Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Enhanced Resolution MALDI TOF-MS Sample Surface
Technical Field
This invention relates generally to mass spectrometry, and in particular to
matrix assisted laser desorption and ionization time-of flight mass
spectrometry.
Specifically, this invention relates to a method and apparatus for improved
sample
desorption by laser excitation that results in greatly enhanced mass
resolution and
sensitivity for a time-of flight system.
2. Background Art
Matrix-assisted laser ionization and desorption time-of flight mass
spectrometry is a recently developed technique which is particularly useful
for the
sensitive analysis of large biomolecules.The matrix is a material that assists
in the
transfer of energy to the analyte molecule, allowing it to be ionized without
significant
fragmentation, and leave the surface of a target that is being irradiated with
a laser.
Typically, a few microliters of a solution containing sample molecules at
concentrations of about 1 ~g/~L are mixed with 10-20 ~L of a solution
containing
matrix molecules at concentrations of about 10 pg/~L. A few microliters of
this
mixture are then deposited on a suitable substrate and dried in air.
As the sample dries, crystals of matrix are formed and the sample is thought
to
be incorporated into the crystal. The substrate is then introduced through a
vacuum
lock into a time-of flight mass spectrometer system. In such systems, a high
voltage
source (often 30 KV or more) will be connected to the substrate.
Once the sample has been introduced into the mass spectrometer, a pulsed laser
is used to irradiate the sample on the substrate. The interaction of the laser
radiation
with the matrix molecules leads, by a process that is only partly understood
today, to
the formation and desorption of largely intact, ionized sample molecules.
Predominantly these ions are of a type known as (M+H)+ ions, that is, the
neutral
sample molecule (M) is ionized by the attachment of a proton.
3o Alternatively, negatively charged ions may be produced, for example by the
removal of a proton. This ionization process has some similarity to the
process called
"chemical ionization" used conventionally in gas chromatography/mass
spectrometry.
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Most frequently these ions are analyzed in so-called linear time-of flight
(TOE)
mass spectrometers. The ions, once formed, are accelerated by an electric
field and
then allowed to travel in straight lines until they are detected. The transit
time between
ion formation and detection can be used to determine the mass of the species
from
which the ions are generated. Typical linear TOE systems are described in U.
S. Pat.
No. 5,045,694 ( Beavis and Chait). Such linear devices provide only modest
mass
resolving power, e.g. 50-800, because they are unable to compensate for
various
known aberrations. A dominant aberration in such linear systems stems from the
fact
that the ions are formed with a wide distribution of initial velocities. This
means that
1 o for an ion of a given mass there will be a distribution of arrival times
at the detector
that will limit the mass resolving power of such a device, since ions with
more initial
velocity in the forward direction will arrive sooner than ions with less
initial velocity.
Techniques for compensating for such aberrations resulting from the initial
velocity distribution in TOE mass spectrometers are well-known. The primary
technique is to provide an electrostatic mirror, called a Reflectron, which
reverses the
direction of travel of the ions in such a way that the effects of these
initial velocity
distributions on ion transit times are eliminated. A recent review article
describing such
devices is "Time-of flight Mass Spectrometry: An increasing Role in the Life
Sciences", R. J. Cotter, Biomed. Env. Mass Spectrom., 18: 513-532 ( 1989). The
practice of matrix-assisted laser desorption and ionization in Reflectron-
based
instruments is also known and typically produces mass resolutions of 2000-4000
for
molecules less than 5000 Daltons in molecular weight.
In both linear and Reflectron-based TOE instruments, it is thought that a
significant factor limiting mass resolution is the interaction of sample ions
with other
desorbed matrix ions and molecules i.e., as the desorbed biomolecules leave
the surface
of the target, they may encounter a plume of matrix ions and molecules.
Interactions
with this plume may change the energy of desorbed biomolecule ions, but not in
a
homogeneous manner. Some biomolecules may gain more kinetic energy, some may
lose kinetic energy. Thus, the time of arrival of the biomolecules is not
exactly the
same because some are flying faster, and some slower, than the mean. The net
result is
band broadening and a concomitant loss in mass resolution. To date, the
inventors are
not aware of efforts to narrow the initial velocity distribution by
manipulation of the
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matrix. Crystal formation occurs as the sample/matrix mixture dries down in a
largely
uncontrolled manner. It is postulated that this leads to variability in the
analysis of the
sample. In addition, the necessity of mixing sample with matrix prior to dry
down often
results in ineffcient use of sample. Accordingly, it would be desirable to
provide
methods and apparatus for providing greater consistency in the sample
preparation
process and more efficient use of sample.
Hillenkamp et al., in British Patent Nos. GB 2236185A, and GB 2236186A,
disclose surfaces and matrices for laser desorption of biomolecules from
surfaces. GB
2236185A discloses a two-dimensional layer comprising a matrix ("absorbing
component") underlying the substrate. The application is aimed at
macromolecular
blotting. GB 2236186A discloses desorption ofbiomolecules using 337 nm or
higher
laser radiation on a similar surface. Sinapinic acid is shown l5as the matrix.
Cottrell, PCT/GB90/00973, discloses a method for preparing a sample for
analysis by LDMS that includes electrospraying the matrix (Nicotinic acid)
onto a
target surface, then sample in TFA is applied and dried down. Finally, the
sample is
introduced into the mass spectrometer and a laser is directed onto the sample,
desorbing the sample.
Cottrell, PCT/GB90/00974, discloses a method for preparing a sample for
analysis by LDMS that includes electrospraying a substrate (Nitrocellulose)
onto a
target, depositing a sample dissolved in aqueous 0.1 % trifluoroacetic acid
(TFA), and
then drying it down. Matrix material (Nicotinic acid, 30 mM in acetone) is
then applied
in droplet form to cover the dried-down sample and dissolve the substrate
Nitrocellulose. Sample, substrate and matrix dry down together in an
intermixed form.
If the sample is a protein, the protein adsorbs to the Nitrocellulose by
hydrophobic
interactions. Loss of mass resolution is caused when excess matrix is
evaporated from
the surface of the target, causing the plasma effect described supra.
Cottrell, PCT/GB90/00975, additionally discloses the use of various matrix
materials such as Cinnamic acid, Benzoic acid, or Coumarin in the methods
disclosed
above.
Beavis, J. Phys. D: Appl. Phys., 26(3), 442-7, has emphasized the desirability
of crystal formation.
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Hutchens (Proceedings of the 41St American Society
of Mass Spectrometry Conference on Mass Spectrometry and
Allied Topics, May 31 - June 4, 1993, pp. 781a-781b) has
described a technique called Surface-Enhanced Neat
Desorption (SEND), in which energy-absorbing molecules are
covalently bonded to an inert substrate, allegedly providing
a matrix-free method for introducing large molecular weight
biopolymers into the gas phase without fragmentation.
However, this technique appears to have limited
reproducibility. Other efforts to laser desorb biomolecules
include those of Tanaka et al. (Rapid Commun. Mass
Spectrom. 2, 151-153, (1988)) who describe a system for
matrix-assisted laser desorption and ionization in which the
sample is dissolved in glycerol containing small Cobalt
particles. Cornett et al, Anal. Chem. 65: 2608-2613 (1993)
has described a system in which various energy absorbing
molecules such as Rhodamine 6G are dissolved along with the
sample molecules in a liquid matrix such as 3-nitrobenzyl
alcohol.
Williams in U.S. Patent No. 5,135,870 and Becker
in U.S. Patent No. 4,920,264 describe systems involving
frozen layers of ice for the desorption and ionization of
DNA. None of these systems have demonstrated surprising
resolution to date.
Thus the. need exists for a laser desorpt on matrix
composition that can decrease the loss in mass resolution
that occurs when using prior art procedures as well as
provide increased sensitivity.
Summary of the Invention
According to the present invention, there is
provided a thin layer for sample analysis by matrix-assisted
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laser desorption mass spectrometry, comprising crystals of a
matrix material dispersed in a support material, wherein the
support material is a solid or is formed from a solid and
said solid support material limits the growth of matrix
material crystals.
Also according to the present invention, there is
provided a thin layer for sample analysis by matrix-assisted
laser desorption mass spectrometry, comprising a matrix
material-solid composition ranging from 70~ solid to 30~
solid, disposed upon a substrate wherein the matrix material
is dispersed within said solid as crystals, said thin layer
promoting resolution and/or reproducibility of mass
spectrometry analysis.
According to the present invention, there is
further provided a method for making a thin layer for sample
analysis by matrix-assisted laser desorption mass
spectrometry, said thin layer comprising a matrix material
dispersed within a support material, comprising the steps
of: (a) depositing a solution containing said matrix
material, said support material and a solvent upon a
substrate; and (b) evaporating said solvent, thereby forming
a dispersion of said matrix material and said support
material in a thin layer on said substrate.
According to the present invention, there is
further provided a method of making a thin layer for sample
analysis by matrix-assisted laser desorption mass
spectrometry, said layer comprising a matrix material-
support material dispersion on a substrate, said layer
having a thickness of not greater than 2 Vim, comprising the
step of depositing a solution containing a matrix material,
a support material and a solvent upon a spinning substrate
at a deposition rate sufficient to allow evaporation of said
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solvent, thereby forming a thin layer comprising the matrix
material dispersed within said support material on said
substrate.
There is also provided a thin film for receiving a
sample for analysis by matrix-assisted laser desorption mass
spectrometry, said thin film comprising a matrix material
dispersion on a substrate, wherein said thin film is formed
by depositing a solution containing a matrix material, a
support material and a solvent upon a spinning substrate at
a deposition rate sufficient to allow evaporation of said
solvent, thereby forming said thin film of said dispersion
of said matrix material within said support material on said
substrate.
3. Brief Description of Drawings
Figs. la and 1b are mass spectra obtained from an
insulin sample deposited on a thin film of cellulose acetate
and matrix on a glass disc substrate; Fig. lc is a
photomicrograph of the same insulin sample on this thin film
on this disc.
Figs. 2a, 2b, 2c and 2d are scanning electron
micrographs of cross-sections of preprepared matrix-
cellulose acetate films.
Figs. 3a and 3b are mass spectra obtained from an
insulin sample prepared by conventional means, deposited and
dried on a glass disc; Fig. 3c is a photomicrograph of the
same insulin sample on this disc.
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Figs. 4a and 4b are mass spectra obtained from an insulin sample deposited on
a thin film on a glass disc substrate (an alternative embodiment); Fig. 4e is
a
photomicrograph ofthe same.insulin sample on this disc.
Fig. 5 is a high-resolution mass spectrum of a sample of mahoheptaose
deposited on a preprepared substrate similar to that one used to generate
Figs. la and
1b.
Fig. 6 is a mass spectrum obtained with insulin on a thin precast film ofi
cellulose nitrate and matrix on a. glass disc substrate.
tp q. ' Description of Embodiments
The present invention overcomes the disadvantages and limitations of the prior
art by providing a surprising enhancement in mass resolution comprising a thin-
layer
for sample analysis by laser desorption mass spectrometry wherein the
band-broadening contribution due to the matrix substrate sample composition is
. miuuimized. In accordance with the invention, mass resolution and
sensitivity are
increased by the use of a thin layer com~prisutg a matrix material in a
supported
dispersion wherein the support is a solid or is formed from a sofid. The solid
support is
preferably a solid that limits matrix crystal growth, most preferably a
polymer.
Thiclcnesses not greater than 1 um are most preferrtd.
The preferred embodiment of the inv~tion is a thin layer for sample analysis
by
matrix assisted laser desorption mess spectrometry, comprising a matrix-
polymer
composition disposed upon a substrate. Any matrix material may be used, but
Sinapinic
acid or a-cyano-4 bydroxycinnamic.acid is preferred. The matrix-polymer
composition
may include a polymer selected from the group consisting of cellulose acetate,
cellulose nitrate, and polycarbonate. However, other polymers are also
possible. The
matrix-polymer composition may preferably range from about 70% polymer to
about
30% polymer. Preferably, it is 50%. The mass resolution of the sample is
significantly
enhanced using this invention, and unexpected mass resolutions of over 8000
have
been demonstrated in a linear 150-cm flight tube machine.
The invention is also directed to a device for performing matrix-assisted
laser
desorption mass spectrometry of sample molecules. comprising a substrate
capable of
receiving on its surface a thin layer as previously described. The device is
combined
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with a solution of sample molecules wherein the sample and matrix are
substantially
coplanar, and then subjected to mass analysis.
The substrate underlying the device is selected from the group consisting of
glass, ceramic, plastic, metal, or similar materials. The thin layer of the
device is
resistant to decreased mass resolution and sensitivity over time, i.e., it has
a substantial
shelf life.
The invention is also directed to a method for making a thin layer for sample
analysis by matrix-assisted laser desorption mass spectrometry, the thin layer
comprising a matrix material in a supported dispersion wherein the support is
a solid,
comprising the steps of depositing a solution containing matrix, support and
solvent
upon a substrate; and then evaporating the solvent, thereby interspersing the
matrix
and support on the substrate in a thin layer. The preferred method is spin
casting,
however other methods are also possible. The product made by this process is
also a
part of this invention.
The invention will be described in part by referring to the attached figures.
The
invention is directed to a thin layer for sample analysis by matrix-assisted
laser
desorption mass spectrometry, comprising a matrix material in a supported
dispersion
wherein the support is a solid or is formed from a solid. The term "supported
dispersion" refers to a solid support for maintaining the matrix in a
dispersed state in
which large crystal formation is inhibited. A preferred embodiment is a thin
film of
matrix comprising a-cyano-4-hydroxycinnamic acid and polymer comprising
cellulose
acetate, having a thickness of not greater than 1 ~.m Other matrices and
solids,
including polymers, may be used. For matrices, these may include, but are not
limited
to, thymine, pyrazinecarboxylic acid, thiourea, nicotinic acid, vanillic acid,
ferulic acid,
caffeic acid, sinapinic sacid, dihydrobenzoic acid, and other derivatives of
these acids.
See GB 2236185A (Hillenkamp et al.) for other matrices.
Polymers that may come within the scope ofthis invention broadly include all
those that may be used in the process of making the matrix-polymer thin film,
as
described below. These polymers may include cellulose acetate, cellulose
nitrate,
3o polycarbonate, nylon, PVDF and any that may conveniently be prepared as
solutions.
The specific combination of matrix and polymer does not appear to be critical.
Sinapinic acid and a-cyano-4-hydroxycinnamic acid are preferred matrices,
while
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polycarbonate, cellulose acetate, and cellulose nitrate are preferred
polymers. One
fimction of the polymer appears to be to inhibit formation of large, thick
crystals of
matrix.
The solvent used to deposit both matrix and solid must be able to solubilize
botli. For cellulose acetate and a-cyano-4-hydroxycinnamic acid, acetone is
preferred.
For cellulose nitrate, 75% ether/25% ethanol is preferred. For polycarbonate,
tetrahydrofuran is preferred. One of ordinary skill will be able to determine
a suitable
solvent for solubilizing both matrix and polymer.
The choice of material for a substrate onto which these films of matrix and
polymer are deposited is not critical and various materials may have different
advantages in different analytical situations. Stainless steel, glass and
quartz discs are
preferred embodiments. Ceramic, plastic and other compositions, porous and
non-porous surfaces, are likely to work. Further, it may be useful to
impregnate matrix
into a thin surface layer, either as a dissolved species or into small voids,
into or onto a
thick material such as a polymer sheet that can readily be punched into
conveniently
shaped substrates.
It is possible that having a substrate that would control the thiclaless of
the
matrix-sample mixtures in these systems would be bencficial. This could be a
porous
polymer substrate, or any substrate that would absorb and retain the matrix-
sample
mixture in a thin layer. Microreticulated surfaces are specifically
contemplated.
The thickness of these films is believed to be an important aspect of the
invention. Increased mass resolution is shown in those films which comprise a
thin
layer, i.e. having thicknesses under 2 ~.m. The range of thicknesses over
which this
invention is operable is from 5 to .005 ~m A preferred range is from 2 to 0. I
Vim, and
the most preferred range is from 1 to 0.05 ~m Fig. 2b shows scanning electron
micrographs of films of the matrix a-cyano-4-hydroxycinnamic acid and
cellulose
acetate. These pictures were obtained by taking glass discs onto which had
been
deposited thin films, and fracturing them so that the films could be observed
in
cross-section. Fig. 2a shows a cross section of a film such as those used to
obtain the
spectra displayed in Figs. la, 1b, and 5. Its thickness can be measured as
approximately
0.37 Vim. Fig. 2b shows a film in cross-section which was prepared in a
similar fashion
except that the substrate was spun at roughly 30,000 rpm instead of 5000 rpm
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resulting in a thinner film. Its thickness can be measured as approximately
0.09 gm.
Spectra obtained from sample deposited on such a film can provide high
resolution, but
signal is sometimes not as uniform across the substrate. Fig. 2c shows a thick
film
approximately 2.2 ~m thick which provides lower resolution and lower
sensitivity
spectra. Fig. 2d is a picture at greater magnification of the same film shown
in Fig. 2b.
The exact reason for this increased performance is not completely evident at
this point, but it is likely that several different factors are relevant.
Although the
invention is not limited to any particular theory, the following may explain
the
observed results. One model for the matrix assisted desorption and ionization
process
proposes that it is necessary for the solid matrix to be converted to a
gaseous plume by
absorbing incident laser radiation which then serves to expel the sample
molecule or
ion into the vacuum (A. Vertes et al, "Expansion Cooling in Matrix Assisted
Laser
Desorption-a Hydrodynamic Study," Proceedings of the 39th American Society of
Mass Spectrometry Conference on Mass Spectrometry and Allied Topics May 19-
24,
1991, pp. 927-928). For the molecules that are absorbing energy from the
heated
matrix, it is usually true that the thickness of matrix crystals will affect
how quickly
their temperature will rise, i.e., thicker objects take longer to get hot.
Accordingly,
having a thin film may help ensure that all ions are created at closer to the
same time.
Thus, it is possible that having the sample and matrix contained in a uniform
thin film,
as opposed to the relatively thick, jagged, three dimensional crystals of the
prior art,
may improve the likelihood that all ions formed start closer to the same place
and at
the same equipotential in the acceleration region. This obviously would help
improve
resolution.
Alternatively the same model proposes that as sample ions are formed and
accelerated in the acceleration region of the mass spectrometer, they undergo
collisions
with neutral and charged matrix molecules and ions and that these collisions
contribute
to an energy "spread" of the ion population. It is therefore possible that
ions formed
from sample molecules in or contiguous to a thin film of matrix will undergo
fewer
collisions and thus suffer less energy spread. It may well be that the
importance of the
relatively inert polymer in this film is to provide mechanical integrity, but
also to
prevent the aggregation of the matrix into large structures when the sample
molecule
solvent is applied and thus to maintain the sample molecules and the matrix
molecules
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in a physically constrained and hence thin environment. This explanation is
consistent
with the improved resolution demonstrated in Figs. la and 1b relative to Figs.
3a and
3b.
The invention also includes a method of making a thin layer for sample
analysis
by matrix assisted laser desorption mass spectrometry, the layer comprising a
matrix
material in a supported dispersion wherein the support is a solid, comprising
the steps
of depositing a solution containing matrix, solid and solvent upon a
substrate, and then
evaporating the solvent, thereby interspersing the matrix and support on the
substrate
in a thin layer. The method is accomplished by depositing roughly 10 ~L of a
solution
of 30 mg/ml of a polymer, most preferably cellulose acetate, and 30 mg/ml of a
matrix,
most preferably a-cyano-4-hydroxycinnamic acid, onto a substrate, preferably a
glass
or metal disc roughly 0.5" in diameter, spinning at roughly 5000 rpm. The
resulting
film dries rapidly (typically less than a few seconds) and is usually less
than 1 ~m thick.
These discs may be stored and used at a later time. Thickness of the film is
controlled
by the concentration of the acyano-4-hydroxycinnamic acid and cellulose
acetate in
solution; the rate of spinning; and the rate of solvent evaporation. Spin
casting is only
one method of making the thin film; others such as electrospray deposition
(See WO
91/02961 ) and chemical vapor deposition are within the knowledge of one of
ordinary
skill in the art.
2o A sample molecule such as Bovine Insulin was applied by dispensing a small
volume, 0.5 ~L is typical, of an Insulin solution onto the substrate and then
allowing it
to dry in air. A sphotomicrograph of such a film, after a sample of 0.5 ~L of
a solution
of 0.001 ~g/~L of Insulin in water and 0.1 % trifluoroacetic acid has been
deposited on
it and allowed to dry, is shown in Fig. 2c. This dried sample may then be
introduced
into a laser desorption time-of flight mass spectrometer.
Mass spectra obtained from such a sample are shown in Example 1 (Fig. la and
1b). The spectrum shown in Fig. la was acquired with two laser shots from a
Nitrogen
laser emitting at 337 nm (see Materials under Examples). Other lasers emitting
at
different frequencies are also possible, and well known to those of ordinary
skill. The
spectrum shown in Fig. 1b was acquired by scanning the laser beam across the
sample
and averaging together only those spectra which had an insulin peak above a
certain
threshold intensity.
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Spectra obtained from such samples show much better mass resolution and
reproducibility than spectra obtained from samples prepared by the most common
prior
art technique, which does not show a thin layer of polymer-matrix. Mass
resolution is
defined as the mass (parent ion mass divided) by the mass represented by the
width of
the peak at half of its height. This may be seen by comparing Figs la and 1b
to Figs. 3a
and 3b respectively. The former and the latter spectra were obtained in the
same
instrument and with the same initial instrument parameters. It should be noted
that 10
times as much insulin was deposited on the disc used to obtain the prior art
Figs. 3a,
3b and 3c as was deposited on the disc used to obtain Figs. la, Ib and lc,
highlighting
the fact that surprizingly increased sensitivity is also a hallmark of this
invention.
A further embodiment is shown by Figs. 4a, 4b and 4c. These figures were
obtained in a manner similar to Figs. la, 1b and Ic, except that no cellulose
acetate was
used. Thus a thin film consisting only of a-cyano-4-hydroxycinnamic acid was
deposited on the quartz substrate. While the mass spectral results obtained
were better
than those obtained by conventional means and displayed in Figs 3a and 3b,
these
results are not as good as those displayed in Figs. la and Ib. Further
evidence of the
increased mass resolution conferred by this invention is shown in Fig. 5. A
similar
linear time-of flight mass spectrometer was used on a sample of the
oligosaccharide
Maltoheptaose, except that in this case the flight tube length was
approximately 1.5 m
and the ion detector used was a dual chevron channel plate assembly such as
the #F
TO-2003 time-of flight detector sold by Galileo Electrooptics. The essential
characteristic of this detector is that it has a very fast response time and a
50 ~ output
impedance. In addition a higher speed digitizer, such as LeCroy #9360 sampling
at 5
GHz with an analog bandwidth of 300 MHz was used.
Sugars typically have a greater proclivity for sodium ions, as opposed to
peptides which have a greater tendency to ionize by the addition of a proton.
Consequently the peaks shown in Fig. 5 are what are known as (M+Na)+ ions.
This
spectrum was the average of those produced by 5 laser shots. There is some
uncertainty with regard to the filll interpretation of this spectrum, i. e.,
there are some
extra peaks that are not readily assigned to a known species. What is clear,
however, is
that this spectrum clearly demonstrates the ability of samples prepared by
this invention
to be analyzed with higher mass resolution than has hitherto been demonstrated
in a
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linear time-of flight machine of this length. The individual Maltoheptaose
species that
result from the distribution of the natural abundance of'~C are resolved. In
addition,
by inspecting the peak labeled with the mass 1177.9 and expanding it as is the
lower
trace, it is evident that the mass 8797 is calculated by measuring the full
width at half
height of the peak relative to the arrival time of the peak. This is roughly
an order of
magnitude greater than has hitherto been observed in any linear machine of
comparable
length for ions created by matrix-assisted laser desorption and ionization and
even
exceeds that obtainable in most reflectron-based instruments.
Other adaptations of this technology to the mass analysis of macromolecules
may develop according to these scenarios. Sellergren et al., J. Chromatogr.
347: l-10,
(1985); Glad etal., J. Chromatogr. 347:11-23, (1985) and Dabulis et al,
Biotechnology and Bioengineering, 39( 2): 176-185 ( 1992) have each proposed
different ways of "molecular imprinting." Molecular imprinting is a method of
synthetically generating binding sites with some degree of specificity for 12
other
molecules by creating a mold or pocket in another material such as a plastic,
so that
sample molecules may be held and hence concentrated. Hutchens et al, Rapid
Commun. Mass Spectrom. 7: 576-580, ( 1993) have demonstrated the use of single
stranded DNA as an affinity capture surface to concentrate species of interest
for laser
desorption. DNA and other large biological molecules such as antibodies,
however, are
often complex and fragile. The methodologies of molecular imprinting, however,
can
produce inexpensive rugged binding sites made from inert robust materials and
such
imprints may have value, particularly in diagnostic applications. Thus it may
be
advantageous to use such binding materials in conjunction with matrix assisted
laser
desorption and ionization. Specifically, small plastic particles can be
prepared by well
known techniques that contain molecular imprints of molecules of interest. It
may thus
be usefizl to incorporate such beads into thin films of matrix and possibly
other inert
materials in ways that do not destroy the molecular imprint such as by
dissolving the
matrix and any other materials in a solvent that does not dissolve the beads.
Thus it is likely that other means of generating a thin sample-matrix
3o combination may be effective. For example, a micro-reticulated surface such
as can
routinely be generated on glass or silicon (or by molding plastics or epoxies
from
masters) when coated with a thin film of matrix, may prove to be an effective
substrate,
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the micro-reticulation serving to prevent the aggregation of matrix and/or
sample when
the sample is deposited. Likewise laminated structures consisting of thin, and
if
necessary porous, layers of matrix and an inert substance may be effective.
Alternatively, it may be possible to form pre-prepared substrates by coating
or
polymerizing the inert material around small preformed matrix crystals or
particles. It
may also be appropriate and convenient to polymerize matrix monomers (or more
generally energy absorbing monomers) around predeposited sample molecules. It
will
also be evident that these thin films need not be continuous, e.g. thin
patches of such
films may work too.
Having now generally described this invention, the same will become better
understood by reference to certain specific examples which are included
lierein for
purposes of illustration only and are not intended to be limiting unless
otherwise
specified. All U.S. patents cited herein are fully incorporated by reference
in their
entirety.
Modes for Carr~rin~ Out the Invention
Materials
All spectra displayed in Figs la, !b, 3a, 3b, 4a and 4b were obtained by
introducing the respective samples into a linear time-of flight mass
spectrometer with a
flight tube length of approximately 55 cm and a fast response time response
discrete
dynode electron multiplier, such as Model # AF820H manufactured by ETP PTY
Ltd.,
with the last few dynodes buffered by extra capacitance. Pulses of laser
radiation at
337 nm from a nitrogen laser such as is Model #VSL337ND manufactured by L.S.I.
were used for the matrix-assisted desorption and ionization. The output of the
electron
multiplier was sampled at 100 Msamples/sec by a nominal 8 bit digitizer with
an analog
bandwidth of 300 MHz such as is offered by several manufacturers such as
LeCroy
Inc. Bovine Insulin was obtained from Sigma (St. Louis, MO). Sinapinic acid
and
a-cyano-4 hydroxycinnamic acid were obtained from Aldrich Chemical Co.
(Milwaukee, Wisconsin).
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Example l: Construction of a Thin Films of a-cyano-4-hydroxvcinnamic acid and
Cellulose Acetate
This example is a preferred embodiment and is discussed in reference to Figs.
la, 1b, lc, 2a and 5. A thin film of a-cyano-4-hydroxycinnamic acid and
cellulose
acetate was prepared by dropping roughly 10 ~.L of a solution of 30 mg/ml of
cellulose
acetate and 30 mg/ml of a-cyano-4hydroxycinnamic acid onto a glass disc
spinning at
roughly 5000 rpm. The glass disc was spun by mounting it onto a Dremel
(Racine, WI)
Moto-tool Model 395 type 3. The resulting film dried rapidly (typically less
than a few
seconds) and is typically less than 1 pm thick. These discs may be stored and
used at a
later time. Typically a sample such as insulin may be applied by dispensing a
small
volume, 0.5 pL is typical, of an insulin solution onto the substrate and tlien
allowing it
to dry in air. A photomicrograph of such a film, after a sample of 0.5uL of a
solution
of 0.001 pg/pL of insulin in water and 0.1 % trifluoroacetic acid has been
deposited on
it and allowed to dry, is shown in Fig. lc. This dried sample was then
introduced into a
laser desorption time-of flight mass spectrometer. Mass spectra obtained from
such a
sample are shown in Fig. la and 1b. The spectrum shown in Fig. la was acquired
with
two laser shots. The spectrum shown in Fig. 1b was acquired by scanning the
laser
beam across the sample and averaging together only those spectra which had an
insulin
peak above a certain threshold intensity. Spectra obtained from such samples
sliow
surprizingly better mass resolution and better reproducibility than spectra
obtained
from samples prepared by the most common prior art technique.
Example 2: Construction of Thin Films of a-cyano-4-hydroxycinnamic acid and
Cellulose Nitrate
These thin films were prepared according to the procedure of Example 1
except that the solution of cellulose nitrate and a-cyano-4-hydroxycinnamic
acid was
prepared in a mixture of 75% anhydrous ether and 25% ethanol by volume. A
spectrum from insulin deposited on such a thin film is shown in Fig. 6.
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Example 3: Construction of Thin Films using Polycarbonate and
a-cyano-4-hydroxycinnamic acid
These thin films are prepared according to the procedure of Example 1 except
that the solution of polycarbonate a-cyano-4-hydroxycinnamic acid was prepared
in
tetrahydrofuran.
Although the invention has been described by way of illustration and example
for purposes of clarity and understanding, it will be obvious that certain
changes and
modifications may be practiced within the scope of the invention, as limited
only by the
1o scope ofthe following appended claims.
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