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Description  |
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FIELD OF THE INVENTION
This invention relates to a method of facilitating DNA/RNA Mass
Spectrometry and more particularly to a method using laser ablation,
ionization and time of flight mass spectrometry to identify, by their
masses, large molecules and molecular fragments in complex mixtures.
BACKGROUND OF THE INVENTION
A need exists for determining the molecular mass of high molecular weight
organic molecules such as nucleic acids, proteins, oligosaccarides, and
like moieties having molecular weights of 3000 daltons (Da) and more, and
for polymer size determinations. Presently no accurate general method for
such determinations exist.
Heretofore, the best known method for the determination of protein and
nucleic acid masses is gel electrophoresis which at best has an accuracy
of .+-.5%. Presently, the only method known for determining polymer size
distribution is a gel permeation method which is recognized as imprecise
and only measures relative sizes. More accurate mass spectrometric methods
have been reported recently for protein mass determination, but this
approach has not been extended to other polymers.
Mass spectrometric analysis of massive biopolymers such as nucleic acids,
proteins, and oligosaccharides requires a means of volatilizing the
molecules without fragmentation or degradation, or with controlled
fragmentation, together with a means of ionizing the gas-phase molecules
efficiently, again without inducing fragmentation. Slow heating of such
molecules typically results in pyrolysis rather than volatilization. Thus,
a number of desorption techniques have been developed which involve a very
rapid input of energy into the target material, either by fast
(mega-electron volt) or slow (kilo-electron volt) heavy-ion impact or by
photon irradiation, to achieve desorption in a time that precludes
complete degradation. Advantages are derived from dissolving the sample to
be volatilized in a liquid or solid matrix, which, in the case of
kilo-electron volt ion impact desorption, can act to minimize ion beam
damage, or, for pulsed laser desorption, can serve as a chromophore,
efficiently coupling the radiative energy into the material to be
volatilized.
The present invention represents a substantial improvement over the prior
art by determining molecular masses through the use of pulsed laser
ablation, multiphoton ionization and time of flight mass spectrometry.
BRIEF SUMMARY OF THE INVENTION
The present invention utilizes a matrix to mediate the volatilization of
large molecules and employs a pulsed laser desorption technique for
biomolecules which is specifically demonstrated by the desorption of
intact DNA molecules of 410,000 Daltons (Da) molecular weight. In
addition, with the ablating laser tuned to a resonant frequency of certain
atomic components of the sample, e.g. alkali and alkali earth metals,
multiphoton ionization of these atoms is induced efficiently producing
ions which attach to the volatilized sample molecules. The resulting
ionized molecules can be accelerated into a mass spectrometer and
identified by accurate determination of their masses.
More particularly the present invention comprises a process, in which a
pulsed laser irradiating the sample stage or the sample can cause complex
molecules such as nucleic acids, polymers and the like to be volatilized,
intact or partially fragmented, which allows accurate determination of the
mass of such intact molecular ions and/or fragments, and the identity and
structure of such complex molecules to be elucidated.
Accordingly a principal object of the present invention is to provide
improved means and methods for the volatilization and consequent mass
spectrometric analysis of involatile, thermally labile high molecular
weight compounds such as nucleic acids, carbohydrates, proteins and like
biopolymers.
Another object of the present invention is to provide improved means and
methods for characterizing non-biochemical polymers by mass spectrometric
analysis.
Still another object of the present invention is to provide a means to
control the fragmentation of volatilized large molecules, suppressing
fragmentation when analysis of complex mixtures is desired, and
controllably inducing fragmentation at structure-specific sites when
structural information is desired for a single molecular species.
These and still further objects as shall hereinafter appear are readily
fulfilled by the present invention in a remarkably unexpected manner as
will be readily discerned from the following detailed description of an
exemplary embodiment thereof especially when read in conjunction with the
accompanying drawing in which like parts bear like numerals throughout the
several views.
BRIEF DESCRIPTION OF THE DRAWING
In the drawing:
FIGS. 1A, 1B and 1C are is a graphic representation of a timed sequence in
practice of the present invention;
FIG. 2 is a five shot laser ablation/ionization Time of Flight mass
spectrum of the single-stranded DNA oligomer dp(A).sub.8 obtained at a
power density of approximately 5.times.10.sup.8 W/cm.sup.2 and wavelength
of 578 nm showing the parent (2600 Da) and dimer (5250 Da) molecular ions;
FIG. 3 is a five shot spectrum of the single-stranded DNA oligomer
dp(A).sub.8, obtained at a power density of approximately 5.times.10.sup.7
W/cm.sup.2 and wavelength of 589 nm showing fragmentation; and
FIG. 4 is a spectrum of the double-stranded DNA oligomer
##STR1##
obtained at a laser power density of about 5.times.10.sup.8 W/cm.sup.2.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to laser ablation/ionization and mass
spectrometric analysis of massive polymers. Effective laser desorption of
massive molecules can be accomplished by ablating a frozen film of
solution containing the molecules. The film, when ablated, produces an
expanding vapor plume which entrains the intact molecules or fragments
thereof.
The use of a volatile frozen solvent having a low boiling point and a low
critical temperature provides several additional advantages as will be
described. First, the critical temperature imposes an upper limit on the
temperature attained before ablation occurs. Second, the free expansion of
the ablated matrix vapor produces a substantial degree of internal cooling
of the entrained macromolecules which stabilizes them against gas phase
dissociation. Cooling can be extremely rapid. For example, with a laser
spot size of 0.1 mm, substantial cooling occurs over a distance of about 1
mm above the surface of its substrate, in about 1 microsecond if gas
velocities are about 10.sup.3 m/s. The matrix is further chosen for its
solvent properties and for its vacuum compatibility as will hereafter
appear in greater detail.
Water, the natural solvent for most biomolecules, is an appropriate solvent
for use in the practice of the present invention. The vacuum compatibility
of the water is assured by freezing the solution to liquid nitrogen
temperature. To produce the ionization needed for mass spectrometry, it is
preferable to use a laser wavelength in the visible region namely between
400 nm to about 600 nm.
The pulsed laser ablation, in vacuum, of DNA molecules from frozen aqueous
solutions has been accomplished. DNA was chosen as a test material because
such large nucleic acids have not previously been volatilized by
desorption techniques, and because sensitive autoradiographic techniques
are available to detect and characterize .sup.32 P-labeled DNA.
To verify the contents of the vapor plume created by laser ablation the
laser target was a thin film of a frozen aqueous TE buffer (10 mm tris, 1
mm EDTA, pH 7.5), solution of an Msp 1 restriction enzyme digest of the
Escherichia coli plasmid pBR322, containing fragments of double-stranded
DNA ranging in size from 9 to 622 base pairs, or from about 7 to 410 kDa.
The solution (50 to 100 microliters, 2 micrograms/mL) was smeared onto a
copper cold finger which was initially cooled to -20.degree. C. to create
a thin ice film. If desired, the cold finger can be acid-cleaned before
each experiment and will exhibit a bright metallic copper surface. After
several days of applications, a visible thin film of corrosion
(greenish-brown in color) appears on the surface of the copper substrate.
Preferably, this corrosion film is left on the cold finger surface because
it improved the efficiency of the ablation process as hereinafter
described.
The cold finger is inserted into an ion-pumped vacuum system and cooled
with liquid nitrogen while the system is evacuated to 10.sup.-6 torr. The
frozen films are then irradiated in vacuum by 20-nanosecond (ns) pulses
from an excimer laser-pumped dye laser operating at 581 nm (wavelength of
maximum laser output for the system used) at power densities ranging from
about 10.sup.6 to about 10.sup.8 W/cm.sup.2. The laser power density at
the film surface is varied by changing the laser spot size at the target
over a range of diameters between 0.15 mm and 1.5 mm using a lens with a
focal length of 150 mm. The spot sizes were estimated visually after
irradiation. At 581 nm both the DNA and the water are transparent, and
energy deposition occurs initially in the copper substrate. Ablated
material is collected on siliconized microscope slides placed 2.0 cm away
from the target. After the slides are removed from the vacuum system,
direct-contact autoradiograms of the collector slides are obtained.
When thin regions of the ice film (10-100 micrometers thick, estimated from
the pressure pulses on the ion pump power supply), are irradiated, most of
the radioactivity collected is concentrated in diffuse but strongly
forward-peaked deposits characteristic of the free expansion of the vapor
from the laser-ablated areas.
Subsequent polyacrylamide gel electrophoresis (PAGE) of material extracted
specifically from the ablation deposits indicated that the material was
fragmented to a variable degree, but that intact DNA molecules as massive
as 410,000 Da had also been ablated from the starting digest.
To demonstrate the efficacy of the present invention a simple linear time
of flight (TOF) mass spectrometer was constructed. A field-free drift
region was created using a section of copper tubing (43 cm in length, 1 cm
i.d.), the ungridded entrance of which was placed 1 cm away from the
cooled sample stage. For positive ion mass spectra, the drift tube was
held at an acceleration potential of -100 eV while the sample stage
remained at ground potential. Terminating the drift tube was a 16-dynode
electron multiplier with the first dynode held at -3.5 kV. The signal from
the electron multiplier was fed through an operational amplifier (time
constant about 5 microseconds) to a Tektronix model 2221 digital storage
oscilloscope (200 ns/channel as used). 20 ns duration pulses from an
excimer laser-pumped dye laser (Lambda Physik EMG50/FL2000) impinged on
the sample at an angle about 45.degree.-50.degree. to the sample normal.
The laser was focussed through a lens of 20 cm focal length to a spot size
on the sample which was variable in area from between about 10.sup.-1 to
about 10.sup.-2 mm.sup.2. The oscilloscope was triggered at the beginning
of the laser pulse, and ion intensities were monitored with respect to
time. Flight times at the maxima of the peaks were determined using the
internal cursor of the oscilloscope. Spectra were output to an X--Y
plotter. The figures were obtained by digitizing the mass spectra from the
raw X-Y plots into a suitable computer (HP 9836 Hewlett-Packard), and then
replotting the data (see FIGS. 2-4). The background signals between peaks
in the mass spectra arose from amplifier noise. No background subtraction
was performed.
Time to mass conversion was performed using an instrumental calibration
equation determined from the linear regression fit of mass vs. time data
obtained by the laser ablation/ionization of cesium iodide samples.
Cluster ions from the cesium iodide were resolved up to (CsI).sub.6
Cs.sup.+. For these peaks, mass determination errors averaged .+-.0.5%,
with errors stemming mainly from the broad peak shapes. Because of the
long time constant of the operational amplifier, operation at the low
accelerating voltage of -100V was used to achieve a mass resolving power
of about 5-15 in the mass range from 1-10,000 Da. Even with this
instrument limitation, resolution of molecular fragments sufficient for
identification was achieved Mass spectra were also obtained from frozen
cesium iodide solutions. Cesium iodide clusters were not seen above
(CsI).sub.2 Cs.sup.+ in this case, nor were water clusters larger than
(H.sub.2 O).sub.3 H.sup.+. The high molecular weight ions observed from
frozen nucleic acid solutions were not massive water cluster ions.
The nucleic acid samples used were obtained in their sodium salt forms and
diluted to about 2 micrograms/ml with a 10mM : 1 mM tris:EDTA (TE) buffer
solution, pH=7.5. Approximately 40 microliters (about 8-30 picomole DNA)
of the solutions were smeared onto a 1 cm.sup.2 area of a pre-cooled
(about 253 K) flat copper sample stage which was cooled in vacuum by means
of a liquid nitrogen cold finger. Prior to application of the sample, the
surface of the copper sample stage was either polished to a shiny
appearance or allowed to corrode (by application of the TE buffer to the
sample stage several days prior to sample preparation). After about 30 min
at 253K and atmospheric pressure, the sample stage was inserted into the
vacuum system and slowly pumped down with a rotary pump as the sample
stage was cooled to liquid nitrogen (LN.sub.2) temperature. After the
sample had achieved LN.sub.2 temperature, the system was evacuated with an
ion pump (120 L/s) to a pressure of about 1.times.10.sup.-6 torr.
During evacuation, the thin ice films slowly sublimed to achieve final
thicknesses ranging from tens to hundreds of micrometers. Film thickness
were estimated by monitoring the current inflections (proportional to the
pressure inflections) of the ion pump power supply during laser
irradiation.
Initially, mass spectra were obtained using a laser wavelength of 581 nm;
this was the laser wavelength at which the maximum power output was
obtained for the laser dye used (Rhodamine 6G). It was found that by
tuning the laser to wavelengths in resonance with electronic transitions
of sodium or copper atoms, which populated the ablated vapor plume, more
intense and much more reproducible spectra were obtained. Under these
conditions, ionization occurs by multiphoton ionization of the sodium or
copper atoms followed by attachment of the resulting ions to the ablated
biomolecules as shown in FIGS. 1A, 1B and 1C.
The mass spectra shown in FIGS. 2 through 4 were obtained at two different
laser wavelengths, namely 578 nm and 589 nm. At 578 nm, atomic sodium
exhibits a resonant 2-photon electronic transition and atomic copper
exhibits a resonant one-photon transition and irradiation at this
wavelength increased the ionization efficiency of the molecular species.
Similarly, sodium exhibits a resonant 1-photon electronic transition at
589 nm. By tuning the laser to this wavelength, molecular ion signals of
comparable intensities and reproducibility to those obtained at 578 nm are
obtained. Compared to the spectra obtained at off-resonant wavelengths
such spectra exhibited an increase in molecular ion intensities of about
an order of magnitude. The ratio of parent molecules to fragments was
previously observed to be dependent on the laser power density and the
absorptivity of the copper substrate, each of which has influence on the
substrate heating rate. In the wavelength range 578-589 nm, the
absorptivity (A) of polished copper is about 0.3, and increases to about
0.9 for a corroded surface. All spectra presented here were obtained from
samples applied to an corroded (A about 0.9) sample stage, which, at a
laser power density of 5.times.10.sup.8 W/cm.sup.2, produced the highest
ratio of parent to fragment ions.
As stated earlier, the resolving power of the mass spectrometer used was
limited to 5-15. The large width of the parent and fragment peaks arises
primarily from the limitations of the amplifier used. Not only does the
long time constant of this amplifier (about 5 microseconds) lead to
intrinsically broad peaks, but also the long time constant dictated
operation at a low accelerating voltage of .sup.- 100V, exacerbating the
effects of initial kinetic energies of the ions.
FIG. 2 is a mass spectrum (sum of 5 laser shots) of the single-stranded DNA
oligonucleotide pd(A).sub.8, laser ablated/ionized from frozen aqueous
solution at a laser power density of 5.times.10.sup.8 W/cm.sup.2 and
wavelength of 578 nm. Peaks are observed at masses 2600 and 5250 Da, which
were identified as the parent monomer and dimer, pd(A).sub.8 + and
2(pd(A).sub.8)+, respectively (MW=2,720 D for the sodium salt of the
molecule). A shift to lower masses should result if the ions acquire
kinetic energies of a few eV, corresponding to expansion velocities of a
few hundred meters/second. Intense ion signals are also present in the
mass region form about 50 to 600 Da, presumably derived from multiple
fragmentation of the parent molecule.
FIG. 3 shows a 5 shot accumulation mass spectrum of single stranded DNA
oligomer pd(A).sub.8 at a laser wavelength of 589 nm, and a power density
of 5.times.10.sup.7 w/cm.sup.2. Peaks indicating partial fragmentation of
the parent molecule are seen. The peaks shown are consistent with removal
of consecutive pd(A) nucleotide units from the parent molecule. Fragment
ions of this sort were typically observed at a laser power density less
than 1.times.10.sup.8 W/cm.sup.2. The relationship between laser power
density and the degree of fragmentation is inverse. The nucleic acid is
transparent in the wavelength region used, so little direct excitation of
the molecules should occur. It is believed that fragmentation occurs in a
transient high temperature liquid phase as the solutions are heated to a
temperature (limited by the critical temperature of the H.sub.2 O matrix,
647K) sufficient for ablation to begin. Once expansion of vapor begins,
cooling occurs, effectively quenching the fragmentation process. Reducing
the power input by a factor of 10 lengthens the heating time by a factor
of 100, allowing more time for fragmentation in the liquid phase. The
absence of a continuous background signal, which would arise from
unimolecular dissociation in the acceleration region, is consistent with
the idea that fragmentation occurs solely in the liquid phase.
FIG. 4 shows a mass spectrum obtained by laser ablation/ionization of the
double-stranded DNA oligomer,
##STR2##
The mass spectrum was obtained using a laser power density of about
5.times.10.sup.8 W/cm.sup.2, and a laser wavelength of 589 nm, and shows a
parent molecular ion signal at mass 10,300 Da. In the low mass region, a
peak corresponding to Na+ is observed. Signals are observed in the mass
region 280 to 390 Da, stemming from fragmentation of the sample molecules.
The calculated mass for the parent molecule (sodium salt, cationized with
Na.sup.+) is 10,619 Da.
Typically, a molecular ion signal is observed from a given target area for
a duration of 1-3 laser pulses, after which only Cu.sup.+ and Na.sup.+
are observed. Signals due to molecular fragmentation, and H.sup.+ and
(H.sub.2 O).sub.n H.sup.+ clusters also disappear after a few laser
shots. During acquisition of multiple-shot spectra, the sample stage is
moved between each laser shot to expose fresh material. For each analysis
a total of between 8-30 pmol of nucleic acid is applied to the substrate.
Assuming uniform coverage over the 1 cm.sup.2 sample area, the total
number of molecules desorbed per pulse was approximately 10.sup.8
-10.sup.9 (spot area 10.sup.-2 -10.sup.-1 mm.sup.2), so that only a few
femtomoles (tens of picograms) of nucleic acid were removed to obtain each
5 shot spectrum. Since the sample received no treatment other than
freezing, unablated sample can be readily recovered when desired.
As will appear, the above described techniques are not limited to the
nucleic acids or proteins. The laser ablation of polymers from films of
frozen solutions as described herein allows the determination of polymer
size distribution per se. Thus, any polymer candidate can be dissolved in
a volatile organic solvent, such as benzene or toluene, frozen onto a
liquid nitrogen-cooled cold finger, and thereafter ablated with a pulsed
laser into a time-of-flight mass spectrometer. By coating the substrate
with a compound containing a readily ionizable metal such as sodium, or
other alkali or alkaline earths, and tuning the laser to the appropriate
resonant transitions such as 578 or 589 nm, for sodium, or by tuning the
laser to a resonant transition in atoms of the substrate material such as
578 nm for copper, ions are produced which attach to the ablated polymer
molecules to allow mass spectrometric separation. The difficulty of
ionizing hydrocarbon polymer molecules, which are not intrinsically
ionized in the solid phase, has previously presented a major impediment to
polymer mass spectrometry. The mass measurement is absolute, in contrast
to gel permeation; mass range should be at least 300,000 daltons,
encompassing many commercial polymers; and accuracy of mass determination
is better than 0.01%, far better than gel permeation.
The pulsed laser ablation of frozen aqueous solutions as described herein
offers a unique volatilization technique for bimolecular and polymer mass
spectrometry. Given the production of vapor-phase molecules, mass
spectrometry requires, in addition, ionization, mass analysis, and
detection steps. The process of resonant multiphoton ionization of atoms
in the ablated plume, followed by attachment of these ions to the ablated
molecules is a new and important process which considerably simplifies
mass spectrometry of ablated massive molecules. Mass analysis by
time-off-light techniques has a mass range limited only by the ability to
detect massive molecular ions. Such detection is vastly improved by
creating more ions in a given laser pulse, using the multiphoton
ionization and attachment process of the present invention. The varying
degree of fragmentation evident in the DNA mass distributions results from
the different rates of energy input into the matrix which may be
controllably induced by varying the laser power density. Because small
oligonucleotides undergo thermal fragmentation preferentially at the
phosphodiester linkage, direct acquisition of sequence information in the
mass spectrometer is now possible.
Time of flight mass spectra of single and double-stranded oligomeric
nucleic acids, at masses up to 10,600 Da, have been shown. Volatilization
is accomplished by pulsed laser ablation of frozen aqueous solutions of
the sample at laser wavelengths of 578 and 589 nm. Fragmentation was
increased when the rate at which energy was deposited in the substrate was
reduced by lowering laser power density. It is therefore possible to
obtain sequence information directly for small single-stranded
oligonucleotides by determining the masses, and therefore the identities,
of individual nucleotides split off sequentially from the terminus of an
oligonucleotide chain.
From the foregoing, it becomes apparent that means and methods have been
herein described and illustrated which fulfill all of the aforestated
objectives in a remarkably unexpected fashion. It is of course understood
that such modifications, alterations and adaptations as may readily occur
to an artisan having the ordinary skills to which this invention pertains
are intended within the spirit of the present invention which is limited
only by the scope of the claims appended hereto.
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