may be
freely chosen. An approximately optimum value for the first
electric Field, E.sub.1 is given by
E.sub.1=5mv.sub.0/.DELTA.t (4)
[0103] where m is the smallest mass of interest in Daltons, v.sub.0
is the most probable initial velocity in meters/second, and
.DELTA.t is the delay time, in nanoseconds, between the ionization
pulse and application of the second field. At this magnitude of the
first electric field applied in the retarding direction, ions of
the selected mass with velocity equal to one half the most probable
velocity will be stopped at the time the second field is applied,
and ions with velocity less than one quarter of the most probable
velocity will be returned to the sample surface and neutralized. In
MALDI only a very small fraction of the ions have velocities less
than one quarter of the most probable velocity, thus ions of the
selected and higher masses will be extracted and detected with high
efficiency. On the other hand, ions of lower mass are partially or
totally suppressed. In particular, ions with masses less than about
one quarter of the selected mass are almost completely suppressed
since they return to the sample and are neutralized before
application of the second electric field.
[0104] The method may also include a computer algorithm for
calculating the optimum values for the electric fields and the time
delay, and the use of a computer and computer interface to
automatically adjust the outputs of the power sources and the delay
generator.
[0105] The method may include measuring a sample comprising at
least one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or selected from the group consisting of peptides,
proteins, PNA, carbohydrates, glycoconjugates and glycoproteins.
The sample may include a matrix substance absorbing at the
wavelength of the laser pulse to facilitate desorption and
ionization of the one or more molecules.
[0106] The most significant improvement of performance was observed
for highly polar biopolymers such as oligo- and polynucleotides.
This improved resolution is essential for the mass spectrometric
evaluation of DNA sequencing ladders.
[0107] FIGS. 4a-b illustrates improvements of mass resolution in
oligonucleotides with a MALDI TOF mass spectrometer incorporating
the principles of this invention. FIG. 4a is a spectrum of a 22mer
DNA sample recorded by conventional MALDI. A mass resolution of 281
was obtained. FIG. 4b is a spectrum of the same 22mer DNA sample
recorded with a MALDI TOF mass spectrometer incorporating the
principles of this invention. The mass resolution in FIG. 4b
corresponds to the isotope limited value. For a small protein of
the same molecular mass 500 or 600 mass resolution with
conventional MALDI mass spectrometry is routine. Thus there are
significant improvements in resolution in MALDI TOF mass
spectrometry of DNA and carbohydrates by incorporating the
principles of this invention.
[0108] One advantage of a MALDI TOF mass spectrometer incorporating
the features of the present invention is the ability to correct for
initial kinetic energy spread to a higher order by utilizing an ion
reflector with the mass spectrometer and correctly choosing the
operating parameters.
[0109] FIG. 5 is a schematic diagram of a laser/desorption
time-of-flight mass spectrometer which incorporates the principles
of this invention and includes a single stage ion reflector 150.
This embodiment includes a two-field ion source 152 with a holder
154 and a first 156 and second 158 element. Power supplies (not
shown) are electrically connected to the holder 154 and the first
156 and second 158 elements such that the electric field between
the first element 156 and the holder 154 is variable before ion
extraction as described in the text associated with FIG. 2. This
embodiment also includes a laser 159 for ionizing and desorbing
sample ions. A sample 160 is proximately disposed to the holder
154. The sample 160 may include a matrix molecule that is highly
absorbing at the wavelength of the laser 158. The matrix
facilitates desorption and ionization of the sample 160.
[0110] The ion reflector 150 is positioned at the end of a
field-free drift region 162 and is used to compensate for the
effects of the initial kinetic energy distribution by modifying the
flight path of the ions. A first detector 164 is used for detecting
ions with the ion reflector 150 de-energized. A second detector 166
is used for detecting ion with the ion reflector 150 energized.
[0111] The ion reflector 150 is positioned at the end of the
field-free drift region 162 and before the first detector 164. The
ion reflector 150 consists of a series of rings 168 biased with
potentials that increase to a level slightly greater than an
accelerating voltage. In operation, as the ions penetrate the
reflector 150, they are decelerated until their velocity in the
direction of the field becomes zero. At the zero velocity point,
the ions reverse direction and are accelerated back through the
reflector 150. The ions exit the reflector 150 with energies
identical to their incoming energy but with velocities in the
opposite direction. Ions with larger energies penetrate the
reflector 150 more deeply and consequently will remain in the
reflector for a longer time. The potentials are selected to modify
the flight paths of the ions such that ions of like mass and charge
arrive at the second detector 166 at the same time.
[0112] FIGS. 6a-b illustrates resolutions of nearly 8,000 mass
resolution for a RNA 12mer sample and about 5,500 mass resolution
for a RNA 16mer sample recorded with a MALDI TOF mass spectrometer
having a reflector and incorporating the principles of this
invention. The observed resolution on these examples represents a
lower limit, since the digitizing rate of the detector electronics
is not sufficient to detect true peak profiles in this resolution
range. Comparable performance could be obtained on peptides and
proteins. This invention thus improves resolution for all kinds of
biopolymers. This is in contrast to conventional MALDI where
resolution and sensitivity on oligonucleotides is considerably
degraded in comparison with peptides and proteins.
[0113] Another advantage of a MALDI TOF mass spectrometer,
incorporating the principles of this invention, is the ability to
reduce the number of high energy collisions. Under continuous ion
extraction conditions, ions are extracted through a relatively
dense plume of ablated material immediately after the ionization
event. High energy (higher than thermal energies) collisions result
in fast fragmentation processes during the acceleration phase which
gives rise to an uncorrelated ion signal. This uncorrelated ion
signal can significantly increase the noise in the mass spectra. By
incorporating the principles of present invention in a mass
spectrometer, parameters such as the electric field before and
during ion extraction, and the extraction time delay can be chosen
such that the plume of the ablated material has sufficiently
expanded to reduce the number of high energy collisions.
[0114] The present invention also features a method of improving
resolution in MALDI TOF mass spectrometry by reducing the number of
high energy collisions during ion extraction. A potential is
applied to a sample holder having a sample proximately disposed to
the sample holder. The sample comprises one or more kinds of
molecules to be analyzed. A potential is applied 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. The sample is
ionized with a laser which generates a pulse of energy to ablate a
cloud of ions and neutrals.
[0115] A second potential is applied at either the sample holder or
the first element at a predetermined time subsequent to the
ionization which, together with the potential on the sample holder
or first element, defines a second electric field between the
sample and the first element. The second electric field extracts
the ions after the predetermined time. The predetermined time is
long enough to allow the cloud of ions and neutrals to expand
enough to substantially eliminate the addition of collisional
energy to the ions during ion extraction. The predetermined time
may be greater than the time in which the mean free path of ions in
the cloud exceeds the distance between holder and the first
element.
[0116] The method may also include the step of applying a potential
to a second element spaced apart from the first element which,
together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
[0117] Parameters such as the magnitude and direction of the first
and second electric fields and the time delay between the
ionization pulse and application of the second electric field are
chosen so that the delay time is long enough to allow the plume of
neutrals and ions, produced in response to application of the laser
pulse, to expand into the vacuum sufficiently so that further
collisions between ions and neutrals are unlikely. Parameters are
also chosen to insure that sample ions of a selected mass are
detected with optimum mass resolution. The parameters may be
determined manually or by use of a computer, computer interface,
and computer algorithm.
[0118] The method may also include analyzing a sample comprising at
least one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one compound of biological interest selected
from the group consisting of peptides, proteins, PNA,
carbohydrates, glycoconjugates and glycoproteins. The sample may
include a matrix substance absorbing at the wavelength of the laser
pulse to facilitate desorption and ionization of the biological
molecules.
[0119] The present invention also features a method of reducing the
intensity of the matrix signal in matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry. The method
includes incorporating a matrix molecule into a sample. A first
potential is applied to the sample holder. A potential is applied
to a first element spaced apart from the sample holder to create a
first electric field between the sample holder and the first
element which reverse biases the sample prior to the extraction
pulse. Reverse biasing is accomplished by making the potential of
the first element with respect to the potential of the sample
holder, more positive for measuring positive ions and more negative
for measuring negative ions.
[0120] A sample proximately disposed to the holder is irradiated
with a laser producing a pulse of energy. The matrix absorbs the
energy and facilitates desorption and ionization of the sample and
the matrix. The first electric field is chosen to retard the ions
generated from the sample. This field decelerates and directs the
ions back toward the sample surface at a nearly uniform initial
velocity. The lightest matrix having the smallest mass-to-charge
ratio will be turned back first and naturalized on the sample
holder while the heavier ions from biomolecules can be extracted
for mass analysis.
[0121] A second potential is applied to the sample holder at a
predetermined time subsequent to the pulse of energy which create a
second electric field between the sample holder and the first
element to accelerate ions away from the sample surface. The time
between the laser pulse and application of the second potential is
chosen so that essentially all of the matrix ions have returned to
the sample surface where they are neutralized. Thus the matrix ions
are suppressed and the sample ions are extracted.
[0122] The method may include the step of applying a potential to a
second element spaced apart from the first element which creates an
electric field between the first and second elements to accelerate
the ions. Parameters such as the magnitude and direction of the
first and second electric fields and the time delay between the
ionization pulse and the application of the second electric field
are chosen so that matrix ions having a mass less than a first
selected mass are suppressed while sample ions having a mass
greater than a second selected mass are detected with optimum mass
resolution. The parameters may be determined manually or by use of
a computer, computer interface, and computer algorithm.
[0123] The method may include analyzing a sample comprising at
least one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one compound of biological interest selected
from the group consisting of peptides, proteins, PNA,
carbohydrates, glycoconjugates and glycoproteins.
[0124] The method may also include the step of energizing an ion
reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0125] FIGS. 7a-c illustrates a reduction and elimination of matrix
signal with a MALDI TOF mass spectrometer incorporating the
principles of the present invention. FIG. 7a illustrates nearly
field free conditions where the electric potential of the sample
corresponds approximately to the potential on the grid.
[0126] Sample peaks are labeled with 2867 and 5734. Peaks below
mass-to-charge ratio 400 correspond to matrix ions. In FIG. 7b, the
sample potential is reverse biased 25V with respect to the first
grid. This results in a visible decrease in the abundance of the
lighter matrix ions below a mass-to-charge charge ratio of 200. In
FIG. 7c the sample potential is reverse biased 50V with respect to
the first grid. This results in complete elimination of the matrix
ion signal.
[0127] Another advantage of a MALDI TOF mass spectrometer
incorporating the principles of this invention is the ability to
eliminate the effects of fast fragmentation on background noise and
mass resolution. Fast fragmentation is defined as a fragmentation
taking place during acceleration under continuous ion extraction
conditions. The time scale of fast fragmentation is typically less
than one .mu.sec. Fast fragmentation results in ions of poorly
defined energies and uncorrelated ion noise (chemical noise).
[0128] The present invention also features a method of reducing
background chemical noise in matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry by allowing
time for substantially all fast fragmentation to complete prior to
ion extraction. A matrix molecule is incorporated into a sample
comprising one or more molecules to be analyzed so that the matrix
substance facilitates intact desorption and ionization. A potential
is applied to the sample holder. A potential is applied 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 and the first element.
[0129] The sample is ionized with a laser which generates a pulse
of energy where the matrix absorbs at the wavelength of the laser.
A second potential is applied to the sample holder at a
predetermined time subsequent to the ionization which, together
with the potential on the first element, defines a second electric
field between the sample holder and the first element to extracts
the ions. The predetermined time is long enough to allow
substantially all fast fragmentation processes to complete.
[0130] The method may include the step of applying a potential to a
second element spaced apart from the first element which, together
with the potential on the first element, defines an electric field
between the first and second elements for accelerating the
ions.
[0131] Parameters such as the magnitude and direction of the first
and second electric fields and the time delay between the
ionization pulse and application of the second electric field are
chosen so that the time delay is long enough to allow substantially
all fast fragmentation processes to complete. The parameters are
also chosen so that ions of a selected mass are detected with
optimum mass resolution. The parameters may be determined manually
or by use of a computer, computer interface, and computer
algorithm.
[0132] The method may include analyzing a sample comprising at
least one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one compound of biological interest molecule
selected from the group consisting of peptides, proteins, PNA,
carbohydrates, glycoconjugates and glycoproteins.
[0133] The method may also include the step of energizing an ion
reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0134] Another advantage of a MALDI TOF mass spectrometer,
incorporating the principles of present invention, is the ability
to generate a correlated ion signal for fast fragmentation This can
be accomplished by delaying ion extraction until substantially all
fast fragmentation processes complete. A correspondence can then be
established between the ion signal and the chemical structure or
sequence of the sample.
[0135] Another advantage of a MALDI TOF mass spectrometer
incorporating the principles of present invention is that the yield
of fragment ions can be increased by correctly choosing
experimental parameters such as the reverse bias electric field
between the sample holder and the first element prior to ion
extraction, the delay time between the laser pulse of energy and
the ion extraction, and the laser energy density. This can be
accomplished either by increasing the residence time of precursor
ions in the ion source prior to extraction or promoting additional
energy transfer to the sample molecules undergoing fast
fragmentation. Residence time of precursor ions can be extended by
the proper adjustment of the extracting electric field. Typically a
lower extraction field permit a longer optimum extraction delay and
hence a longer residence time. Energy transfer to the sample can be
enhanced by utilizing very high laser energy densities. Delayed ion
extraction is much more tolerant to excessive laser irradiance than
conventional MALDI. A proper selection of matrix material and
possible additives can also influence energy transfer to the sample
molecules.
[0136] The present invention also features a method of increasing
the yield of sequence defining fragment ions of biomolecules
resulting from fast fragmentation processes using matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry. The
method includes incorporating a matrix molecule into a sample
comprising one or more molecules to be analyzed, to facilitate
desorption, ionization, and excitation of the molecule. A potential
is applied to a sample holder. A potential is applied 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.
[0137] The molecules are ionized and fragmented with a laser which
generates a pulse of energy absorbed by the matrix. A second
potential is applied to the sample holder at a predetermined time
subsequent to the ionization which together with the potential on
the first element, defines a second electric field between the
sample holder and the first element. The second electric field
extracts the ions after the predetermined time. The predetermined
time is long enough to allow substantially all fast fragmentation
to complete.
[0138] The method may include the step of applying a potential to a
second element spaced apart from the first element which, together
with the potential on the first element, defines an electric field
between the first and second elements for accelerating the
ions.
[0139] Parameters such as the magnitude and direction of the first
and second electric fields and the time delay between the
ionization pulse and application of the second electric field are
chosen so that the time delay is long enough to allow substantially
all fast fragmentation to complete. These parameters are also
chosen to detect the selected mass with optimum mass resolution.
The parameters may be determined manually or by use of a computer,
computer interface, and computer algorithm.
[0140] The method may include the step of detecting the
mass-to-charge ratio of the sequence specific fragments generated
and the step of identifying a sequence of at least one biomolecule
in the sample wherein the biomolecule is selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one biomolecule selected from the group
consisting of peptides, proteins, PNA, carbohydrates, and
glycoproteins.
[0141] The method may also include the step of increasing the yield
of fragments generated by increasing the energy transfer to the
biomolecule during ionization. The energy transfer may be increased
by selecting a laser wavelength approximately equal to the
wavelength at which the biomolecule absorbs. The energy transfer
may also be increased by incorporating an additive to the
matrix.
[0142] The matrix may be selected to specifically promote
fragmentation of biomolecules. The biomolecule may be an
oligonucleotide and the matrix may comprise at least one of
2,5-dihydroxybenzoic acid and picolinic acid. A second substance
may be added to the matrix to promote fragmentation. The additive
may absorb at the wavelength of the laser but it is not necessarily
effective as matrix in itself. Alternatively the additive may not
absorb at the wavelength of the laser, nor be efficient as a matrix
in itself, but may promote energy transfer from the matrix to the
sample and thus promoting fragmentation.
[0143] The method may also include the step of energizing an ion
reflector spaced apart from the first or second element.
Application of the reflector provides a higher order correction for
energy spread in the ion beam, and when included in this method
provides even higher mass resolution.
[0144] FIG. 8a illustrates an 11mer DNA sample generating mostly
singly and doubly charged intact ions recorded with a MALDI TOF
mass spectrometer incorporating the principles of present
invention, where the objective is to suppress fragmentation and
obtain high resolution and high sensitivity with minimal
fragmentation.
[0145] FIG. 8b illustrates an 11mer DNA sample recorded with a
MALDI TOF mass spectrometer incorporating the principles of the
present invention for increasing the yield of fragment ions. The
sample is measured with a reverse bias electric field between the
sample holder and the first element prior to ion extraction which
allows a relatively long extraction delay (500 ns), and a
relatively high laser energy density. Fragmentation is further
promoted by the use of 2,5-dihydroxybenzoic acid matrix. These
experimental parameters result in the generation of abundant
fragment ions. The interpretation of this fragment ion spectrum
yields the sequence of the oligonucleotide. The "w" ion series is
almost complete and defines the sequence up to the two rightmost
residues and also provides the composition (but not the sequence)
of that dinucleotide piece. FIG. 8c describes the nomenclature of
the fragment ions.
[0146] There are important applications of MALDI TOF mass
spectrometry in the art where it is advantageous to use infrared
lasers for ionization. Unfortunately, a number of infrared lasers
with desirable characteristics, such as the CO.sub.2. laser, have
pulse widths longer than 100 ns. Typically, the use of such long
pulses in conventional MALDI TOF mass spectrometry is undesired
since the mass spectral peaks can be excessively wide due to the
longer ion formation process. The use of delayed extraction MALDI
TOF mass spectrometer, however, can eliminate the undesirable
effects of a long ionizing laser pulse. Ions formed in an early
phase of the laser pulse are emitted from the sample surface
earlier than those formed in a late phase of the laser pulse During
extraction, the early phase ions will be farther away from the
sample surface than the late phase ions. Consequently, the late
phase ions will be accelerated to a slightly higher energy by the
extraction pulse. Under optimized conditions the late phase ions
will catch up with the early phase ions at the detector
position.
[0147] Another advantage of a MALDI TOF mass spectrometer
incorporating the features of the present invention is the ability
to achieve high mass resolution utilizing a long-pulse infrared
laser. A long pulse is defined as a pulse with a length longer than
the desirable peak width of an ion packet when detected. With
pulsed ion extraction instruments, desirable peak widths are
typically 5-100 ns. The desirable peak width varies with the
mass-to-charge ratio of the ions, for example, 5 ns for an
isotopically resolved small peptide and 100 ns for a protein of
mass-to-charge ratio of 30,000.
[0148] The present invention also features a method of improving
resolution in long-pulse laser desorption/ionization time-of-flight
mass spectrometry. A first potential is applied to a sample holder.
A second potential is applied 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. A sample proximately disposed to the sample
holder is ionized to form ions with an infrared laser which
generates a pulse of energy with a long time duration. The time
duration of the pulse of energy is greater than 50 ns.
[0149] The potential on the first element with respect to the
sample holder may be more positive for measuring positive ions and
more negative for measuring negative ions to spatially separates
ions by their mass prior to ion extraction. At least one of the
first or second potentials is varied at a predetermined time
subsequent to ionization to define a second different electric
field between the sample holder and the first element which
extracts ions for a time-of-flight measurement. The predetermined
time may be greater than the duration of the laser pulse.
[0150] The method may include the step of applying a potential to a
second element spaced apart from the first element which, together
with the potential on the first element, defines an electric field
between the first and second elements for accelerating the
ions.
[0151] The sample may comprise a matrix substance absorbing at the
wavelength of the laser pulse to facilitate desorption and
ionization of sample molecules. The sample may also comprise at
least one compound of biological interest selected from the group
consisting of DNA, RNA, polynucleotides and synthetic variants
thereof or at least one compound of biological interest selected
from the group consisting of peptides, proteins, PNA,
carbohydrates, glycoconjugates and glycoproteins.
[0152] FIGS. 9a-c illustrates the ability to analyze very complex
oligonucleotide mixtures with a MALDI TOF mass spectrometer
incorporating the principles of this invention. FIG. 9a is a mass
spectrum of a 60mer DNA sample containing sequence specific
impurities recorded with conventional MALDI TOF mass spectrometer.
The sequence is not readable.
[0153] FIG. 9b is a mass spectrum of a 60mer DNA sample containing
sequence specific impurities recorded with a MALDI TOF mass
spectrometer incorporating the principles of this invention. More
than half of its sequence can be read from the spectrum. FIG. 9c
presents an expanded portion the mass spectrum presented in FIG.
9b. The level of performance indicated by FIG. 9c is adequate to
analyze DNA sequencing ladders all in one vial. Thus by using a
MALDI TOF mass spectrometer incorporating the principles of this
invention, one can analyze a single Sanger mixture with all the
four series present. The ability to sequence DNA with impurities is
essential to the possibility of profiling DNA sequencing
mixtures.
[0154] The present invention also features a method of sequencing
DNA by mass spectrometry. The method includes applying a first
potential to a sample holder comprising a piece of DNA of unknown
sequence. A second potential is applied 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. The sample is ionized to form
sample ions. At least one of the first or second potentials is
changed at a predetermined time subsequent to ionization to define
a second different electric field between the sample holder and the
first element which extracts ions for a time-of-flight measurement.
The measured mass-to charge ratio of the ions generated are used to
obtain the sequence of the piece of DNA.
[0155] The DNA in the sample is cleaved to produce sets of DNA
fragments, each having a common origin and terminating at a
particular base along the DNA sequence. The sample may comprise
different sets of DNA fragments mixed with a matrix substance
absorbing at a wavelength substantially corresponding to the
quantum energy of the laser pulse which facilitates desorption and
ionization of the sample. The mass difference between the detected
molecular weight of a peak of one of the sets of DNA fragments
compared to a peak of another of the sets of DNA fragments can be
determined.
[0156] The present invention also features a method of improving
resolution in laser desorption/ionization time-of-flight mass for
nucleic acids by reducing high energy collisions and ion charge
exchange during ion extraction. A potential is applied to a sample
holder comprising a nucleic acid. A potential is applied 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. A sample is
ionized to form a cloud of ions with a laser which generates a
pulse of energy. A second potential is applied to the sample holder
at a predetermined time subsequent to the ionization which,
together with the potential on the first element, defines a second
electric field between the sample holder and the first element, and
extracts the ions after the predetermined time. A potential may be
applied to a second element spaced apart from the first element
which, together with the potential on the first element, defines an
electric field between the first and second elements for
accelerating the ions.
[0157] The predetermined time is chosen to be 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. The predetermined time can be chosen to be
greater than the time in which the mean free path of ions in the
cloud approximately equals the distance between the holder and the
first element. The predetermined time can also be chosen to be
greater than the time it takes for substantially all fast
fragmentation to complete.
[0158] The sample may comprise a matrix substance absorbing at the
wavelength of the laser pulse to facilitate desorption and
ionization of the sample.
[0159] The present invention also features a method of obtaining
accurate molecular weights of MALDI TOF mass spectrometry. A major
problem with MALDI TOF mass spectrometry is that it is difficult to
obtain accurate molecular weights without the use of internal
standards consisting of known compounds to a sample containing an
unknown compound. Unfortunately, different samples respond with
widely different sensitivities and often several attempts are
required before a sample containing the correct amount of internal
standard can be prepared. Also, the internal standard may interfere
with the measurement by producing ions at the same masses as those
from an unknown sample. Thus for many applications of MALDI TOF
mass spectrometry it is important to be able to convert the
measured time-of-flight to mass with very high precision and
accuracy without using internal standards.
[0160] In principle, it is possible to calculate the time-of-flight
of an ion of any mass as accurately as the relevant parameters,
such as voltages and distances. But in conventional MALDI TOF mass
spectrometry accurate calculations are generally not possible
because the velocity of the ions after acceleration is not
accurately known. This uncertainty occurs because of collisions
between ions and neutrals in the plume of material desorbed from
the sample surface. The energy lost in such collisions varies with
parameters such as laser intensity and mass. Thus, the relationship
between measured flight time and mass is different from one
spectrum to the next. To obtain accurate masses it is necessary to
include known compounds with masses similar to those of the unknown
sample to accurately calibrate the spectrum and determine the mass
of an unknown.
[0161] In the present invention, the ions are produced initially in
a region in which the electrical field is weak to zero. The initial
field may accelerate ions in the direction opposite to that in
which they are eventually extracted and detected. In this method,
application of the extraction field is delayed so that the plume is
sufficiently dissipated such that significant energy loss due to
collisions is unlikely. As a result, the velocity of any ion at any
point in the mass spectrometer can be precisely calculated and the
relationship between mass and time-of-flight is accurately known so
that internal calibration of spectra is not required.
[0162] With pulsed ion extraction, the mass of an ion is given to a
very high degree of approximation by the following equation:
M.sup.1/2=A.sub.1(t+A.sub.2)(1-A.sub.3.DELTA.t+A.sub.4t+A.sub.5t.sup.2)
(5)
[0163] where t is the measured flight time in nanoseconds A.sub.1
is the proportionality constant relating mass to flight time when
the initial velocity of the ions is zero. A.sub.2 is the time delay
in nanoseconds between the laser pulse and start of the transient
digitizer. A.sub.3 is small except when delayed sweeps are
employed. The time delay .DELTA. is the time between the laser
pulse and the application of the drawout field. The other terms are
corrections which depend only on the initial velocity, the voltage
on the first element and the geometry of the instrument.
[0164] The above coefficients can be described in terms of
instrument parameters in the following way:
A.sub.1=V.sub.s.sup.1/2(1+.alpha.G.sub.W)/[4.569D.sub.e] (6)
where
D.sub.e=Dzg(y), (7)
[0165] V.sub.s is the source voltage in kilovolts, D is the
field-free distance in mm, G.sub.W is the guide wire setting (% of
source voltage), .alpha. is a constant to be determined
empirically,
g(y)=1+2y.sup.1/2[d.sub.a/D+(d.sub.o/D)(1/{y.sup.1/2+1})], (8)
z=1+(2d.sub.m/D)(V.sub.s/V.sub.m){[1+(V.sub.m/V.sub.s)]1/2-1},
(9)
[0166] d.sub.a is the length of the first ion accelerating region
in mm, d.sub.o is the length of the second accelerating region in
mm, d.sub.m is the length of the accelerating region in front of
the electron multiplier in mm, V.sub.m is voltage applied to the
front of the electron multiplier in kilovolts,
y=V.sub.s/(V.sub.s-V.sub.g)=100/(100-G.sub.R), and (10)
[0167] G.sub.R is the grid setting in percent of source voltage.
The guide wire correction depends on the most probable trajectory
of ions about the wire. The maximum value of .delta. is 0.005 which
corresponds to the ions traveling through the drift tube at
precisely the guide wire potential. Note the actual value of
.delta. will be somewhat less than this, depending on laser
alignment.
[0168] The higher order correction terms are given by
A.sub.3=v.sub.owy.sup.1/2/2D.sub.e (11)
A.sub.4=v.sub.od.sub.ay/2D.sub.e.sup.2 (12)
A.sub.S=v.sub.o.sup.2d.sub.awy.sup.3/2/8D.sub.e.sup.3 (13)
[0169] where v.sub.o is the initial velocity in
millimeters/nanosecond, and w is given by
w=y.sup.-3/2[(D/2d.sub.a-(d.sub.o/d.sub.a)(1+x)]+x(d.sub.o/d.sub.a)-1
(14)
where
x=(V.sub.s-V.sub.g)/V.sub.g=(100-G.sub.R)/G.sub.R. (15)
[0170] These values strictly apply only to operating with the first
field at zero before the application of the drawout pulse.
[0171] When employing an ion detector, the effective drift distance
becomes
D.sub.e=Dzg(y)+4d.sub.R/R (16)
[0172] where d.sub.R is the length of the mirror in mm and R is the
ratio of the mirror voltage to source voltage. Under normal
operation of the reflector, the quantity w becomes
w=x(d.sub.o/d.sub.a)-1. (17)
[0173] With these changes the calibration equations are exactly the
same as those used for the linear analyzer. It should be noted that
y and w are generally much smaller for the reflector, thus the
correction terms are also smaller.
[0174] Equivalents
[0175] While the invention has been particularly shown and
described with reference to specific preferred embodiments, it
should be understood by those skilled in the art that various
changes in form and detail may be made therein without departing
from the spirit and scope of the invention as defined by the
appended claims. For example, although a pulsed laser is described
as the ion source, it is noted that other pulsed ion sources can be
used without departing from the spirit and scope of the
invention.
* * * * *