U.S. patent application number 10/458847 was filed with the patent office on 2003-11-13 for methods and apparatus for external accumulation and photodissociation of ions prior to mass spectrometric analysis.
This patent application is currently assigned to ISIS Pharmaceuticals, Inc.. Invention is credited to Griffey, Richard, Hofstadler, Steven.
Application Number | 20030211628 10/458847 |
Document ID | / |
Family ID | 22885869 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211628 |
Kind Code |
A1 |
Griffey, Richard ; et
al. |
November 13, 2003 |
Methods and apparatus for external accumulation and
photodissociation of ions prior to mass spectrometric analysis
Abstract
The present invention discloses novel methods and apparatuses
for mass spectrometry. In the methods and apparatuses of the
invention, ions are accumulated in an ion reservoir and dissociated
with coherent radiation prior to mass analysis. These methods and
apparatuses are amenable to mass spectrometric analysis of
biomolecules and are particularly useful for the sequencing of
oligonucleotides, peptides and oligosaccharides.
Inventors: |
Griffey, Richard; (Vista,
CA) ; Hofstadler, Steven; (Oceanside, CA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Assignee: |
ISIS Pharmaceuticals, Inc.
|
Family ID: |
22885869 |
Appl. No.: |
10/458847 |
Filed: |
June 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10458847 |
Jun 11, 2003 |
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09927666 |
Aug 10, 2001 |
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09927666 |
Aug 10, 2001 |
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09235528 |
Jan 22, 1999 |
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6342393 |
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Current U.S.
Class: |
436/173 |
Current CPC
Class: |
Y10T 436/24 20150115;
H01J 49/0059 20130101; Y10T 436/143333 20150115 |
Class at
Publication: |
436/173 |
International
Class: |
G01N 024/00 |
Claims
What is claimed is:
1. A method of inducing dissociation of molecular ions prior to
analysis in a mass analyzer of a mass spectrometer, comprising the
steps of: a) generating said molecular ions by ionization; b)
accumulating said ions in an ion reservoir; and c) exposing said
ions in said ion reservoir to coherent radiation to dissociate said
ions prior to mass analysis in a mass analyzer of a mass
spectrometer.
2. The method of claim 1 further comprising transporting said
dissociated ions to a mass analyzer of a mass spectrometer.
3. The method of claim 1 wherein said accumulating of ions occurs
in a m/z range of from about 50 to about 100,000.
4. The method of claim 1 wherein said ion reservoir is a rf trap or
Penning trap.
5. The method of claim 4 wherein said rf trap is a rf-multi-pole
ion reservoir.
6. The method of claim 5 wherein said rf-multi-pole ion reservoir
is a quadrupole, hexapole or octapole ion reservoir.
7. The method of claim 1 wherein said ion reservoir is evacuated to
10.sup.-3 to 10.sup.-6 torr.
8. The method of claim 1 wherein said coherent radiation is emitted
from a laser.
9. The method of claim 8 wherein said laser is an infrared
laser.
10. The method of claim 2 wherein said transporting is effected by
either removing or reversing an electric field generated by gate
electrodes on either side of said ion reservoir.
11. The method of claim 2 wherein said mass analyzer is a trapped
ion cell of a Fourier transform ion cyclotron mass spectrometer, a
time-of-flight mass spectrometer, a quadrupole ion trap mass
spectrometer, a quadrupole mass analyzer, or a magnetic/electric
sector mass spectrometer.
12. An apparatus for dissociation of ions in mass spectrometry
comprising: a) an ion reservoir for receiving and accumulating ions
produced from an ion source; and b) a coherent radiation source in
operative association with said ion reservoir and which emits
coherent radiation to dissociate ions within said ion reservoir
prior to analysis in a mass analyzer of a mass spectrometer.
13. The apparatus of claim 12 wherein said ion reservoir is a rf
trap or Paul trap.
14. The apparatus of claim 13 wherein said rf trap is a
rf-multi-pole ion reservoir.
15. The apparatus of claim 14 wherein said rf-multi-pole ion
reservoir is a quadrupole, hexapole or octapole ion reservoir.
16. The apparatus of claim 12 wherein said ion reservoir is
evacuated to 10.sup.-3 to 10.sup.-6 torr.
17. The apparatus of claim 12 wherein said coherent radiation is
emitted from a laser.
18. The apparatus of claim 17 wherein said laser is an infrared
laser.
19. A mass spectrometer comprising: a) ion source; b) ion
reservoir; c) first conduit connecting said ion source to said ion
reservoir for transporting ions from said ion source to said ion
reservoir; d) coherent radiation source in operative association
with said ion reservoir which emits coherent radiation to
dissociate ions within said ion reservoir; e) mass analyzer; and f)
second conduit connecting said ion reservoir to said mass analyzer
for transporting ions between said ion reservoir and said mass
analyzer.
20. The apparatus of claim 19 wherein said ion reservoir is a rf
trap or Paul trap.
21. The apparatus of claim 20 wherein said rf trap is a
rf-multi-pole ion reservoir.
22. The apparatus of claim 21 wherein said rf-multi-pole ion
reservoir is a quadrupole, hexapole or octapole ion reservoir.
23. The apparatus of claim 19 wherein said ion reservoir is
evacuated to 10.sup.-3 to 10.sup.-6 torr.
24. The apparatus of claim 19 wherein said coherent radiation is
emitted from a laser.
25. The apparatus of claim 24 wherein said laser is an infrared
laser.
26. The apparatus of claim 19 wherein said transporting ions from
said ion reservoir to said mass analyzer is effected by either
removing or reversing an electric field generated by gate
electrodes on either side of said ion reservoir.
27. The apparatus of claim 19 wherein said mass analyzer is a
trapped ion cell of a Fourier transform ion cyclotron mass
spectrometer, a time-of-flight mass spectrometer, a quadrupole ion
trap mass spectrometer, a quadrupole mass analyzer, or a
magnetic/electric sector mass spectrometer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to improved methods and
apparatus for mass spectrometry. In particular the invention
provides methods and apparatus that dissociate ions in an ion
reservoir prior to mass spectrometric analysis. The methods and
apparatus of the invention can be used in the analysis of ions of
peptides, proteins, carbohydrates, oligonucleotides, nucleic acids,
and small molecules as prepared by combinatorial or medicinal
chemistry.
BACKGROUND OF THE INVENTION
[0002] Mass spectrometry (MS) is a powerful analytical tool for the
study of molecular structure and interaction between small and
large molecules. The current state-of-the-art in MS is such that
less than femtomole quantities of material can be readily analyzed
using mass spectrometry to afford information about the molecular
contents of the sample. An accurate assessment of the molecular
weight of the material may be quickly obtained, irrespective of
whether the sample's molecular weight is several hundred, or in
excess of a hundred thousand, atomic mass units or Daltons (Da).
Mass spectrometry can elucidate significant analytical aspects of
important biological molecules. One reason for the utility of MS as
an analytical tool is the availability of a variety of different MS
methods, instruments, and techniques which can provide different
pieces of information about the samples.
[0003] A mass spectrometer analyzes charged molecular ions and
fragment ions from a sample molecule. These ions and fragment ions
are then sorted based on their mass to charge ratio (m/z). A mass
spectrum is produced from the abundance of these ions and fragment
ions that is characteristic of every compound. In the field of
biotechnology, mass spectrometry can be used to determine the
structure of a biomolecule. Of particular interest is the ability
of mass spectrometry to be used in determining the sequence of
oligonucleotides, peptides, and oligosaccharides.
[0004] Various mass spectrometric techniques can be used to deduce
the sequence of an oligonucleotide. Murray, K. K., J. Mass Spec.,
1996, 31, 1203-1215, which is incorporated herein by reference in
its entirety. Two commonly used ionization methods are electrospray
ionization (ESI) and matrix-assisted laser desorption/ionization
(MALDI). Mass spectrometry is also commonly used for the sequencing
of peptides and proteins (see, in general, Biemann, K., Annu. Rev.
Biochem., 1992, 61, 977-1010, which is incorporated herein by
reference in its entirety).
[0005] In principle, mass spectrometers consist of at least four
parts, (1) an inlet system, (2) an ion source, (3) a mass analyzer
and (4) a mass detector/ion-collection system. Skoog, D. A. and
West, D. M., Principles of Instrumental Analysis, Saunders College,
Philadelphia, Pa., 1980, 477-485. The inlet system permits the
sample to be introduced into the ion source. Within the ion source,
molecules of the sample are converted into gaseous ions. The most
common methods for ionization are electron impact (EI),
electrospray ionization, chemical ionization and matrix-assisted
laser desorption/ionization (MALDI). A mass analyzer resolves the
ions based on mass-to-charge ratios. Analyzers can be based on
magnetic means (sector), time-of-flight, quadrupole and Fourier
transform mass spectrometry (FTMS). A mass detector collects the
ions as they pass through the detector and records the signal. Each
ion source can potentially be combined with each type of mass
analyzer generating a wide variety of mass spectrometers.
[0006] The field of mass spectrometry is rapidly evolving.
Improvements in mass spectrometric instrumentation and
methodologies are needed to address increasingly challenging
applications in a number of research arenas including the physical,
biological, and medical sciences. In many implementations of mass
spectrometers based on Penning and Paul traps, ion formation,
isolation, and detection take place in the same region of a vacuum
chamber and are temporally, rather than spatially, separated. In a
typical pulse, sequence ions are alternatively formed and detected;
the ionization duty cycle is defined as the fraction of time ions
are formed compared to the overall experiment time. Thus, in high
resolution measurements, which may take several seconds to perform
yet require ionization intervals of only a few milliseconds, the
overall ionization duty cycle is only a few percent. A number of
approaches have been explored to improve the ionization duty cycle
including schemes in which ions are formed and continuously
accumulated in an external ion reservoir and periodically gated
into the mass analyzer. For example, a Penning trap in the fringing
magnetic field of an Fourier transform ion cyclotron resonance
(FTICR) mass spectrometer was used to accumulate ions formed by EI
during high resolution measurements in the FTICR cell. Hofstadler,
S. A. and Laude, D. A, Jr., Anal. Chem., 1991, 63, 2001-2007, which
is incorporated herein by reference in its entirety. Senko, M. W.
et al. (J. Amer. Soc. Mass Spectrom., 1997,8,970-976) demonstrated
that an external ion reservoir formed by an rf-only multipole
bounded by two electrostatic elements can efficiently accumulate
ions generated by electrospray ionization and the ion ensemble can
be periodically pulsed into the FTICR cell for mass analysis.
[0007] Another means of improving mass spectra is the use of
dissociation to fragment the molecular ions. Dissociation
strategies for tandem ESI-MS can be separated into two general
categories: those which take place in the ESI source prior to mass
analysis, and those which take place after the ESI source and often
rely on some form of m/z dependent ion manipulation. For example,
Loo, J. A. et al. (Anal. Chim. Acta, 1990, 241, 167-173)
demonstrated that large multiply charged proteins could be
effectively dissociated by employing a relatively large voltage
difference between the exit of the desolvating capillary and the
skimmer cone. Similarly, Rockwood, A. L. et al. (Rapid Comm. Mass
Spectrom., 1991, 5, 582-585) demonstrated that ions could be
thermally dissociated in the ESI source by heating the desolvation
capillary to extreme temperatures. Both of these "in-source"
dissociation schemes produce mass spectra which are rich in
fragment ions and can provide sequence information for peptides,
proteins, or oligonucleotides. Alternatively, a number of
post-source dissociation schemes have been presented which are now
widely employed. In general, scanning MS/MS instruments such as
triple quadrupoles and magnetic sector instruments employ
collisionally activated dissociation (CAD) to effect the
dissociation of an m/z selected parent ion. Dagostino, P. A., et
al., J. Chrom., 1997, 767, 77-85. In addition to employing various
forms of CAD (Gauthier, J. W., et al., Chim. Acta, 1991, 246,
211-225; and Senko, M. W:, et al., Anal. Chem., 1994, 66,
2801-2808), FTICR instruments have successfully demonstrated the
use of UV-photodissociation (Williams, E. R., et al., J. Amer. Soc.
Mass Spectrom., 1990, 1, 288-294), infrared multiphoton
dissociation (IRMPD) (Little, D. P., et al., Anal. Chem., 1994, 66,
2809-2815), surface induced dissociation (SID) (Ijames, C. F. and
Wilkins, C. L., Anal. Chem., 1990, 62, 1295-1299; and Williams, E.
R., et al., J. Amer. Soc. Mass Spectrom., 1990, 1, 413-416),
blackbody infrared radiative dissociation (BIRD) (Price, W. D., et
al., Anal. Chem., 1996, 68, 859-866), and more recently, electron
capture dissociation (ECD) (Zubarev, R. A., et al., J. Am. Chem.
Soc., 1998, 120, 3265-3266) to fragment precursor ions.
[0008] Infrared multi-photon dissociation (IRMPD) uses
photodissociation generally in combination with FTICR or quadrupole
ion trap mass analyzers. In this method, ions are collected in the
FTICR analyzer cell and the laser interacts with ions within the
cell. In IRMPD, the laser dissociates ions into fragment ions, as
opposed to an ionization method involving lasers, e.g. MALDI. The
most common method of ionization used in IRMPD methods is
electrospray ionization as this provides more highly charged ions
that are more easily dissociated, as compared to MALDI. Little, D.
P., et al., Anal. Chem., 1994, 66, 2809-2815. Little, D. P., et al.
used IRMPD for protein and nucleotide sequencing. IRMPD has also
been used with quadrupole ion trap mass spectrometers. Colorado,
A., et al., Anal. Chem., 1996, 68, 4033-4043.
[0009] Currently, IRMPD methods are limited to mass spectrometers
based on FTICR and QIT. With FTICR methods the kinetic energy
release which accompanies the dissociation event can cause a
redistribution of the ions in the trapped ion cell. Upon
excitation, these ions can obtain a range of cyclotron radii, which
precludes high performance mass measurements. Also, the laser
irradiation interval is identical for each ion, which limits the
dissociation pathways available to the ion.
[0010] Although improvements have been made in the mass
spectrometric analysis of biomolecules, especially with the use of
IRMPD, there remains a need for improved mass spectrometric methods
and apparatuses.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic representation of one embodiment of a
mass spectrometer using external ion reservoir accumulation of
ions. Also shown is a representation of the electrostatic
potentials used.
[0012] FIG. 2 is a series of mass spectra for a 20-mer
oligonucleotide. FIG. 2a represents non-IRMPD mass spectrometry.
FIG. 2b represents traditional in-cell IRMPD. FIG. 2c represents
IRMPD in which the dissociation was performed in the external ion
reservoir concurrent with ion accumulation.
[0013] FIGS. 3a and 3b are expanded views of the mass spectra from
FIGS. 2b and 2c, respectively, showing the region from 625 to
660.
[0014] FIG. 4 is a series of mass spectra for ubiquitin. The top
spectrum represents non-IRMPD mass spectrometry, while the bottom
spectrum represents IRMPD in which the dissociation was performed
in the external ion reservoir concurrent with ion accumulation.
SUMMARY OF THE INVENTION
[0015] The present invention describes methods and apparatuses for
inducing controlled dissociation of molecular ions external to a
mass analyzer of a mass spectrometer prior to mass measurement. The
methods and apparatuses comprise generating said ions, collecting
said ions in an ion reservoir, and dissociating said ions by
application of coherent radiation prior to analysis in a mass
analyzer of a mass spectrometer. In preferred embodiments, the ion
reservoir is a rf multi-pole ion reservoir and the coherent
radiation is applied by a laser. The apparatuses of the invention
may be self-contained units that are capable of being retrofitted
to many types of mass spectrometers or may be complete mass
spectrometers in themselves.
[0016] The present invention provides methods and apparatuses that
dissociate ions external to a mass analyzer of a mass spectrometer
prior to mass spectrometric analysis. These methods and apparatuses
can be used for the analysis of singly or multiply charged ions of
peptides, proteins, carbohydrates, oligonucleotides, nucleic acids,
and small molecules as prepared by combinatorial or medicinal
chemistry. The present invention is especially useful for the
sequence analysis of peptidas and proteins, synthetic
oligonucleotides, chemically modified oligonucleotides, RNA, DNA,
and oligosaccharides. For modified oligonucleotides that are
refractory to conventional enzymatic degradation, the methods of
the invention provide a rapid means of sequence analysis. In
addition, the identity of small molecules and their presence in
tissue extracts, for example, may be determined by this method.
[0017] The methods and apparatuses of the invention provide
enhanced sensitivity and unexpected ion abundances compared to
dissociation of ion in the mass analyzer, as exemplified by IRMPD
using Fourier transform ion cyclotron (FTICR) or quadrupole ion
trap (QIT). This is due to enhanced ion statistics during the
accumulation process in the external ion reservoir. Additional
advantages include an improved duty cycle and enhanced ease of
operation.
[0018] In the present invention, ions of interest are first
generated using conventional ionization techniques. These ions are
collected in an ion reservoir, where dissociation of the ions
occurs. Mass analysis of the dissociated ions is then performed.
The ion reservoir is preferably driven at a frequency that captures
the ions of interest. These collected ions are dissociated by
application of coherent radiation from, for example, an infrared
laser. In a preferred embodiment, the gas pressure around the ion
reservoir is reduced to 10.sup.-3-10.sup.-6 torr by vacuum pumping.
Following dissociation, the ensemble of ions is transported into
the mass analyzer such as by removing or reversing the electric
field generated by gate electrodes on either side of the ion
reservoir.
[0019] Ionization of molecules results in charged particles that
can be manipulated by electrostatic potentials. Many ionization
methods used with mass spectrometry are amenable to the invention.
These include electrospray, chemical, MALDI, laser desorption
ionization (LDI), fast atom bombardment (FAB), electron ionization,
thermospray ionization, secondary ion mass spectrometry (SIMS),
liquid SIMS, field desorption (FD), and .sup.252Cf desorption (see
Constantin, E. and Schnell, A., Mass Spectrometry, Ellis Horwood,
New York, 1990). Other types of ionization methods are also
amenable to the present invention.
[0020] The ion reservoir may be a quadrupole, hexapole, octapole or
other rf-multi-pole ion reservoir (rf is a shorthand notation for
radio frequency). In a rf-multi-pole, a field is formed by pairs of
parallel, electrically conducting rods. Each pair of electrodes is
electrically connected. A rf oscillator supplies a positive signal
to one electrode in each pair and a signal of opposite charge and
equal strength to the other electrode in each pair. Another ion
trap based on radio frequency is a Paul trap. Cooks, R. G., et al.,
Acc. Chem. Res., 1994, 27, 315. Other possible ion reservoirs
include Penning traps (Vartanian, V. H., et al., Mass Spectrometry
Reviews, 1995, 14, 1-19), electrostatic lenses, jet expansion and
electrostatic ion reservoirs (White, F. M., et al., Rapid Comm. in
Mass Spec., 1996, 10, 1845-1849). The ion reservoir may be used to
collect negatively or positively charged ions generated by the ion
source. The ion reservoir preferably has a gated electrode to allow
the accumulation of ion fragments prior to their mass
measurement.
[0021] Ions are preferably collected in the ion reservoir in a
generally mass-inselective manner. This permits dissociation over a
broad mass range, with efficient retention of fragment ions. By
"mass-inselective", it means that ions are not collected based on
their mass to charge ratio. Theoretically, all ions are collected
regardless of their mass. From a practical standpoint, those
skilled in the art will recognize that there are lower and upper
limits to the size of ions that are collected. With the limitation
of current instruments, this m/z range is from about 50 to about
100,000 m/z. The ion reservoir also provides a spatial separation
which results in a more time-efficient method of mass spectrometry.
Thus, the dissociation and measurement take place concurrently in
spatially distinct regions of the spectrometer. Mass measurement
requires lengthy times. Thus, an improved ionization duty cycle
results, which enables improved analysis of on-line separations,
e.g. capillary electrophoresis (CE), or liquid chromatography (LC).
With the methods of the present invention, an
accumulation/dissociation efficiency of near unity can be
achieved.
[0022] In preferred embodiments of the present invention,
dissociation occurs in a relatively high pressure
(10.sup.-3-10.sup.-6 torr). This results in two advantages over
traditional IRMPD as exemplified in FTICR and QIT mass
spectrometry. Under high pressures, collisions with neutrals damp
the ion cloud to the center of the well and stabilize fragment
ions, resulting in significantly improved fragment ion retention.
In addition, the fragment ion coverage is significantly improved,
giving more sequence information.
[0023] The expanded range of fragment ions observed may be the
result of at least two contributing factors. First, performing
IRMPD during the external ion accumulation event means that ions
accumulated in the external ion reservoir can experience a range of
irradiation intervals if the laser is activated concurrent with ion
injection. For example, in a 500 ms accumulation/dissociation
interval, an ion trapped within the first 10 ms of the event will
have the opportunity to be irradiated for nearly 500 ms while an
ion trapped near the end of the event may be exposed to the laser
beam for only a few milliseconds. Additionally, performing the
dissociation in the high pressure region of the ion reservoir
allows collisional focusing, and potentially collisional
stabilization of metastable fragments, and mitigates the potential
for a spatially defocused ion cloud in the trapped ion cell.
[0024] The coherent radiation used in the invention interacts with
the molecular ions to dissociate them into fragments. Any coherent
radiation source can be used with the invention provided the
molecular ions absorb photons at the wavelength emitted by the
coherent radiation source. The preferred coherent radiation of the
invention is emitted by a laser. Infrared lasers, operating from 1
to about 12 mm, both continuous wave (CW) and pulsed, are amenable
to the invention. Ultraviolet lasers, operating from about 150 to
400 nm, generally pulsed, are also amenable to the invention. In a
preferred embodiment the laser operates at a wavelength in the
infrared region. Typical lasers that may be used in the invention
include CO.sub.2 lasers, CO lasers and Nd-YAG lasers.
[0025] In one embodiment, the coherent radiation emitted from the
laser is parallel to the rf-poles and its beam is centered within
the rf-multi-pole, i.e, is coaxial relative to the ion reservoir.
In another embodiment, the coherent radiation emitted by the laser
interacts with the ion volume in an orientation other than a
coaxial orientation, e.g. at an oblique angle. The laser may be
placed at an angle that permits multiple passes through the ion
cloud with the walls of the ion reservoir, e.g. a White cell
(Watson, C. H., et al., J. Phys. Chem., 1991, 95, 6081-6086), or a
single pass perpendicular to the flight path of the ions. When
multiple passes through the ion cloud are desired, the walls of the
ion reservoir have reflective surfaces. It is known by those
skilled in the art to provide optics in the ion reservoir to permit
the coherent radiation to interact with the fragmented ions.
[0026] Ions are accumulated in the ion reservoir external to the
mass analyzer of a mass spectrometer. A preferred gated electrode
prevents ions from entering the mass analyzer until desired. Once a
sufficient ion population is accumulated in the ion reservoir, the
voltage potential can be shifted or removed, to allow the ions to
enter the mass analyzer.
[0027] The present invention modifies traditional IRMPD methods in
mass spectrometers based on FTICR and QIT. It also permits the use
of IRMPD methods in mass spectrometers based on detection schemes
other than FTICR and QIT. Many other types of mass analyzers are
also amenable to the invention. The invention is equally applicable
to Fourier transform ion cyclotron, quadrupole ion trap (i.e. Paul
trap), time-of-flight, electric/magnetic sector, quadrupole, and
hybrid mass spectrometers. In preferred embodiments, the mass
analyzer is composed of a Fourier transform ion cyclotron mass
spectrometer, a time-of-flight mass spectrometer, or a quadrupole
ion trap (i.e. Paul trap) mass spectrometer.
[0028] In an embodiment of the invention, an apparatus for ion
dissociation comprises an ion reservoir that is capable of
receiving and accumulating ions from an ion source and transporting
ions or dissociated ions to a mass analyzer, and a coherent
radiation source capable of emitting coherent radiation for
dissociating ions within the ion reservoir prior to mass analysis.
It is envisioned that this apparatus will be self-contained and
capable of being retrofitted to present generation mass
spectrometers. Thus, a mass spectrometer can be adapted to transfer
dissociated ions to a mass analyzer e.g. by providing a conduit
from the ion reservoir to the mass analyzer. No additional
elaborate optics are required. This provides a cost-effective means
of implementing IRMPD.
[0029] Alternatively, a mass spectrometer may comprise the
apparatus for dissociation in combination with an ion source and a
mass analyzer.
[0030] Mass spectrometry ion sources are well known in the art. One
such ion source uses electrospray ionization mass spectrometry
(ESI-MS). Smith et al., Anal. Chem., 1990, 62, 882-899; Snyder, in
Biochemical and biotechnological applications of electrospray
ionization mass, American Chemical Society, Washington, DC, 1996;
and Cole, in Electrospray ionization mass spectrometry:
fundamentals, instrumentation, Wiley, N.Y., 1997. ESI produces
highly charged droplets of the sample being studied by gently
nebulizing the sample solution in the presence of a very strong
electrostatic field. This results in the generation of highly
charged droplets that shrink due to evaporation of the neutral
solvent and ultimately lead to a "Coulombic explosion" that affords
multiply charged ions of the sample material, typically via proton
addition or abstraction; under mild conditions. ESI-MS is
particularly useful for very high molecular weight biopolymers such
as proteins and nucleic acids greater than 10 kDa in mass, for it
affords a distribution of multiply-charged molecules of the sample
biopolymer without causing any significant amount of fragmentation.
The fact that several peaks are observed from one sample, due to
the formation of ions with different charges, contributes to the
accuracy of ESI-MS when determining the molecular weight of the
biopolymer because each observed peak provides an independent means
for calculation of-the molecular weight of the sample. Averaging
the multiple readings of molecular weight so obtained from a single
ESI-mass spectrum affords an estimate of molecular weight that is
much more precise than would be obtained if a single molecular ion
peak were to be provided by the mass spectrometer. Further adding
to the flexibility of ESI-MS is the capability to obtain
measurements in either the positive or negative ionization
modes.
[0031] Matrix-Assisted Laser Desorption/Ionization Mass
Spectrometry (MALDI-MS) is another ion source method that can be
used for studying biomolecules. Hillenkamp et al., Anal. Chem.,
1991, 63, 1193A-1203A. This technique ionizes high molecular weight
biopolymers with minimal concomitant fragmentation of the sample
material. This is typically accomplished via the incorporation of
the sample to be analyzed into a matrix that absorbs radiation from
an incident UV or IR laser. This energy is then transferred from
the matrix to the sample resulting in desorption of the sample into
the gas phase with subsequent ionization and minimal fragmentation.
One of the advantages of MALDI-MS over ESI-MS is the simplicity of
the spectra obtained as MALDI spectra are generally dominated by
singly charged species. Typically, the detection of the gaseous
ions generated by MALDI techniques, are detected and analyzed by
determining the time-of-flight (TOF) of these ions. While MALDI-TOF
MS is not a high resolution technique, resolution can be improved
by making modifications to such systems, by the use of tandem MS
techniques, or by the use of other types of analyzers, such as
Fourier transform (FT) and quadrupole ion traps.
[0032] Fourier transform mass spectrometry (FTMS) is mass detection
technique and is especially useful because of its ability to make
mass measurements with a combination of mass measurement accuracy
and resolution that is superior to other MS detection techniques,
in connection with ESI or MALDI ionization. Amster, J. Mass
Spectrom., 1996, 31, 1325-1337. Further it may be used to obtain
high resolution mass spectra of ions generated by any of the other
ionization techniques. The basis for FTMS is ion cyclotron motion,
which is the result of the interaction of an ion with a
unidirectional magnetic field. The mass-to-charge ratio of an ion
(m/q or m/z) is determined by a FTMS instrument by measuring the
cyclotron frequency of the ion. The insensitivity of the cyclotron
frequency to the kinetic energy of an ion is one of the fundamental
reasons for the very high resolution achievable with FTMS. FTMS is
an excellent detector in conventional or tandem mass spectrometry,
for the analysis of ions generated by a variety of different
ionization methods including ESI and MALDI, or product ions
resulting from collisionally activated dissociation (CAD).
[0033] Collisionally activated dissociation (CAD), also known as
collision induced dissociation (CID), is a method by which analyte
ions are dissociated by energetic collisions with neutral or
charged species, resulting in fragment ions which can be
subsequently mass analyzed. Mass analysis of fragment ions from a
selected parent ion can provide certain sequence or other
structural information relating to the parent ion. Such methods are
generally referred to as tandem mass spectrometry (MS or MS/MS)
methods and are the basis of the some of MS based biomolecular
sequencing schemes being employed today.
[0034] FTICR-MS, like ion trap and quadrupole mass analyzers,
allows selection of an ion that may actually be a weak non-covalent
complex of a large biomolecule with another molecule (Marshall and
Grosshans, Anal. Chem., 1991, 63, A215-A229; Beu et al., J. Am.
Soc. Mass Spectrom., 1993, 4, 566-577; and Winger et al., J. Am.
Soc. Mass Spectrom., 1993, 4, 566-577); (Huang and Henion, Anal.
Chem., 1991, 63, 732-739), and is compatible with hyphenated
techniques such as LC-MS (Bruins, Covey and Henion, Anal. Chem.,
1987, 59, 2642-2646; Huang and Henion, J. Am. Soc. Mass Spectrom.,
1990, 1, 158-65; and Huang and Henion, Anal. Chem., 1991, 63,
732-739) and CE-MS (Cai and Henion, J. Chromatogr., 1995, 703,
667-692) experiments. FTICR-MS has also been applied to the study
of ion-molecule reaction pathways and kinetics.
[0035] Typically, tandem mass spectrometry (MS.sup.n) involves the
coupled use of two or more stages of mass analysis where both the
separation and detection steps are based on mass spectrometry. The
first stage is used to select an ion or component of a sample from
which further structural information is to be obtained. This
selected ion is then fragmented by (CID) or photodissociation. The
second stage of mass analysis is then used to detect and measure
the mass of the resulting fragments or product ions. The advent of
FTICR-MS has made a significant impact on the utility of tandem,
MS.sup.n procedures because of the ability of FTICR to select and
trap specific ions of interest and its high resolution and
sensitivity when detecting fragment ions. Such ion selection
followed by fragmentation routines can be performed multiple times
so as to essentially completely dissect the molecular structure of
a sample. A two-stage tandem MS experiment would be called a MS-MS
experiment while an n-stage tandem MS experiment would be referred
to as a MS.sup.n experiment. Depending on the complexity of the
sample and the level of structural detail desired, MS.sup.n
experiments at values of n greater than 2 may be performed.
[0036] Ion trap-based mass spectrometers are particularly well
suited for such tandem experiments because the dissociation and
measurement steps are temporally rather than spatially separated.
For example, a common platform on which tandem mass spectrometry is
performed is a triple quadrupole mass spectrometer. The first and
third quadrupoles serve as mass filters while the second quadrupole
serves as a collision cell for CAD. In a trap based mass
spectrometer, parent ion selection and dissociation take place in
the same part of the vacuum chamber and are effected by control of
the radio frequency wavelengths applied to the trapping elements
and the collision gas pressure. Hence, while a triple quadrupole
mass analyzer is limited to two stages of mass spectrometry (i.e.
MS/MS), ion trap-based mass spectrometers can perform MS.sup.n
analysis in which the parent ion is isolated, dissociated, mass
analyzed and a fragment ion of interest is isolated, further
dissociated, and mass analyzed and so on. A number of MS.sup.4
procedures and higher have appeared in the literature in recent
years. Cheng et al., Techniques in Protein Chemistry VII, Academic
Press, Inc., 1996, 7, 13-21.
EXAMPLES
Example 1
Instrumentation
[0037] All experiments were performed on a Bruker DALTONICS
(Billerica, Mass.) Apex 70e Fourier transform ion cyclotron
resonance mass spectrometer. The spectrometer is equipped with an
Analytica (Branford, Conn.) electrospray source utilizing a
grounded ESI emitter, a counter current drying gas, a glass
depolvation capillary, a single skimmer cone, and an rf-only
hexapole ion-reservoir. Ions are accumulated in the external ion
reservoir for 500 ms and pulsed into the INFINITY.TM. trapped ion
cell where they are analyzed by FTICR. All aspects of the
experiment including data acquisition, processing, and plotting
were performed using Bruker XMASS version 4.0 running on a Silicon
Graphics R5000 workstation. A 17 mM solution of Isis 2302, a 20-mer
phosphorothioate oligonucleotide (synthesized as described in U.S.
Pat. No. 5,514,788, herein incorporated by reference) was
electrosprayed from a 50:50 isopropanol:water solution containing
0.1% tripropyl amine through an off-axis electrospray probe at a
flow rate of 1.5 mL/minute.
[0038] IRMPD. As shown in FIG. 1, the external ion accumulation
region is comprised of a biased skimmer cone, an rf-only hexapole
operating at 5 MHz (500 Vpp), and an auxiliary "gate" electrode at
the low pressure end of the hexapole. The capillary exit voltage is
maintained at -68 V to avoid fragmentation due to conventional
nozzle-skimmer dissociation, or -180 V to induce nozzle-skimmer
dissociation. For operation in the negative ionization mode, the
potential of the skimmer cone is typically held at -15 V while the
gate electrode toggles between -15 V during accumulation and 0.2 V
during injection; the polarity of these electrodes is reversed for
operation in the positive ionization mode. IRMPD of ions in the
external ion reservoir was effected by irradiation at 10.6 mm from
a Synrad (Mukitelo, Wash.) 25 W CW CO.sub.2 laser. A lab-built
aluminum optical bench was positioned approximately 1.5 m from the
actively shielded superconducting magnet such that the laser beam
was aligned with the central axis of the magnet. Using standard IR
compatible mirrors and kinematic mirror mounts, the unfocused 3 mm
laser beam was aligned to traverse directly through the 3.5 mm
holes in the trapping electrodes of the INFINITY.TM. trapped ion
cell and longitudinally traverse the hexapole region of the
external ion reservoir finally impinging on the skimmer cone.
Alignment was accomplished by a preliminary visual alignment with a
visible diode laser such that light could be seen exiting the glass
desolvation capillary at the source-end of the spectrometer.
Subsequent alignment was optimized by the mass spectral
fragmentation response with the IR laser triggered during the ion
accumulation interval. The laser was operated at an output of 28
watts as measured at the entrance to the mass spectrometer.
Example 2
Comparison of Mass Spectrometry Methods with an Oligonucleotide
[0039] ESI-FTICR spectra of a 20-mer phosphorothioate
oligonucleotide were acquired from a 17 mM 50:50
H.sub.2O:isopropanol solution with 0.1% tripropylamine. Each
spectrum was acquired following a 500 ms ion accumulation interval
in the external ion reservoir, a) employing a standard detection
sequence, b) employing an in-cell IRMPD pulse sequence which
incorporates a 100 ms laser pulse from a CO2 laser (10.6 mm), c)
identical conditions as in a) except the laser is traversing the
external ion reservoir during the 500 ms ion accumulation interval
effecting IRMPD in the external ion reservoir.
[0040] FIG. 2a contains a typical ESI-FTICR spectrum of a 20-mer
phosphorothioate oligonucleotide obtained by externally
accumulating ions for 500 ms prior to injection and detection in
the FTICR cell. FIG. 2b contains an ESI-FTICR spectrum obtained
from in-cell IRMPD effected by externally accumulating ions for 500
ms in the external ion reservoir, transferring them to the trapped
ion cell, and then irradiating them for 100 ms. FIG. 2c contains an
IRMPD ESI-FTICR spectrum obtained from externally accumulating ions
for 500 ms concurrent with IR irradiation in the external
reservoir. Following the accumulation/irradiation interval, the
ions-are-transferred to the trapped ion cell where they are mass
analyzed. While a significant degree of fragmentation is observed
from the in-cell IRMPD as shown in FIG. 2b, the majority of the
assignable fragment ions correspond to relatively low molecular
weight singly charged fragments consistent with w and a-base ions
and their respective decomposition products including neutral base
loss and dehydration. From the collection of fragment ions
detected, only 6 bases from the 5' end and 6 bases from the 3' of
the analyte can be unambiguously assigned. Very few fragment ions
are present at multiple charge states and there is insufficient
fragmentation to determine the entire sequence of the
oligonucleotide. As evidenced by FIGS. 3a and 3b, the spectrum in
FIG. 2c is rich in fragment ions corresponding to a wide range of
charge states (1- to 5-) and molecular weights and provides greater
information than in-cell IRMPD. Full coverage of the
oligonucleotide sequence is observed with most w and a-base ions
observed at multiple charge states. In addition to an improvement
in fragment ion abundance and sequence coverage, the spectrum
acquired with the external IRMPD scheme exhibits improved resolving
power and signal-to-noise relative to the spectrum-acquired
utilizing in-cell IRMPD.
[0041] Table 1 compares the fragment ions observed for the 20-mer
phosphorothioate oligonucleotide employing three different
dissociation techniques, nozzle-skimmer dissociation, in-cell
IRMPD, and external IRMPD, all acquired under otherwise similar
conditions. Note that while both nozzle-skimmer dissociation (FIG.
2a) and in-cell IRMPD (FIG. 2b) result in 5' fragments extending
only to the a.sub.7-base ion, the external IRMPD scheme provides
multiple charge states of fragment ions extending to the
a.sub.13-base. Similarly, from the 3' end of the molecule,
nozzle-skimmer and in-cell IRMPD provide fragment ions out to the
w.sub.9 and 2.sub.7 species, respectively, while the external IRMPD
scheme provides w ions as large as the w.sub.13 ion. In general the
external IRMPD scheme provides more charge states of each fragment
than the other methods. For example, while nozzle-skimmer
dissociation produces the 2- charge state for the w.sub.7, w.sub.8,
and w.sub.9 species, the external IRMPD scheme produces the 2-, 3-,
and 4-charge state for each of these species providing further
confirmation of these sequence specific ions.
Table 1: Fragment Ions Observed for a 20-mer Phosphorothioate
Oligonucleotide Using Three Dissociation Techniques: Nozzle-skimmer
(DNS), IRMPD in the Trapped Ion Cell of a FTICR Mass Spectrometer
(In-cell IRMPD), and IRMPD Effected in the External Ion Reservoir
With Subsequent Detection by FTICR (Hexapole IRMPD)
[0042] The numbers in each column correspond to the charge state(s)
of the fragments observed. All ions are negatively charged.
1 in-cell Hexapole Fragment DNS IRMPD IRMPD a2-base 1 1 1 a3-base 1
1 1 a4-base 1 1,2 1,2 a5-base 1 1,2 1,2 a6-base 1 2 2,3 a7-base 1 2
2,3 a8-base 2,3 a9-base a10-base 2,3 a11-base 3,4 a12-base 4
a13-base 3,4 w2 1 1 w3 1 1,2 1,2 w4 1 2 1,2 w5 1 2 2,3 w6 3 w7 2
2,3 2,3,4 w8 2 2,3,4 w9 2 2,3,4 w10 3,4 w11 w12 w13 3,4
Example 3
Comparison of Mass Spectrometry Methods With Ubiquitin
[0043] ESI-FTICR spectra of ubiquitin acquired from a 10 mM 50:50
H.sub.2O:solution with 1% HOAc. The spectrum in a) was acquired
following a 500 ms ion accumulation interval in the external ion
reservoir, the spectrum in b) was acquired employing a 500 ms ion
accumulation interval during which the CO.sub.2 laser was
traversing the ion reservoir. The spectra obtained are shown in
FIG. 4.
* * * * *