U.S. patent application number 12/206450 was filed with the patent office on 2009-03-19 for multi-beam ion mobility time-of-flight mass spectrometer with bipolar ion extraction and zwitterion detection.
Invention is credited to Thomas F. Egan, Valeriy V. Raznikov, J. Albert Schultz, Michael V. Ugarov.
Application Number | 20090072133 12/206450 |
Document ID | / |
Family ID | 37482180 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090072133 |
Kind Code |
A1 |
Schultz; J. Albert ; et
al. |
March 19, 2009 |
MULTI-BEAM ION MOBILITY TIME-OF-FLIGHT MASS SPECTROMETER WITH
BIPOLAR ION EXTRACTION AND ZWITTERION DETECTION
Abstract
The present invention relates generally to instrumentation and
methodology for the characterization of chemical samples in
solutions or on a surface which is based on modified ionization
methods with or without adjustable pH and controllable H-D exchange
in solution, an improved ion mobility spectrometer (IMS), a
multi-beam ion pre-selection of the initial flow, and coordinated
mobility and mass ion separation and detection using a single or
several independent time-of-flight mass spectrometers for different
beams with methods for fragmenting ion mobility-separated ions and
multi-channel data recording
Inventors: |
Schultz; J. Albert;
(Houston, TX) ; Raznikov; Valeriy V.; (Moscow,
RU) ; Egan; Thomas F.; (Houston, TX) ; Ugarov;
Michael V.; (Houston, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY, SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
37482180 |
Appl. No.: |
12/206450 |
Filed: |
September 8, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11441768 |
May 26, 2006 |
7429729 |
|
|
12206450 |
|
|
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60685240 |
May 27, 2005 |
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Current U.S.
Class: |
250/282 ;
250/287 |
Current CPC
Class: |
H01J 49/40 20130101;
H01J 49/009 20130101; G01N 27/622 20130101 |
Class at
Publication: |
250/282 ;
250/287 |
International
Class: |
H01J 49/40 20060101
H01J049/40; B01D 59/44 20060101 B01D059/44 |
Claims
1-85. (canceled)
86. A method of analyzing a sample comprising the steps of:
creating a flow of gaseous ions or a mixture of gaseous ions and
gaseous neutral species from said sample wherein said sample
comprising isotopically-labeled chemical species and a
corresponding non-isotopically-labeled chemical species wherein
said isotopically-labeled chemical species and the corresponding
non-isotopically-labeled species have a nearly identical mobility
cross-section and wherein said method further comprises using
shifts of mass-to-charge ratio related to said isotopic labeling to
analyze said sample; directing said flow into a collection region;
injecting said flow from said collection region into an ion
mobility assembly; and, detecting said flow exiting said ion
mobility assembly.
87. The method of claim 86 wherein said sample is a biological
sample.
88. The method of claim 86 wherein said sample comprises a drug
bound to a least one biomolecule.
89. The method of claim 86, wherein said sample comprises a drug
bound to a metabolite.
90. The method of claim 86, wherein the ion mobility assembly
comprises a plurality of mobility tubes.
91. The method of claim 86, wherein the collection region is an
orthogonal collection region.
92. The method of claim 86, wherein said isotopically-labeled
chemical species and the corresponding non-isotopically-labeled
species form an isotopic pair.
93. The method of claim 86, wherein the step of detecting said flow
exiting said ion mobility assembly further comprises an algorithm
for identifying an isotopic pairs by horizontal shifts of
mass-to-charge ratio related to said isotopic pair.
94. The method of claim 86, wherein said sample comprises a
non-exchangeable isotopically-labeled chemical species and a
corresponding non-isotopically-labeled chemical species.
95. The method of claim 87, wherein said biological sample is a
cell culture.
96. The method of claim 87, wherein said biological sample
fluid.
97. The method of claim 87, wherein said biological sample is a
tissue sample.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. patent Ser. No.
11/441,768 filed on May 26, 2006 which claims priority to U.S.
provisional application Ser. No. 60/685,240, filed on May 27,
2005.
TECHNICAL FIELD
[0002] The present invention relates generally to instrumentation
and methodology for the characterization of chemical samples in
solutions or on a surface and is based on modified ionization
methods with or without adjustable pH and controllable
hydrogen-deuterium (H-D) exchange in solution, an improved ion
mobility spectrometer (IMS), a multi-beam ion pre-selection of the
initial flow, and coordinated mobility and mass ion separation and
detection using a single or several independent time-of-flight mass
spectrometers (TOFMS) for different beams with methods for
fragmenting ion mobility (IM) separated ions and multi-channel data
recording.
BACKGROUND OF THE INVENTION
[0003] Among a variety of ionization techniques applied to mass
spectrometry, electrospray ionization (ESI) has evolved into a
powerful and widely practiced tool for the analysis of high
molecular weight biological molecules. The success of ESI in the
analysis of biomolecules lies in the method's ability to extract
fragile chemical species intact from solution in an ionized form,
and transfer them to the gas phase for mass analysis. A unique
characteristic of the electrospray (ES) ion source is the ability
to form multiply-charged ions, which facilitates the analysis of
extremely high molecular weight molecules with mass analyzers
having relatively low nominal upper mass limits. Electrospray
ionization methods have been extensively reviewed. See, for
example, reviews by Banks, Jr. and Whitehouse in Methods in
Enzymology, Vol. 270, 1996, pp. 486-519; and Smith, R. D., et al.,
Analytical Chemistry, Vol. 62, 1990, pp. 882-899. In an ES ion
source, a liquid sample is introduced through a small bore tube
that is maintained at several kilovolts at or near atmospheric
pressure into a chamber containing a bath gas. A strong
electrostatic field at the tip of the tube charges the surface of
the emerging liquid generating Coulomb forces sufficient to
overcome the liquid's surface tension and to disperse the liquid
into a fine spray of charged droplets. After passing through the
atmospheric-low pressure interface and desolvation region, ions are
injected into a mass spectrometer. For analysis of complex samples,
the multicharged ion formation characteristics of Electrospray
Ionization Mass Spectrometry (ESI-MS) complicate mass spectral
analysis, particularly for high mass biomolecules. Under the
current understanding in the art, it is unclear why multicharged
ion distributions observed in electrospray mass spectra are so
different from the charge distributions of the corresponding ions
in solution. For example, ESI mass spectra of positive ionized
peptides or proteins are usually collected under pH conditions such
that all or nearly all basic amino acid residues inside this
peptide are be protonated with a probability extremely close to 1.
Essentially, only ions with maximal possible charge are expected to
exist in solution but ESI mass spectrum exhibits a wide
distribution of multicharged ions. Since charge distributions of
ions in solution are well established and since these distributions
can be controlled by changes of the solution pH (properly
controlling other experimental conditions), it would be highly
valuable analytically to develop methods of extracting ions from
solution while conserving their equilibrium solution charge
distribution. The important property of biomolecules in solution is
the isoelectric point, which is determined by the solution pH when
the total charge of the biomolecule is zero. Using so called
isoelectric focusing, it is possible to achieve good separations of
biomolecules in gel electrophoresis techniques, where a difference
in isoelectric points of about 0.01 is sufficient. Additional
separation techniques for analysis of multicharged large ions would
also be useful. Ion mobility is a technique of great interest as
ion mobility resolving power increases proportionally to the square
root of ion charge, yielding not only improved peak separation in
the mobility cell but in addition, the mobility peak width may
provide information about the ion charge state.
[0004] An IMS is typically composed of an ionization source, a
drift cell, and an ion detector; examples of the latter include a
sampling plate, an electron multiplier, or a mass spectrometer. Ion
mobility spectrometry separates ions in terms of their mobility
with reference to a drift/buffer gas by measuring the equilibrium
velocity of the ions. When gaseous ions in the presence of a drift
gas experience a constant electric field, they accelerate until a
collision occurs with a neutral molecule. This acceleration and
collision sequence is repeated continuously. Over time, this
scenario averages out over the macroscopic dimensions of the drift
tube to a constant ion velocity based upon ion size, charge, and
drift gas pressure. The ratio of the velocity of a given ion to the
magnitude of the electric field experienced by it is the ion
mobility. In other words, the ion drift velocity (.nu..sub.d) is
proportional to the electric field strength (E) where the ion
mobility K=.nu..sub.d/E is a function of the ion volume/charge
ratio. Thus IMS is a technique similar to mass spectrometry, having
a separation component to it. The IMS technique is generally
characterized as having high sensitivity with moderate separation
power. Separation efficiency is compromised when "bands" of the
various ions spread apart as opposed to remaining together in a
tight, well-defined beam. This efficiency or resolving power for
what is considered "classic" ion mobility (using uniform or
quasi-uniform electric field to effect a separation due to the
Einstein relationship between mobility coefficient and diffusion
coefficient for ions for given ion charge) increases as the square
root of applied voltage along mobility cell. This maximum voltage
for a given length of mobility cell is restricted by the
possibility of glow discharge and decomposition of ions due to
heating from rapid velocities in the buffer gas. Increasing the
buffer gas pressure does allow application of higher cell voltages
and improved mobility resolving power.
[0005] Another possible analytical technique, using a new
continuous flow technique for separation of gas-phase ions at
atmospheric pressure, and referred to as high-field asymmetric
waveform ion mobility spectrometry (FAIMS), has recently been
described. (see R. W. Purves, R. Guevremont, S. Day, C. W. Pipich,
M. S. Matyjaszczyk, Rev. Sci. Instrum. 69, 1094-4105 (1998); R.
Guevremont, R. W. Purves, Rev. Sci. Instrum. 70, 1370-1383 (1999)).
This technique is simply a further development of the cylindrical
geometry case of the method implemented for the plane geometry and
described earlier. (see I. A. Buryakov, E. V. Krylov, E. G.
Nazarov, U. K. Rasulev Int. J. Mass Spectrom. Ion Processes 128,
143-148 (1993)). Adequate separation capability of this method for
isomeric compounds was demonstrated. see D. A. Barnett, B. Ells, R.
Guevremont, R. W. Purves "Separation of leucine and isoleucine by
elecrtospray ionization-high field asymmetric waveform ion mobility
spectrometry-mass spectrometry"; J. Am. Soc. Mass Spectrom. 10,
1279-1284 (1999)). This approach is more suitable for coupling with
continuous ionization methods such as electrospray. Its main
difference from classic ion mobility spectrometry is focusing and
recording of only one type of the ions from continuous ion flow for
each time moment. All other ions are usually lost. The situation is
the same as for all instruments of scanning type which may be
adequate when the amount of the sample is not so important or when
determination of only one or few known components is necessary.
However, use of multi-beam ion pre-selection as proposed in the
present invention partially overcomes this drawback and finds
general use. Herein we describe the specific embodiment of the
modified FAIMS for analysis of aerosol particles.
[0006] The combination of an ion mobility spectrometer (IMS) with a
mass spectrometer (MS) is well known in the art. In 1961, Barnes et
al. were among the first to combine these two separation methods.
Such instruments allow for separation and analysis of ions
according to both their mobility and their mass, which is often
referred to as two dimensional separation or two dimensional
analysis. Young et al. realized that an orthogonal time-of-flight
mass spectrometer (oTOFMS) is the preferred mass spectrometer type
to be used in such a combination because of its ability to detect
simultaneously and very rapidly (e.g., with a high scan rate) all
masses emerging from the mobility spectrometer. Their combination
of a mobility spectrometer with an oTOFMS is herein referred to as
an Ion Mobility-oTOFMS or IM-oTOFMS. This instrument comprised
means for ion generation, a mobility drift cell, and an oTOFMS with
a small orifice for ion transmission coupling the mobility cell to
the oTOFMS.
[0007] Use of MS as a detector allows for resolution based on
mass-to-charge ratio after separation based upon ion mobility.
Shoff and Harden pioneered the use of Mobility-MS in a mode similar
to tandem mass spectrometry (MS/MS). In this mode, the mobility
spectrometer is used to isolate a parent ion and the mass
spectrometer is used for the analysis of fragment ions (also called
daughter ions) which are produced by fragmentation of the parent
ions. Herein, this specific technique of operating a Mobility-MS is
referred to as Mobility/MS, or as Mobility/TOF if the mass
spectrometer is a TOFMS-type instrument. Other instruments and
methods using sequential IMS/MS analysis have been described (see,
e.g., McKight, et al. Phys. Rev., 1967, 164, 62; Young, et al., J.
Chem. Phys., 1970, 53, 4295; U.S. Pat. Nos. 5,905,258 and 6,323,482
of Clemmer et al.; PCT WO 00/08456 of Guevremont) but none combine
the instrumental improvements disclosed presently. When coupled
with the soft ionization techniques and the sensitivity
improvements realizable through use of the drift cell systems
herein disclosed, the IMS/MS systems and the corresponding
analytical methods of the present invention offer analytical
advantages over the prior art, particularly for the analysis of
macromolecular species, such as biomolecules.
[0008] The challenging issue when constructing an IMS-MS device is
to achieve a high ion transmission from the mobility region into
the MS region of the tandem instrument. It is at this interface
that the earlier approaches of ion mobility technology using a
linear field appear incongruous with the goal of maximizing ion
throughput across the IMS/MS interface. The mobility section is
operating at a pressure of typically between 1 mTorr and 1000 Torr
whereas the MS is typically operating at pressures bellow 10.sup.-4
Torr. In order to maintain this differential pressure it is
necessary to restrict the cross section of the opening that permits
the ions to transfer from the mobility section to the MS section.
Typically this opening cross section is well below 1 mm. Hence it
is desirable to focus the ions into a narrow spatial distribution
before this interface transmission occurs. Another important
property of the ion beam arriving into the MS is the divergence of
this beam in the kinetic energy for ion motion in the plane
orthogonal to the direction of their insertion into the MS. Ion
beam energy divergence is the main factor responsible for the
resolution properties of the mass spectra for orthogonal TOFMS. In
2004, Loboda U.S. Pat. No. 6,744,043 described several versions of
using of radio frequency (RF) ion guide for focusing of ions inside
the mobility cell. However, this approach is suitable for low
pressure ion mobility separation not more than a few Torr.
Furthermore, RF focusing of ions decreases with increasing of m/z
of ions so this method has some important restrictions. As
discussed herein, RF focusing of ions in interface region just
after the exit orifice of the mobility cell and before the entrance
orifice of TOFMS is free from these drawbacks.
[0009] H. H. Hill, in the late 1980's, developed methods for
introducing large biomolecules from aqueous samples directly into
IMS using electrospray ionization techniques. (see Hill, H. H.; and
Eatherton, R. L., "Ion Mobility Spectrometry after
Chromatography-Accomplishments Goals, Challenges", J. Research of
the National Bureau of Standards, Accuracy in Trace Analysis,
93(3), 1988, 425; see Shumate, C. B.; and Hill, H. H., "Coronaspray
Nebulization and Ionization of Liquid Samples for Ion Mobility
Spectrometry", Analytical Chemistry, 61, 1989, 601. Recently, Hill
and co-workers have interfaced a high resolution atmospheric
pressure ion mobility spectrometer to a time-of-flight mass
spectrometer and obtained rapid 2-D separations of amphetamines
(Steiner, W. E.; Clowers, B. H.; Fuhrer, K.; Gonin, M.; Matz, L.
M.; Siems, W. F.; Schultz, A. J.; and Hill, H. H., "Electrospray
Ionization with Ambient Pressure Ion mobility Separation and Mass
Analysis by Orthogonal Time-of-Flight Mass Spectrometry", Rapid
Commun. Mass Spectrom., 15, 2001, 2221-2226), PTH-amino acids
(Steiner, W. E.; Clowers, B. H.; Hill, H. H., "Rapid Separation of
Phenylthiohydantoin Amino Acids: Ambient Pressure Ion Mobility Mass
Spectrometry (IMMS)", Anal. and Bioanal. Chem., accepted October
2002), and chemical warfare degradation products (Steiner, W. E.;
Clowers, B. H.; Matz, L. M.; Siems, W. F.; Hill, H. H., "Rapid
Screening of Aqueous Chemical Warfare Agent Degradation Products:
Ambient Pressure Ion Mobility Mass Spectrometry (IMMS)", Anal.
Chem., 2002, 74, 4343-4352). At the interface between the IMS and
the TOF, collision-induced dissociation of mobility separated ions
can be turned on and off by varying the interface voltage to
provide an added dimension of analysis. This and other known
approaches for coupling of electrospray ion source with IMS/MS all
suffer from large losses of ions in all stages of their transport
and some decreases in mobility resolving power due to significant
width of initial ion package formed by interruption (pulse-forming)
of the continuous ion flow from the electrospray ion source. The
typical sensitivity of these measurements is in the range of .mu.M,
which is far worse than that for typical non-IMS electrospray and
matrix-assisted laser desorption ionization (MALDI) measurements.
MALDI sensitivities in the femto-molar range are typical (a
difference of up to nine orders of magnitude). As the continuous
electrospray ion source direct is chopped (or pulsed) for
introduction of the ion package into mobility cell only
approximately 1% of the initial ion source production is utilized
in the mobility cell. The relative time width of this ion package
to the time between such introductions should be less than the
inverse of expected mobility resolving power. Thus, increasing
mobility resolving power would lead in this case to additional
losses of ions and a further decrease in sensitivity. This
pulse-forming condition is related to that with coupling of
continuous ion source with TOFMS before the invention of orthogonal
injection of ions into TOFMS. Herein, a method of ion injection
into mobility cell is demonstrated which is free from the
beam-chopping limitations of usual coaxial introduction of
ions.
[0010] In 2004, Eriksson U.S. Pat. No. 6,683,302 described an
electrospray ion source where heating of droplets emerging from the
electrospray capillary under the influence of a strong electric
field was provided by microwave energy directed between the spray
tip and mass analyzer.
[0011] In 2003, Ranasinghe, et al. U.S. Patent Application
2003/0001090 described splitting the liquid flow from a separation
device into two approximately equal streams and directing them into
two ion spray sources; the first one producing positive ions and
the second one producing negative ions. Two TOFMSs were used for
recording of these positive and negative ions. In 2004, Van Berkel
U.S. Pat. No. 6,677,593 described partial separation of ions in a
liquid phase by applying electric or magnetic fields or their
combination. Enriched positive ion flow is directed into one
capillary whereas the flow with negative ions is sent through
another capillary. Due to the large electric field near the tips of
the capillaries during operation of the electrospray ion source
from solution phases, charge distribution of ions are "spoiled" in
the ion formation and extraction process.
[0012] In 2004, Berggren, et al. U.S. Pat. No. 6,797,945 described
some versions of using piezoelectric formation of charged droplets
for electrospray ion source. This approach may be promising for
several reasons. ESI coupled with pulsed techniques of ion analysis
in classic ion mobility spectrometers is simplified because it is
possible to form droplets in controllable short time intervals. It
is also appears to be important that droplets may be produced
having well known and narrow size distributions. Berggren teaches
that it is possible to get ions with less spread in their charges
by applying less voltage to the tip of the capillary from where the
droplets emerge. However, application of any voltage (to the
piezoelectric element located inside investigated solution) may
change, to some extent, the conditions for ion formation.
Therefore, the charge distribution inside large ions of interest
may still be changed from that in the solution at given pH and
without additional influences.
[0013] An idea to mix microwave voltage for heating with
quasi-periodic signal with frequency band 10-10000 kHz for
splitting of combustion kernels in internal combustion engine was
suggested in 1999 by Gordon, et al. U.S. Pat. No. 5,983,871.
[0014] In 2004, Apffel, et al. U.S. Pat. No. 6,797,946 described
the nebulizing of solutions and ionization of the neutral species
contained in the solutions by atmospheric pressure ionization (API)
and atmospheric pressure chemical ionization (APCI) as well as
suggesting orthogonal injection of resulting ions into the vacuum
part of mass spectrometer. The described version of orthogonal
injection of ions may be considered as a further development of the
widely used approach for removing of large and low charged droplets
from electrospray flow by a gas curtain. Some advantages of this
approach may be expected: lower "curtain" gas flow as it is
injected in the same direction as electrospray flow, and perhaps,
some better sensitivity of measurement and less evaporated solvent
flow inside mass spectrometer. However, Apffell nowhere suggests
using gas counterflow, ion accumulation in traps, and pulse
inserting of ions for analysis which are aspects of the present
invention discussed herein.
[0015] In 2005, Takats, et al. U.S. Patent Application 2005/0029442
described ion spray from solution using increased speed (more than
sound) of nebulizing gas flow assisted with voltage applied to the
sample capillary. The experimental data were presented showing very
narrow distribution of multicharged ions, sometimes showing
reduction to one type of ion. Changes of average ion charge and
peak width with applied voltage and the distance from the sample
capillary tip to the input heated capillary for inserting ions into
mass analyzer for different sample flows were measured. It was
shown that ions with relatively low number of charges and low
intensity may be detected for zero voltage applied to the sample
capillary. The data given for nanoelectrospray for different spray
voltages indicate more average charges for the same voltages after
some onset voltage below which no ions are detected.
[0016] One issued U.S. patent and two pending U.S. patent
applications of Schultz et al. (pending U.S. application Ser. No.
10/861,970, filed Jun. 4, 2004; pending U.S. application Ser. No.
11/231,448, filed Sep. 21, 2005; and U.S. Pat. No. 6,989,528)
describe a system whereby massive cluster ions or massive cluster
ions neubulized in a solvent may be impinged upon a surface both to
liberate and ionize surface bound molecules or elements (SIMS) as
well as simultaneously providing for nondestructive implantation of
a portion of this droplet into the near surface region of a
biopolymer which can thereafter be irradiated with a energetic
particle source such as a laser (MALDI) for liberation of the
molecules within the surface region. These U.S. patent applications
are incorporated by reference as though fully described herein). A
recently published variant of this approach was called Desorption
Electrospray Ionization (DESI) (see Z. Takats, J. M. Wiseman, B.
Gologan, R. Graham Cooks; Science Vol. 306, 15 Oct. 2004, pp
471-473). These techniques appears to be a useful tool for the
investigation of a variety of surfaces of natural origin including
in vivo analyses. The essence of these approaches involves
directing the flow of solvent droplets acquired by
nebulizer-assisted electrospray to the surface under investigation
which is held under usual ambient conditions and insertion of the
resulting flow from the surface into a mass spectrometer through an
atmospheric pressure interface. Interesting experimental results
were demonstrated including the mass spectrum from the finger of a
person 50 min after taking 10 mg of the over-the counter
antihistamine Loratadine (m/z 383/385). The corresponding peaks are
clearly seen in the spectrum. It is stated in the paper that
"changes in the solution that is sprayed can be used to selectively
ionize particular compounds." However use of high voltage applied
to the solvent in the spraying capillary would change the
conditions for formation of ions from the sample compared to those
for initial solvent. Thus, for example, the control of pH in the
solvent for producing of ions with corresponding charge
distribution is impossible in this case as is the case for a
typical electrospray ion source. A method free from this drawback
is an aspect of the present invention.
[0017] Attempts to perform fast three dimensional separation of
ions are also known. In 2001, Clemmer, et al. U.S. Pat. No.
6,323,482 described an approach whereby a quadrupole mass filter is
located between mobility cell and time-of-flight instrument and is
used for separation of non-resolved mobility peaks for providing
collision-induced dissociation for selected ions. In 2003, also
Clemmer U.S. Pat. No. 6,559,441 suggested the performance of two
consecutive separations of ions before mass analysis due to two
different molecular characteristics.
[0018] In 2004, Woods and Virgil, in U.S. Pat. No. 6,797,482,
described the approach for high-resolution identification of
solvent-accessible amide hydrogens in protein binding sites.
Exchange in solution of "open" hydrogen atoms for heavy hydrogen
atoms--tritium and deuterium--is used. Therefore, hydrogen atoms
buried inside folded proteins are not exchanged. To reveal the
corresponding amino acid residues with substituted and
non-substituted H-atoms, proteolysis by special enzymes working
under low temperature (close to 0.degree. C.) and in strong acidic
conditions (for pH about 2, 7) is used. Such low pH values and low
temperatures significantly suppress isotopic exchange of H-atoms so
it is possible to conserve information about initial structure of
the protein in solution. Further HPLC separation is performed in
such severe conditions for the same reason. The number of
substituted H-atoms in different fractions is estimated by
scintillator counting for the case of tritium exchange and mass
spectrometry measurements for the case of deuterium exchange. The
'482 patent gives a detailed overview of this field. It teaches
that using mass spectrometry for solving these problems is
restricted to overall determination of the number of substituted
H-atoms for corresponding ions without further attempts to locate
the sites having these atoms. Using the approach described therein,
it is difficult to find locations of substituted H-atoms very
precisely.
[0019] All of the above-referenced U.S. patents and published U.S.
patent applications are incorporated by reference as though fully
described herein.
[0020] Although much of the prior art resulted in improvements in
ion production, focusing, separation, and in ion throughput from
ion source to the mobility cell and to the mass spectrometer in
tandem instruments, there is room for additional improvement in all
these directions. The inventors describe herein a concept and
designs of a new type electrospray ion source, multi-beam ion
mobility and mass separations with multi-channel data recording
which result in instrumental embodiments to provide improved ion
production from investigated samples, their separation and
measurements.
BRIEF SUMMARY OF THE INVENTION
[0021] The present invention is directed instrumentation and
methodology for the characterization of chemical samples in
solutions or on a surface which is based on modified ionization
methods with or without adjustable pH and controllable H-D exchange
in solution, an improved ion mobility spectrometer (IMS), a
multi-beam ion pre-selection of the initial flow, and coordinated
mobility and mass ion separation and detection using a single or
several independent time-of-flight mass spectrometers for different
beams with methods for fragmenting ion mobility-separated ions and
multi-channel data recording.
[0022] In one aspect of the present invention, there is an
apparatus for analyzing a sample, the apparatus comprising a source
for the generation of a flow of gaseous ions or a mixture of
gaseous ions and gaseous neutral species from the sample, the
source producing the flow in a first direction; an orthogonal
collection region fluidly coupled to the source; and, at least one
ion mobility assembly fluidly coupled to the source, the ion
mobility assembly comprising a plurality of mobility tubes, wherein
the ion mobility assembly has a separation axis which is orthogonal
to the first direction.
[0023] In some embodiments, the ion mobility assembly further
comprises a plurality of CID tubes and a plurality of exit tubes,
the CID tubes being fluidly coupled to the mobility tubes and the
exit tubes being fluidly coupled to the CID tubes. In some cases,
the ion mobility assembly further comprises at least one
multichannel RF interface fluidly coupled to at least one of the
CID tubes. In some embodiments, the at least one multichannel RF
interface comprises pairs of rods and confining plates. The ion
mobility assembly may further comprise at least one multichannel RF
interface fluidly coupled to at least one of the mobility tubes. In
some embodiments, the at least one multichannel RF interface
comprises pairs of rods and confining plates. In some embodiments,
the apparatus further comprises at least one TOFMS fluidly coupled
to the ion mobility assembly. In some embodiments, the TOFMS
comprises a position sensitive detector. The at least one TOFMS may
be an oTOFMS. The at least one TOFMS may be a LoTOFMS. In some
cases. the TOFMS may comprise a detector comprising a plurality of
anodes in which two or more anodes of the plurality are each linked
to single detector channels. In such cases, the single detector
channel is a TDC channel. In some embodiments of the apparatus, the
orthogonal collection region comprises one or more voltage grids.
In some embodiments, the apparatus further comprises an ion
trapping region fluidly coupled to the orthogonal collection region
and to the ion mobility assembly, the ion trapping region
comprising at least one ion trap. The ion traps may be DC field
traps. The ion traps may be RF voltage traps. In some embodiments
having an ion trapping region, the ion trapping region comprises a
variable size exit orifice. In some embodiments, the apparatus
further comprises a laser positioned to excite the gaseous ions or
mixture of gaseous ions and gaseous neutral species in the ion
trapping region, in the orthogonal collection region, or in both
the ion trapping region and in the orthogonal collection region. In
some embodiments, the apparatus further comprises means for a
variable gas flow in the source, or in a region between the source
and the ion mobility assembly, or in both. In some embodiments, the
apparatus further comprises one or more mirrors in the region
between the source and the ion mobility assembly In some
embodiments, the apparatus further comprises a laser positioned to
excite the gaseous ions or mixture of gaseous ions and gaseous
neutral species in the orthogonal collection region. In some
embodiments, the orthogonal collection region comprises at least
one voltage grid for each mobility tube In some embodiments, the
source is selected from the group consisting of a laser desorption
source, a cluster bombardment source, a secondary ion source, a
desorption electrospray ionization source an electrospray
ionization source, photoionization source, and any combination
thereof. Preferably where a laser desorption source is used, it is
a matrix assisted laser desorption ionization source. In some
cases, the source comprises a droplet generator and is selected
from the group consisting of electrospray source, a pneumo-spray
source, an atmospheric pressure ionization source, a laserspray
source, a vibrating orifice aerosol generator, and any combination
thereof. In some embodiments, the apparatus further comprises means
for a variable gas flow in one or more components of the ion
mobility assembly. In some embodiments, the apparatus further
comprises at least one funnel, the at least one funnel comprising
electrode structures providing variable high and low electric
fields, the at least one funnel positioned immediately before the
at least one mobility tube. In some embodiments wherein the
apparatus further comprises at least one funnel comprising
electrode structures providing variable high and low electric
fields, the variable high and low electric fields comprise
spatially alternating high and low electric fields. In some
embodiments wherein the apparatus further comprises at least one
funnel, the apparatus further comprising means for a variable gas
flow in the at least one funnel. In some embodiments the apparatus
further comprises at least one funnel, the at least one funnel
comprising electrode structures providing variable high and low
electric fields; at least one capillary electrode assembly; or,
both the at least one funnel and the at least one capillary
electrode assembly, wherein the at least one funnel and the at
least on capillary electrode assembly are positioned at the exit
of, or immediately after the at least one mobility tube. In some
embodiments of the apparatus, the plurality of mobility tubes
comprise electrode configurations producing periodic electric
fields, hyperbolic electric fields or a combination of periodic and
hyperbolic electric fields. In some embodiments of the apparatus,
one or more of the plurality of mobility tubes comprises an
entrance cone electrode. In some embodiments of the apparatus, the
at least one ion mobility assembly comprises a plurality of ion
mobility assemblies and wherein the plurality comprises at least
one pair of ion mobility assemblies and wherein one ion mobility
assembly of the pair is opposed to the other ion mobility assembly
of the pair. In some embodiments of the apparatus, the source
further comprises means to deliver a pH adjustor composition to the
sample. In some embodiments of the apparatus, the apparatus further
comprises a pH measuring device positioned in the source. In some
embodiments of the apparatus, the source further comprises means to
deliver a deuterated composition to the sample. In some
embodiments, the apparatus further comprises a microwave voltage
source coupled to the source. In some embodiments, the apparatus
further comprises a sound frequency voltage source coupled to the
source. In some embodiments of the apparatus, the source comprises
an aerosol sampler, the aerosol sampler comprising a capillary and
a chamber containing a radioactive element, the chamber operable to
hold opposite charges near opposing walls of the chamber.
[0024] In another aspect of the present invention, there is a
method of analyzing a sample comprising the steps of creating a
flow of gaseous ions or a mixture of gaseous ions and gaseous
neutral species from the sample; directing the flow into an
orthogonal collection region; orthogonally injecting the flow from
the orthogonal collection region into at least one ion mobility
assembly, the at least one ion mobility assembly comprising a
plurality of mobility tubes; and, detecting the flow exiting the
ion mobility assembly.
[0025] In some embodiments of the method, the ion mobility assembly
further comprises a plurality of CID tubes and a plurality of exit
tubes. In some embodiments of the method, the ion mobility assembly
further comprises at least one multi-channel RF interface. In some
embodiments of the method, the ion mobility assembly further
comprises at least one multi-channel RF interface. In some
embodiments of the method, the step of detecting comprises
detecting with at least one TOFMS, the TOFMS comprising a position
sensitive detector. In some cases, the TOFMS is an oTOFMS. In some
cases, the TOFMS is a LoTOFMS In some embodiments of the method,
the step of detecting comprises detecting with at least one TOFMS
comprises detecting with at least one TOFMS comprising a detector
comprising a plurality of anodes in which two or more anodes of the
plurality are each linked to single detector channels. In some
cases wherein the TOFMS comprises a detector comprising a plurality
of anodes in which two or more anodes of the plurality are each
linked to single detector channels, the single detector channel is
a TDC channel. In some cases, the step of directing the flow into
an orthogonal collection region comprises directing the flow near
or through one or more voltage grids. In some embodiments of the
method, the method further comprises the step of directing the flow
of gaseous ions or mixture of gaseous ions and gaseous neutral
species through an ion trapping region comprising at least one ion
trap, the ion trapping region being located between the orthogonal
collection region and the ion mobility assembly. The ion traps may
be DC field traps, RF voltage traps or a combination thereof. In
some embodiments involving an ion trapping region, the step of
directing the flow into the ion trapping region comprises directing
the flow through a variable size exit orifice. In some embodiments
involving an ion trapping region, the method further comprises the
step of irradiating the flow of gaseous ions or mixture of gaseous
ions and gaseous neutral species with a laser, the step of
irradiating being preformed in the ion trapping region, in the
orthogonal collection region, or in both the ion trapping region
and the orthogonal collection region. In some embodiments, the
method, further comprises the step of applying a variable gas flow
to the flow of gaseous ions or mixture of gaseous ions and gaseous
neutral species during the steps of creating, orthogonally
injecting, or during both the steps of creating and orthogonally
injecting. In some cases, the method further comprises the step of
directing the flow of gaseous ions or mixture of gaseous ions and
gaseous neutral species through one or more mirrors during the
steps of creating, orthogonally injecting, or during both the steps
of creating and orthogonally injecting. In some embodiments of the
method, the step of creating comprises creating with a source
selected from the group consisting of a laser desorption source, a
cluster bombardment source, a secondary ion source, a desorption
electrospray ionization source an electrospray ionization source,
photoionization source, and any combination thereof. Preferably, in
cases using a laser desorption source, the laser desorption source
is a matrix assisted laser desorption ionization source. In some
cases, the step of creating comprises creating droplets with a
source selected from the group consisting of an electrospray
source, a pneumo-spray source, an atmospheric pressure ionization
source, a laserspray source, a vibrating orifice aerosol generator,
and any combination thereof. In some embodiments wherein droplets
are created, the method further comprises the step of splitting the
droplets into positively and negatively charged droplets by
quasi-resonant sound electric field or ultrasound frequency
electric field. In some embodiments wherein droplets are created,
the method further comprises the step of drying the droplets by
ambient gas heating and microwave absorption. In some embodiments
of the method, the method further comprises the step of applying
and varying a gas flow in one or more components of the ion
mobility assembly. In some embodiments of the method, the method
further comprises the step of directing the flow through at least
one funnel, the funnel positioned immediately before the at least
one mobility tube, the at least one funnel comprising electrode
structures providing variable and/or spatially alternating high and
low electric fields. In some embodiments of the method described in
the preceding sentence, the method, the method further comprises
varying a flow of gas in the at least one funnel; varying polarity
and/or magnitude of voltage across the funnels; or, varying both
the flow of gas and the polarity and/or magnitude of voltage. In
some embodiments of the method, the method further comprises the
step of irradiating the flow of gaseous ions or mixture of gaseous
ions and gaseous neutral species with laser radiation, the step of
irradiating being preformed before the step of directing the flow
into the orthogonal collection region. In some embodiments of the
method which comprises irradiation of the flow with laser
radiation, the method further comprises the step of varying a flow
of gas during the step of creating the flow of gaseous ions and
neutral species. In some embodiments of the method using a step of
laser irradiating, the step of irradiating comprises reflecting the
laser radiation from one or more mirrors In some embodiments of the
method, the method further comprises the step of applying periodic
electric fields, hyperbolic electric fields of a combination of
periodic and hyperbolic electric fields in one or more of the
plurality of mobility tubes. In some embodiments of the method, one
or more of the plurality of mobility tubes comprises an entrance
cone electrode. In some embodiments of the method, the step of
orthogonally injecting the flow into the at least one ion mobility
assembly comprises orthogonally injecting the flow into a plurality
of ion mobility assemblies and wherein the plurality comprises at
least one pair of ion mobility assemblies wherein one ion mobility
assembly of the pair is opposed to the other ion mobility assembly
of the pair In some embodiments of the method, the method further
comprises the step of delivering a pH adjustor composition to the
sample. In some embodiments of the method wherein a pH adjustor
composition is delivered, the step of delivering a pH adjustor
comprises mixing the sample with flows of acid or base buffers or a
combination of acid and base buffers. In some embodiments of the
method wherein a pH adjustor composition is delivered, the step of
delivering is regulated by a feedback signal. The feedback signal
may be generated by a pH measuring device. In some embodiments of
the method wherein a pH adjustor composition is delivered, the step
of detecting comprises detecting for samples at specific pH values.
In some embodiments of the method, the method further comprises the
step of delivering a deuterated composition to the sample. In some
embodiments of the method, the method further comprises the step of
applying a microwave voltage to the flow of gaseous ions or mixture
of gaseous ions and gaseous neutral species. In some embodiments of
the method, the method further comprises the step of applying a
sound frequency voltage to the flow of gaseous ions or mixture of
gaseous ions and gaseous neutral species. In some embodiments of
the method, the method further comprises the step of collecting
intensity data and correlating the intensity data from positive and
negative ions to identify positive ion/negative ion pairs, wherein
the intensity data is acquired from the step of detecting. In some
embodiments of the method, the method further comprises the step of
collecting intensity data and correlating intensity data with the
ion charge distribution of the sample, wherein the intensity data
is acquired from the step of detecting. In some embodiments of the
method, the step of creating further comprises generating an
aerosol. In some embodiments of the method involving creation of an
aerosol, the step of creating the flow of gaseous ions or mixture
of gaseous ions and gaseous neutral species from the sample
comprises creating the flow from an aerosol. In some embodiments of
the method, the sample comprises a biological sample comprising
non-exchangeable isotopically-labeled and non-isotopically-labeled
chemical species and the method further comprises using shifts in
mass-to-charge ratio related to the isotopic labeling to analyze
the biological sample. In some embodiments of the method described
in the preceding sentence, the chemical species is a drug. In
another embodiment of the method comprising the use of
non-exchangeable isotopically-labeled and non-isotopically-labeled
chemical species, the chemical species is a known mixture of
istotopically-labeled and unlabeled chemical species and the method
further comprises correlating the shifts in mass-to-charge ratio to
determine the mass of a chemical complex comprising the chemical
species and one or more other unknown chemical species; and, the
mass of the one or more other unknown chemical species.
[0026] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawing, in which:
[0028] FIG. 1A. Schematic of a measuring unit for multi-beam ion
mobility drift cell TOFMS with multi-channel data recording, common
for most embodiments of the invention.
[0029] FIG. 1B. Schematic view of ion and neutral trapping,
postionization and orthogonal IM injection region common for
different embodiments of the present invention.
[0030] FIG. 2. Schematic view of the proposed electrospray
interface.
[0031] FIG. 3. Schematic view of the proposed electrospray
interface with bombardment of the sample surface by solvent
droplets (for DESI version).
[0032] FIG. 4. Section A-A from FIG. 1B and FIG. 2. Neutrals are
trapped in the center, positive ions are trapped to the left, and
negative ions to the right.
[0033] FIG. 5. Simulation results for short mobility cell with
different focusing of ions at the exit of mobility cell.
[0034] FIG. 6. More detailed schematic view (including
cross-section orthogonal to IM ion beam) from the orthogonal
direction of the RF-guide IM/TOF interface.
[0035] FIG. 7. Schematic view showing recording of separate ion
beams in the TOFMS.
[0036] FIG. 8 Possible distribution of counts on the TDC channels
contributed by the fifth ion beam.
[0037] FIG. 9. Schematic cross-section of trapping region for
multi-beam profiling of a sample surface.
[0038] FIG. 10. Schematic view from the top of trapping region for
multi-beam profiling of a sample surface.
[0039] FIG. 11. Schematic view of specific part of interface for
investigation of aerosol particles.
[0040] FIG. 12. Illustration of separation of charged aerosol
particles before IM-TOFMS measurements.
[0041] FIG. 13. Illustration of separation of neutral aerosol
particles before IM-TOFMS measurements.
DETAILED DESCRIPTION OF THE INVENTION
[0042] As used herein, "a" or "an" means one or more, unless
otherwise expressly indicated or obvious from the context. This is
particularly true when reference is made to instrumental
apparatuses or individual components of the same.
[0043] As used herein, a "plurality" means two or more.
[0044] As used herein, "IM" is defined as ion mobility. As used
herein, "IMS" is defined as "ion mobility spectrometry" when used
in the context of a technique or "ion mobility spectrometer" when
used in the context of an instrument or apparatus.
[0045] As used herein, a "zwitterion" is a molecule with one or
more positively and one or more negatively charged structural
groups in which the total positive charge is equal to the total
negative charge. Thus the total charge of zwitterion is zero. The
"isoelectric point" is the pH value (pI) at which the average
electric charge is zero on the molecule.
[0046] A charged zwitterion may be a zwitterion with one or more
excess positive or negative charges. For example, at some pH
bradykinin can exist as a zwitterion which is charged during a
MALDI desorption as MH.sup.+.
[0047] As used herein, a "mobility cell assembly" is defined as a
single or multi-channel device which performs mobility separation
of ions and comprises at least one mobility tube, a collision
induced dissociation (CID) tube wherein collision-induced
ionization occurs, and optionally, final ion transport with cooling
gas flow through "exit tubes" into multi-channel RF-ion guide. In
the multi-channel embodiment, the mobility cell assembly comprises
a plurality of first mobility tubes, CID tubes, exit tubes and
RF-ion guides, preferably with each of the aforementioned component
in series with one another and each series in parallel with at
least one other series. Multi-bore and Multichannel are used
interchangeably.
[0048] As used herein, "mobility tube" is an ion mobility cell; the
terms ion mobility cell and mobility tube are synonymous herein.
The term "exit tube" is defined as the final mobility tube in a
series of mobility tubes.
[0049] As used herein, the term "funnel", when used in reference is
defined as a conical device comprising electrode pairs (of
descending open area along the direction from the ion source to the
ion detector) to which attractive or repulsive voltages may be
applied linearly or to individual electrodes. The funnel may
optionally contain an exit tube comprising a capillary exit end
formed by alternating electrode pairs.
[0050] As used herein "collision induced dissociation tube" or "CID
tube" is a mobility tube assembly which may also contain a funnel
electrode assembly and a capillary exit tube electrode assembly in
which high electric fields may be created sufficient either to
further focus ions onto the axis of the mobility tube or, at higher
voltages, to provide collision-induced dissociation of ions into
structural fragments.
[0051] As used herein, an "orthogonal collection region" is defined
by the volume between at least one electrode and/or voltage grid
pair through which ions (possibly of both signs) and neutrals which
are mixed with a carrier gas pass orthogonally in front of the
entrance of at least one IM tube. Neutrals which are formed within
the gas flow through this region are transformed into ions by an
ionization or fragmentation process (such as by a laser) within
this region. This region may also be referred to as an "orthogonal
IM injection region".
[0052] As used herein, an "orthogonal collection region" is defined
by the volume between at least one electrode and/or voltage grid
pair through which ions (possibly of both signs) and neutrals which
are mixed with a carrier gas pass orthogonally in front of the
entrance of at least one IM tube. Neutrals which are formed within
the gas flow through this region are transformed into ions by an
ionization or fragmentation process (such as by a laser) within
this region. This region may also be referred to as an "orthogonal
IM injection region".
[0053] As used herein, an "orthogonal collection region" is defined
by the volume between at least one electrode and/or voltage grid
pair through which ions (possibly of both signs) and neutrals which
are mixed with a carrier gas pass orthogonally in front of the
entrance of at least one IM tube. Neutrals which are formed within
the gas flow through this region are transformed into ions by an
ionization or fragmentation process (such as by a laser) within
this region. This region may also be referred to as an "orthogonal
IM injection region".
[0054] As used herein, the term "separation axis" as it relates to
an ion mobility assembly or any individual component of an ion
mobility assembly is the axis defining the direction of travel of
ions and/or neutral species traversing or being transported through
the ion mobility assembly or any individual component of the ion
mobility assembly.
[0055] As used herein. IM-oTOFMS refers to a combination of an ion
mobility spectrometer with an orthogonal time of flight mass
spectrometer. An IM-TOFMS more generally refers to a combination of
an ion mobility spectrometer with a time of flight mass
spectrometer.
[0056] As used herein the term "DESI" refers to desorption
electrospray ionization.
[0057] The present invention is mainly directed to a system and
methods consisting of an ion mobility drift cell transporting ions
in a gas at high pressures from any ion source (e.g., a MALDI
(matrix assisted laser desorption ionization) or other laser
desorption source, a cluster bombardment source, a secondary ion
source, a desorption electrospray ionization source an electrospray
ionization source, photoionization source, or any combination of
the foregoing) into a mass spectrometer. FIG. 1A shows a schematic
of an embodiment of a combined multichannel IM-TOFMS analyzer
assembly (400). The multichannel IM-TOFMS analyzer assembly (400)
comprises an ion mobility assembly and an orthogonal TOFMS. The
various components of the ion mobility assembly have entrance and
exit openings to allow beams of ions an/or beams of ions and
neutrals to enter and exit. The use of static nonlinear periodic
fields (see U.S. Pat. Nos. 6,639,213; 6,897,437; and 6,992,284 to
Schultz et al., incorporated by reference as though fully described
herein) to funnel ions from a large area (even at moderately high
pressures--including atmospheric pressure) into a small bore
multichannel ion mobility cell and still retain high mobility
resolution is the counterintuitive concept which is an aspect of
the present invention. The electrode configurations of mobility
cells capable of producing periodic fields, hyperbolic fields and
combinations of periodic and hyperbolic fields are now known in the
art through the aforementioned patent references. By use of an
electrostatic funneling of the ions at the beginning of the IM
cell, a large volume of ions is collected and compressed and passed
into a subsequent smaller bore section of the mobility cell. Such
an arrangement can still maintain an overall high mobility
resolution after transport through the entire mobility cell. This
is because the funnels (53) can be constructed of electrode
structures which provide a spatially alternating high and low field
which acts to focus and randomize the ion path lengths in the
funnel (and in subsequent smaller bore sections of the mobility
cell). This even works at pressures near atmospheric pressure. Thus
ions near the entrance edges of the funnels are mixed with ions
which enter near the center region of the funnels and the result is
that all the ions irrespective of where they enter the entrance
funnel experience the same randomized path length through the
funnels. Furthermore, by making the length of the funnel (53) small
compared to the length of the IM tube (55), the effect of unequal
path lengths can be further minimized. The exit end of the IM tube
may also contain a funnel and/or capillary electrode assembly to
further reduce the size of the ion beam, reduce gas flow into and
increase the efficiency of pumping out of the interface region
(70). By placing numerous such multi-bore IM-TOFMS assemblies (400)
opposite one another (see FIG. 1B), one may construct opposing
multi-bore arrays of IM cells whereby oppositely charged ions can
be extracted from a long column of ions mixed with a near
atmospheric pressure gas flow (40) which is orthogonal to the axes
of the mobility cell arrays. Pumping (49) provides the gas flow
inside the orthogonal collection region (41). It is thus possible
to collect ions from a large rectangular or cylindrical volume (41)
of ions or post-ionized atoms or molecules entrained in a gas flow
which is orthogonal to the axis of the multi-bore IM cell or of one
or more opposed multi-bore cell arrays. By intermittently applying
voltages on grids (61) and (62) (which may be independent pairs of
grids individually biased in front of each funnel (53)), it is
possible to create a field (63) which moves ions (22) orthogonal to
the direction of ion/gas flow motion to the entrances of the
funnels (53) restricted by collimating electrodes (44). The
entrained ions are thus forced to deviate orthogonally from the gas
stream and into the IM arrays, effecting orthogonal injection into
the IM arrays (see FIG. 1A). The injection is said to be orthogonal
because the path of travel in the IM arrays is orthogonal to the
path of travel in the preceding gas train. The manipulation and
further insertion of the ions (22) can be achieved by controlling
the polarity and/or magnitude of voltages across the funnels (53),
the IM tubes (55) and by the independent gas flows (46) and (47)
into the funnels (53) and the section (45) of IM tubes (55) using
variable gas pressures and control of flows through variable
pumping orifices (17). Thus the gas flow can be out of the funnel
into the orthogonal collection region (41) or the flow can be
reversed so that some gas comes into the funnel from the orthogonal
collection regions (41) as desired. The type of gas introduced (46)
can also be different in the funnel (53)(e.g., Xe) from the gases
in the source beam (40) (e.g. atmosphere or He) and the gases
introduced (47) (e.g., He) into the IM section (45). Thus IM
spectra acquisition from a nearly continuous source of ions is
possible (or from a continuous stream of neutrals which are
periodically ionized by, for example, a line focused pulsed laser).
After exiting the IM channels through relatively small apertures or
capillary tube electrodes (48), ions enter the interface region
(70) which is at a lower gas pressure than the IM channels. This is
achieved by differential pumping (51). DC voltages are applied to
rings of CID tubes (the exit tube portion of which may also be a
funnel and capillary tube electrodes) (48) to prevent ions from
diverging from the axis by the gas flow. The main function of the
CID tubes is to collect ions coming from corresponding IM channels
and transport them to the multi-channel RF ion guide (70). However,
high electric field inside CID tubes may optionally be applied to
provide collision induced dissociation of some chosen ions. The CID
and exit tube (48) is shown in FIG. 1A for illustration purposes as
a separate unit which is detached from the IM tube (55); however,
the entire continuous assembly may contain an IM tube, CID tube and
funnel exit tube which comprise one entire continuous assembly.
Furthermore, the exit tube may contain a capillary structure
comprising biased electrode pairs which also provides the formation
of a supersonic gas expansion of IM carrier gas containing analyte
ions into the RF interface region (70). To focus each ion beam
(54), a multi-channel RF-ion guide (70) is used. This ion guide
shown in detail in the top part of FIG. 6 consists of pairs of rods
(58) and confining plates (57) between each pair. RF-voltage of the
same phase is applied to rods. DC voltages of rods and confining
plates are the same. The voltage difference between the confining
plates and the TOFMS (50) is adjusted to give ions the energy they
need to enter the TOFMS and to be detected (determined by TOFMS
geometry). These plates allow ion confinement (59) between rods.
Ions (73) entering the orthogonal TOFMS (50) have some divergence
and different velocities. Due to RF-focusing and cooling they are
entering the TOFMS through small orifices fairly below 1 mm
diameter, thus a single pump (52) is sufficient for good operating
pressure. In the instant apparatus, the ion mobility assembly may
comprise at least one mobility tube only. Alternatively, it may
comprise at least one mobility tube and at least one CID tube and
at least one exit tube, and optionally, at least one multichannel
RF interface. Alternatively, it may comprise at least one mobility
tube and at least one multichannel RF interface. The TOFMS is
preferably an oTOFMS.
Simultaneous Orthogonal Insertion of Ions from the Gas Stream (40)
into Opposed Parallel Channel IM Mobility Arrays (FIG. 1b) and the
Addition of Trapping Regions (21, 22) Between the Orthogonal
Accumulation Region (41) and the Entrance of the Funnels (53)
[0058] Two (or four) multichannel ion mobility oTOFMS measuring
units (400) may be opposed as, for example, shown in FIG. 1B.
"Opposed" in this sense includes, for example, "vertically
opposed", "horizontally opposed", "diagonally opposed", etc.; all
that is required is that the opposing measuring units are
configured 180.degree. with respect to one another. FIG. 1B shows a
pair of ion mobility assemblies in which each assembly of the pair
is opposed to the other assembly of the pair. In addition to this
difference from FIG. 1A we also incorporate the capability to use
variable electric fields (16) of increasing strength from the left
to the right orthogonal to the direction of ion (or droplet) motion
within the trapping region provide. These variable fields can
provide some mobility size selection of ions as they are directly
injected into the entrance funnels or alternatively as they are
introduced into specific trapping regions in front of the funnels.
Since the ions of smaller cross-section can be easily deflected
from the from gas flow (40) this leaves only the ions of
successively larger cross-sections remaining in the gas flow and
these heavier ions will subsequently appear before the entrance of
successive funnels (from left to right in FIG. 1B). Here positive
ions are directed to the traps (22) (in the top of FIG. 1B) when
they are under an electric field (16) force, (which is higher than
the force from the gas counter flow (19) coming from the
multi-channel mobility cells). The corresponding negative ions will
be trapped in traps (21) shown in the bottom half of FIG. 1B. As a
result, the increments with which the electric field is increased
from the first trap (close to the entrance of the orthogonal IM
injection region) to the next should be chosen such as to provide
close to uniform ion density over the traps for a given type of
samples. Once trapped in front of the IM channels, ions are
introduced inside cell channels either by a pulsed increase of each
of the fields (16) or, this insertion process may be further
assisted, by additional pulsed electric fields applied across the
entrance cone of each mobility channel. The amplitude of the field
(16) varies for each trap and is adjusted to force ions of a
certain size range into an IM channel (increasing ion sizes from
the first trap to the next ones). (It should also be understood
that some modified form of the grids (61) and (62) shown in FIG. 1A
might be added to aid in localizing and injecting the ions into the
funnels). The time that ions spend in the orthogonal IM injection
region (including optional ion trapping) should be slightly longer
than the time they spend in the mobility cell. Thus the next
portion of ions will not be mixed with the previous one and very
few of the ions from the continuous source will be lost. The gas
pressure inside the interface between the ion source and the
mobility array trapping regions may be about 100 Torr. Then the gas
pressure inside mobility cells may be close to 150 Torr. Such
pressure is sufficient to obtain relatively high mobility
resolution (about 100 even for singly charged ions). Computer
simulations suggest that it is possible to effectively focus ions
at such pressure. This pressure in the mobility cell is suitable
for providing the TOFMS operation. The velocity of the gas flow
(18) along the axis of the trapping region should be such that the
distance traveled by the gas during the time that ions spend in the
orthogonal IM injection region is slightly longer than the length
of the orthogonal IM injection region (for estimations, we used
about 5 cm). It may be done by choosing an appropriate "size" for
the exit orifice (17) at the end of the trapping region. This may
be a physical orifice with variable size or it may be the orifice
interior to a flow controller or variable leak valve whose size can
be varied. After introducing trapped ions inside the IM channels
the electric fields (16) moving ions into the traps are switched to
zero. The fields are switched on in orthogonal direction. A laser
pulse (24) for decomposing neutral zwitterions located on the axis
of the trapping region (23) is applied. The apparatus in FIG. 1A
can also be used at higher pressures near or above atmosphere as no
RF trapping in front of each entrance (21, 22) is used. The counter
gas flow (19) from each mobility cell may be made extremely weak by
appropriate manipulation of the size of the exit orifices (17) and
the speed of pumping after the exit orifices (17). The orthogonal
region between the opposed multi-bore arrays is then filled with
ions and neutrals. After some filling time which is ideally similar
to the transit time of the ions through the multi-bore mobility
assemblies, the electric fields (16) are applied to extract ions
from the orthogonal stream into the nearest mobility cell array.
After the ions are removed from the region and have entered the
mobility cell assemblies, an energetic ionization source (24)
(which may be a laser) is applied to the center region of the
mobility cell to either ionize neutrals or to create ions from
preformed neutral zwitterions.
[0059] In case this arrangement of the laser beam (24) (along axis
of the gas flow) is not suitable (as it can, in some cases, produce
undesirable ions in the region of initial flow from the sample
(40)), it is possible to arrange the laser beam in the orthogonal
direction (29) shown in FIG. 4 (view from section A-A of FIG. 1B).
Using two mirrors (39) shown in the top of FIG. 4 allows multiple
passing by the laser beam the region of desired ion production. The
zwitterions and other neutral species (33) are focused along the
axis of the trapping region by counter gas flows (37) from the four
multi-channel mobility cells (400) located at positions (31), (35),
(36) and (38) as shown in FIG. 4. The electric fields at the
entrance of the mobility cell channels (30) for trapping and
inserting of positive (32), negative (34) initial ions and ions
from zwitterions are also shown. Under increasing electric field,
the positive and negative ions formed from zwitterions in (33)
travel to the top and the bottom mobility cells, respectively.
Other neutral molecules in (33) do not form ions if the photon
energy in laser pulse (somewhat more than 2 eV, far below the
ionization potential of most chemical substances) is only
sufficient to fragment zwitterions and separate complimentary
positive and negative ions.
[0060] The additional features of the invention are (i)
controllable variation of the solution pH to form zwitterions
and/or the controlled variations of the concentration of D.sub.2O
or some other deuterated substance for providing H-D exchange in
solution, (ii) extraction of both positive and negative ions,
followed by selective fragmentation of zwitterions at a given pH to
create simultaneously (and in co-incidence) oppositely charged
fragments from the neutral zwitterion, (iii) ion and neutral
pre-selection by flow characteristics of the molecular movement in
the gas flow prior to formation and injection of the ions into the
multi-bore or opposing multi-bore IM structures (iv) coordinated
mobility and mass ion separation and detection using a single or
several independent TOFMS (for different beams) with on demand and
controllable fragmentation (e.g., collision-induced dissociation
(CID) or photo-ionization/fragmentation, or photofragmentation) of
selected ions without losing other ions for analysis, and (v)
multi-channel data recording. These implementations aim at making a
more efficient use of sample and obtaining maximum useful possible
information about the sample in a reasonably short time.
Specifically, the improvements lie in providing a three-dimensional
separation of the solution constituents based on (i) charge balance
in the biomolecule at the isoelectric point pI (at the
corresponding pH=pI, the average charge of the molecule is 0), (ii)
ion mobility separation, and (iii) mass analysis. Additional
information about ions or even additional separation may be
supplied by controllable H-D exchange in solution since the shifts
in isoelectric points for differently deuterated biomolecules of
the same biopolymer may be different in the presence of deuterated
solvent molecules. Higher sensitivity and more effective sample use
are achieved by maximizing ion production and extraction
(preferably both negative and positive) from the sample. This
includes accumulation and decomposition of zwitterions, multiple
ion beam trapping, high transmission orthogonal injection into a
high gas pressure mobility cells, high transmission mobility
cell/TOFMS interface comprising original multi-channel RF-ion
guide. To reduce the acquisition time and the sample consumption, a
special procedure will be used to predict the isolectric point of a
given biopolymer from the detected distribution of multicharged
ions. Thus no multiple acquisitions at different pH values will be
necessary when this prediction is valid. Multi-channel data
recording not only allows for obtaining single-channel data for
each ion beam but also provides sufficiently large dynamic range
and better description of the mobility peak profiles. These
improvements may be used to increase the throughput from an ion
source to downstream instruments/methods and they also provide
additional information about the investigated samples complimentary
to the mere summing of the data from different ion beams. Namely,
processing intensity distributions of multi-charged ions as a
function of the solution pH provides structural information of the
biomolecule based on variations of pK.sub.a (or pK.sub.b) values
for the specific sites which are able to retain (or remove) protons
or other charges species. Computer analysis of intensity
distributions of deuterium-substituted ions provides additional
information of this kind. Recording complimentary positive and
negative ions formed during the decomposition of zwitterions would
provide unambiguous sequence information for corresponding
biomolecules which may be effectively expanded by collision or
photo-induced dissociation of chosen ions. The resulting
instruments and methods are useful for quantitative and/or
qualitative, structural chemical and biological analysis.
[0061] In one aspect of the present invention, one introduces,
under computer control, pH adjustors (such as, for example,
acid/base buffers) and deuterated solvents directly into a
capillary tube in which the sample solution (or solvent for DESI
and aerosol particles measurements embodiments) is moving. The
addition of pH adjustors may be regulated by a downstream feedback
signal, such as the signal from a downstream pH measuring device.
At the end of this capillary, essentially neutral droplets are
formed by the assistance of a nebulizer gas flow. Their splitting
(or that for droplets from the surface in case of DESI) into
smaller charged droplets and further evaporation of these split
droplets are provided by sound frequency resonant electric field
and by microwave heating. Additional flow of hot gas would be
introduced to prevent ion cluster formation after ions exit the
microwave heating and splitting region. Such an approach is quite
different from approaches whereby charged droplets are extracted by
a strong electric field. Field penetration inside the solution
(significantly increasing near the sharp edges of the capillary) is
likely the main reason why charge distributions of recorded ions in
a typical electrospray mass spectrum contain many highly charged
ions which are substantially different from the charge
distributions of the ions in the bulk solution. Extracting positive
and negative ions and forming charged droplets in softer conditions
coupled with their fast evaporation will likely result in ion
charge distributions similar to that of the ions initially in
solution. It is also possible to accumulate positive and negative
ions from the initial flow in gas dynamic electric ion traps. This
allows for the collection of ions almost continuously while a
previous portion of ions is moving through the ion mobility cell
and being recorded. It gives significantly higher sensitivity.
[0062] Orthogonal ion mobility injection also provides a narrower
initial ion package entering the individual mobility cell channel
(compared to single coaxial ion injection from an electric gating
mechanism or from a co-axial trap) and this assures a significantly
improved resolving power even as the continuously produced ions are
being mobility and mass analyzed and recorded. A small gas counter
flow coming from the mobility cell channels may optionally be used
to prevent neutral species and very large singly charged ions from
entering the mobility cell. Thus, wall contamination of ion optics
and cluster ion formation during their motion through mobility
cells will be significantly reduced. Also lower background signal
and chemical noise will result. The most advanced version of the
proposed system comprises four sets of mobility cells and four
multi-beam TOFMS instruments (for the aerosol particles
measurements embodiment this number may be even increased to 6).
One IM/MS pair analyzes positive and negative ions formed in the
initial ESI flow. The other pair (orthogonal to the first pair)
measures ions formed from the neutral species of the initial ESI
flow. In an IM/MS pair, the positive IM/MS and negative IM/MS goes
orthogonally from the initial axis in two opposite directions. The
four multichannel IM cell arrays generate four weak gas flows
orthogonal and pointing to the axis of initial sample flow. Ions
present in the initial ESI beam are going to traps under balancing
forces from electric fields and gas flows. The four gas flows
constrain the neutral species form the ESI beam close to the
initial ESI beam axis. Among the neutral species, zwitterions may
be of most interest as their formation will be governed by the
controlled pH value of the sample solution. Zwitterions are formed
from biomolecules whose isoelectric points close to the given pH
value. Ions can be formed in this case by internal bond breaking of
neutral zwitterions. Thus, a relatively low fluence laser beam
could produce such ions and avoids formation of ions from other
neutral species. Other types of chain breaking ionization
techniques could also be used such as low energy electron
attachment. After ion accumulation in traps, positive and negative
ions are introduced against the buffer gas flow into the two
multi-channel mobility cells. Once the largest desired ions reach
an ion mobility (IM) channel entrance the electric field moving
ions to these traps is switched to zero and the entrance fields
allowing ions to penetrate the other IM channels (whose axes are
orthogonal to the initial ESI beam and orthogonal to the plane of
the previous pair of IM cells) are switched on and the laser beam
for decomposing of zwitterions is pulsed. After introduction of
produced ions into corresponding mobility cells, a new ion
accumulation/trapping cycle starts. With suitable statistical
treatments the negative and positive fragments from the intact
neutral zwitterions may be detected in coincidence in each set of
opposing mobility cells so that additional structural information
is simultaneously achieved.
[0063] Another embodiment uses the pH-controlled electrospray to
deposit solutions providing a specific isoelectric point separation
of biomolecules on a surface from which the molecules may later be
desorbed by an energetic source such as a laser, or particle beam
before, during, and after the solution comes to dryness. This
surface may be one comprising known MALDI matrices including
nanoparticulates or it may be specially engineered to enhance
desorption of neutrals which may then be fragmented to create
oppositely charged ions if the desorbed neutral is zwitterionic.
Electron attachment of hydrogen-insertion or other negative or
positive ion attachment reactions are also possible ways to create
a gas phase ion containing only one negative, or one positive
charge overall.
[0064] In one possible application, elemental or alloy cluster ions
or elemental or alloy cluster ions within a nebulized droplet are
impinged upon a surface to generate ions from the molecules or
atoms present at the surface. These secondary ions and neutrals are
carried into the IM cell where they can be analyzed. In another
application, pure solvent droplet aerosols or other aerosolized
nanoparticulates are used to impinge the surface layer to desorb
analyte atoms or molecules. In application, pneumo-sprayed droplets
of solution (with or without acceleration of the droplets) are
directed to the surface sample and after "reflection" from the
surface enriched by the sample species are inserted into
desolvation region. In still another application, an on demand
droplet generator or a vibrating orifice generator may be used to
form aerosolized droplets, which may contain analyte or analyte and
nanoparticulate matrices, and these droplets are supplied at a rate
which will place a train of equally spaced droplets into the gas
stream so that each droplet can simultaneously be in front of two
(or four) opposing IM channels at which time all particles can be
simultaneously desorbed by energetic particle beams which may
include a laser. This was described in co-pending U.S. application
Ser. No. 11/025,640 filed Dec. 29, 2004 and published as U.S.
Published Patent Application 2005/0230615 A1 and incorporated by
reference as though fully described herein).
[0065] In an additional embodiment, a surface is located beneath
the opposed multi-bore IM cells and multiple spots of the surface
are alternately (or simultaneously) irradiated with multiple laser
beams (see co-pending U.S. application Ser. No. 11/056,852, filed
Feb. 11, 2005 of Russell et al, and published as U.S. Published
Patent Application 2005/0242277 A1), incorporated by reference as
though fully described herein) so that ions and post-ionized
neutrals which are desorbed from individual regions on the surface
are all registered in their own IM channel of the multi-bore IM
array. Such a surface might be a biological tissue, or a synthetic
surface, or a structured surface such as a microarray. Another
application of this configuration could be the direct analysis of
neutrals, ions, and zwitterions directly desorbed from an
electrophoretically separated and heavy metal stained 2D gel. In
yet another embodiment the surface or microarray may be located
outside the opposed multi-bore IM structure and a gas stream can be
used to entrain neutrals and ions for transporting through the
region orthogonal to the axes of the multi-bore IM arrays which is
between the IM multi-bore arrays.
[0066] The apparatus may also be applied to the analysis of
atmospheric aerosols. These atmospheric aerosols can include whole
cells either within solvent droplets or as isolated aerosolized
cells. Other nanoparticulates or micron-sized particulates either
within a droplet or as an isolated particulate can also be
analyzed. The analysis can be assisted if the solvent droplets
contain desirable matrices to assist in particle desorption from
the aerosols. The apparatus could be used for analysis of
isotopically-labeled drugs or other desired isotopically-labeled
analytes.
[0067] In applications where ion mobility cells filled with a
buffer gas are used as a volume/charge separation stage before
analysis in a mass spectrometer, the cooled ions exit through a
small aperture into a differentially pumped low pressure region
before high vacuum part of the mass spectrometer. To minimize
transmission ion losses at the exit orifice of the ion mobility
cell, the ion beam inside the mobility cell should be focused. In
the region between mobility cell and the high vacuum TOFMS, a
narrow beam allows for the use of a very small aperture to limit
the gas flow. The ion beam should also be cooled as much as
possible and have a low divergence for optimum TOFMS operation
conditions. If this divergence is small in both directions
orthogonal to the direction of the main motion of ions, it is
possible to introduce into the TOFMS, not one, but multiple ion
beams which should be separated from the ion source to the detector
to increase the instrument throughput proportionally to the number
of ion beams. Such approach is feasible because: (i) multi-channel
data recording (multi-channel time-to-digital (TDC)) devices are
widely produced and used and (ii) it is possible to transport ions
after mobility cell inside multi-channel RF-ion guide without
noticeable losses and to focus ions into small entrance apertures
in front of TOFMS thus having an applicable pressure inside it. The
concept of multi-beam ion separation and measuring naturally
incorporates the idea of orthogonal injection of ions coming from a
continuous ion source, which proved to be so fruitful in TOF
instrumentation, to the case of ion mobility spectrometry. However,
here it is possible to enhance the efficient use of sample by
manipulating gas flows and electric fields. Namely, it is possible
to simultaneously insert and use positive and negative parent ions
(wherein the ion source can simultaneously produce them) as well as
the post-ionized neutral species of the initial sample flow. This
is all the more beneficial for the analysis of zwitterion
biopolymers whose presence is controlled by the pH of the solution
and appear often as neutral molecules comprising equally numbers of
spatially distributed positive and negative charge. Due to
differences in isoelectric points only some of the biopolymers
present in the sample could be neutral in the form of zwitterions
at a given pH value. A relatively low energy (about 2 eV) is
sufficient to cause bond breakage in the zwitterions and create
ions (additional few eV may be necessary for separation of created
ions of opposite sign), whereas direct ionization of organic
molecules may demand the energy close to 10 eV. Thus high
selectivity in producing ions from biomolecules of interest may be
achieved. In addition, it is possible to trap ions before the
entrances of multi-channel mobility cell by balancing forces from
the electric field and the counter gas flow. Using different
electric field strengths allows trapping of different type ions in
different traps. Thus some additional ion pre-separation prior to
the mobility channels may be achieved. This pre-separation will
enhance the efficiency of the overall final ion separation.
New Source for Microwave Manipulation of Solvent Droplets in a Gas
Flow
[0068] FIG. 1B schematically illustrates the method of getting ions
from droplets, trapping of ions and neutrals, post-ionization of
neutrals and orthogonal injection of ions into multichannel ion
mobility detection units (400) common for different embodiments of
the present invention. An initial gas flow entraining quasi-neutral
droplets (40) from a solution containing analytes is directed
through the capillary which his surrounded by a solenoid (10). In
one embodiment, a microwave voltage source may be coupled to the
source. Microwave voltage (MV) (11) is inserted through a capacitor
to the central coil of this solenoid. Due to capacitive coupling
between the coils of the solenoid MV would be transferred to them
producing the field inside the solenoid. To prevent irradiation of
this field outside the solenoid a grounded shield (26) is located
around it. The length of the solenoid is equal to the half
wavelength of the microwave field. Thus, a standing wave would be
formed inside the solenoid so that the maximum absolute value of
field strength would be in the middle of the solenoid and zero
field strength at its ends. The same solenoid is used for inserting
(15) DC voltage (through a resistor (300) and sound or ultrasound
frequency AC voltages (through a capacitor (500)) to the left most
coil of the solenoid (10) (as shown in the figure). The last
(right-most) coil of the solenoid is grounded (25). Thus, the gas
flow heating, as well as the droplet oscillation and microwave
heating are provided inside the solenoid. To achieve high
efficiency the resistance of the solenoid and its inductance should
be sufficiently large so that a realistic current for heating and
an AC field strength for droplet splitting can be applied. The
influence of resistance and inductance of the solenoid on the
microwave voltage is small because the capacitive coupling between
its coils is much stronger for high frequency field. For an
approximate average radius r of droplets it is possible to choose
the frequency of AC voltage to provide resonant splitting of the
droplets inside the solenoid. Due to heating, the droplets
evaporate and their sizes becomes smaller. When a droplet size
approaches the optimal size for resonant frequency splitting,
increasing the oscillations under high AC field results in
splitting of the droplet into two droplets. Each of these two
droplets may contain some excess of electric charge of opposite
sign. Estimates show that opposite influence of droplet surface
tension .sigma. and viscosity .eta. of the liquid results in two
resonant radii of the droplet for a given AC frequency. The
resonance frequency .omega. of the droplet oscillations for liquid
of density .rho. may be estimated using the following equation
(obtained using approaches described in L. D. Landau and E. M.
Lifschits, "Mechanics of continuum" Moscow, 1954):
.omega. = 8 .sigma. .rho. r 3 - 64 .eta. 2 .rho. 2 r 4 .
##EQU00001##
[0069] Therefore for each droplet it is possible to have two
chances for resonant splitting during its evaporation inside the
solenoid under influence of a single harmonic AC voltage. As the
energy of microwave droplet heating is proportional to the square
of the field strength, small droplets in the region close to the
middle of the solenoid may explode due to the high vapor pressure
inside them. Therefore the formation of ions of both signs may be
possible as these droplets are normally charged before the
explosion. The resulting species are mixed with hot gas (typically,
nitrogen) which prevents cluster formation and folding of
zwitterions under influence of room temperature gas (preferably
helium) flow (19) from mobility cells, and come inside the trapping
region along the gas flow axis (23). The ions can be analyzed as
previously discussed using the two opposed multi-channel IM units
(400) shown.
[0070] A new approach for electrospray ionization of the sample
solution is suggested to produce both negative and positive ions.
It is schematically shown in FIG. 2. The sample solution (1) moves
towards the end of the sample capillary tube located inside the
nebulizer tube (13) and is mixed with the flows of acid or base
buffers coming from syringes (7) and (9). Also, or alternatively,
some flow of D.sub.2O (or another deuterated compound) may be added
from syringe (5). These syringes have magnetic plungers (6) which
can be moved by electromagnetic coils (8) controlled by computer. A
higher current in the coil provides stronger pressure to the
plunger, which increases the flow of the buffer liquid or
deuterated substance directed to the sample capillary. Thus, the pH
of the investigated solution and/or concentration of species
containing deuterium can be varied. A pH measuring device is
located downstream of the capillary. The measured pH value is read
by computer, and can be used as part of a feedback loop. The
nebulizer gas flow (14) forms a flow of fairly neutral droplets
(12) from the sample solution. No DC electric field is applied in
this region in contrast to conventional electrospray ion source
where only positive or negative ions are extracted. The use of a
high DC electric field, perhaps, is the main reason for the drastic
difference in charge distribution of ions in solution and finally
in the gas phase. (see Kelly, M. A., Vestling, M. M., Fenselau C.
C., Smith P. B.; "Electrospray Analysis of Proteins--a Comparison
of Positive-Ion and Negative-Ion Mass Spectra at High and Low pH"
Org. Mass Spectrom. 1992, 27, 1143-1147). The nebulizer gas may be
heated up to a temperature slightly below the boiling point of the
solution so that ions in solution can rapidly reach the charge
equilibrium state. Just after the tip of the sample capillary, a
sound frequency voltage close to resonance is applied for droplet
splitting (15). According to the calculations for water droplets of
about 0.1 mm diameter, this frequency should be about 4.5 kHz with
an amplitude of a few hundred volts. Such conditions should be
adequate to rapidly (about 1 msec) split these droplets into
smaller ones having some excess positive or negative charge. The
accepted mechanism of droplet evaporation and further splitting
proposed in conventional ESI sources through electrostatic
explosion may be also valid after such initial droplet splitting.
The plates where the sound frequency voltage is applied, also
prevent penetration of microwave voltage inside the sample
capillary and overheating the liquid. The capillary could be made
of glass and not have sharp conducting edges that would produce
strong electric fields inside the capillary. Further evaporation of
the solvent from these droplets is stimulated by heating of these
droplets by microwave influence (11) and hot gas flow (10). Hot gas
is introduced from two opposite directions orthogonal to the flow
of droplets. A microwave electric field is applied in these
directions too. Heating the droplets with a microwave has
significant advantages in comparison to conventional single hot gas
flow heating. Deposition of the energy from a hot gas to droplets
is proportional to the droplet surface area to volume ratio so it
becomes less effective for evaporation of large droplets. In
contrast, the microwave energy deposited to the droplet for small
droplets is proportional to the volume of the droplet. So it has
the same or close efficiency for evaporation of each droplet. The
microwave energy flow is easily controlled, has low power
requirements, and does not transfer the heat to other components of
the system, where it may be undesirable. Nevertheless hot gas flow
(10), dry nitrogen, for example, would also be useful to prevent
undesirable cooling of ions, possible cluster formation and folding
of zwitterions after they exit the microwave heating region. Some
modulation of microwave voltage by sound or ultra-sound frequency
voltages would be useful to split evaporated droplets (when their
size reaches resonance). This will accelerate the process of
droplets evaporation. It is reasonable also to apply some DC
voltage to the plates (11) to separate positive and negative
droplets and ions and to prevent their recombination. The direction
of this field should be the same as further in the trapping region
and the strength being enough to move only light ions formed from
the solvent to the plates (11) only light ions formed from the
solvent. Thus the flow of ion and neutral species (40) would be
formed and directed to the trapping region (it may be referred also
as orthogonal IM injection region).
[0071] Although the examples provided for introduction of pH
modifiers and dueterated compositions to the sample have been
limited to syringes, it should be understood that the means for
introduction of these compositions are not so limited and include
any and all such techniques and manual and automated apparatuses
(including all flow injection techniques and apparatuses) known to
those of ordinary skill in the art as well as any such methods yet
to be developed.
[0072] If the charge distribution of the ions formed in the ESI
interface is close to the initial charge distribution in solution,
it will not be necessary in each case to collect data for a large
number of different pH values. For example if the problem is to
determine the presence and possibly the concentration of a known
(small) set of biopolymers whose isoelectric points have been
previously measured, it is possible to simply collect data at these
isoelectric points, i.e., at the corresponding pH values using
adjustable syringe pumps (7) and (9). These pumps should be
calibrated beforehand. For each isoelectric point, zwitterions
should be concentrated along the axis of the orthogonal IM
injection region and "cleaned" from positive and negative ions as
described before. After decomposition of the zwitterions, the
complimentary positive and negative ions (whose sum of masses gives
the mass of the biopolymer under study) should be searched. To
reliably identify a positive-negative daughter pair, their
intensity distribution over the ion beams should be proportional to
each other within the experimental errors (the difference in the
absolute intensities may be due to different ion transmissions).
These ion intensity distributions depend on gas flow force applied
to the zwitterion and its diffusion coefficient. Further, it would
be useful to compare ions generated from the same pulse. It is
possible to change the amount of the given zwitterions in solution
and in the sample flow by changing slightly the pH of the solution.
The intensities of the true complimentary ion pair should change
proportionally. Tuning the energy of photons in the laser beam
should result in a similar change.
[0073] The characterization of unknown biopolymers in solution may
also be simplified if the ion charge distribution in solution is
measured as previously demonstrated in the art (see, M. O.
Raznikova, V. V. Raznikov: "Determination of the extent of activity
of H-atoms in ions of polyfunctional compounds by H/D exchange mass
spectra" Chimicheskaya fizika, v. 24, N1, c. 3, 2005 (in Russian)).
This method allows one to determine the probabilities of charge
retention (positive and negative) on each site in the biopolymer
using the intensity distribution of the multi-charged ions of the
particular biopolymer. For a given pH value of the solution, the
corresponding pK.sub.a values for a given biopolymer could be
calculated using the probabilities of charge retention so that its
isoelectric point (pI) could be predicted (sum of pKas divided by
two). Besides the distribution itself, the maximum numbers of
positively and negatively charged sites in the given biopolymer
molecule should be determined. This information can be obtained by
doing measurements at extremely low and at extremely high pH values
followed by determination of ion peak with maximum charge for given
polymer. The first measurement will give the maximum number of
positive charges of ions from the given biopolymer, i.e., the
maximum number of positively charged sites ("negative" sites will
be neutralized). The second measurement would give the maximum
number of negatively charged sites.
[0074] The biopolymer conformation, and thus its pKa values, are
likely to change over a wide pH range. In this case, the previous
method would not be reliable for such "long distance" prediction of
pI values. It may then be better to use multi-charged ion
distributions with shorter predicted distance to isoelectric point
or gradually approach the true isoelectric point by changing the pH
around the predicted starting point and find the pH giving maximum
intensity to confirm the isoelectric point. At the isoelectric
point, collision-induced dissociation of some or all found
complimentary ions separated in multi-channel IM cell may give
unique structure information which would be more reliable than that
provided by existing methods using a comparable analysis time and
with comparable amount of the sample. Our three (or
four)-dimensional separation method (isoelectric point, ion
mobility and TOF mass analysis (or TOFMS/MS) gives extremely large
space for characterization of the components in the sample. With
this approach, the use of sample is optimized. The isoelectric
point separation can be performed in a controllable, dedicated way.
If the pK.sub.a values are calculated for all possible charged
sites in the biopolymer, the possibility of erroneous
interpretation of the data will be reduced. It would indicate the
types of residues which carry charge in the biopolymer and,
perhaps, provide information about their environment. Additionally,
mobility measurements can provide information about the
conformation of the molecule. Fairly good mobility resolution of
multi-charged ions and their selective collision induced
dissociation can be important to solve some structural problems
also. In necessary cases, additional information or even
supplementary ion separation may be provided by controllable
addition of deuterated solvent into the sample flow by the syringe
(5). The intensity distributions for peaks with different number of
H-atoms substituted for D are different not only for different
molecules but for different conformations of the same molecule.
Using an approach similar to that mentioned above for the method of
analysis of distribution of multi-charged ions (see M. O.
Raznikova; V. V. Raznikov; "Estimation of Probabilities of
Protonation of Amino Acid Residues in Peptides and Proteins by
their Electrospray Mass Spectra" Chimicheskaya fizika, vol. 20, N.
4, c 13, 2001) it is possible also to interpret the measured
intensity distribution of deuterated ions in order to estimate the
probability of H-D substitution for separate sites in the molecule.
This gives an opportunity to determine the numbers of different
functional groups having labile H-atoms (--NH.sub.2, >NH, --OH
and so on) and, perhaps, draw some conclusions about their
structural orientation in solution (see M. O. Raznikova, V. V.
Raznikov, "Determination of the Extent of Activity of H-atoms in
Ions of Polyfunctional Compounds by H/D Exchange Mass Spectra"
Chimicheskaya fizika, vol. 24, N. 1, c. 3, 2005). The distributions
of ions may be also modified if an additional syringe is used to
add a specific fast acting enzyme to the solutions which would
cause cleavage of biomolecules (and subsequent ion formation of
these fragments according to equilibrium conditions in solution)
prior to the droplet formation as the solution exits the
capillary.
[0075] The previously described approach will work not only for
direct analysis of solution but also for bombardment of the sample
surface by cluster ions, or solvent droplets containing
nanoparticulates (see pending U.S. application Ser. No. 10/861,970,
filed Jun. 4, 2004; pending U.S. application Ser. No. 11/231,448,
filed Sep. 21, 2005; and U.S. Pat. No. 6,989,528) or in DESI mode
of operation using a droplet source (110) which is a modification
of the electrospray interface as is shown in FIG. 3. Many parts of
this interface are the same as those shown in FIG. 1 and FIG. 2,
the exception being the nebulizer capillary (113) is open to the
ambient air and the fact that a pure solvent stream (101) is
employed. The injector tube (13) to the desolvation region is
marked as in FIG. 2 and the remainder of the assembly is identical
to FIG. 2. Instead of investigating a solution containing the
analyte (as in FIG. 2) we are using a flow of solvent (101) which
is inserted into the capillary. Droplets of pH adjusted solvent
(112), emerging into the nebulizer gas (114) are directed to the
moveable surface sample (116) under atmospheric pressure. These
droplets may be neutral or they may be charged by appropriate
biasing of the capillary and appropriate electrodes to accelerate
the droplets toward the surface. "Reflected" droplets (115)
enriched by species taken from the surface sample (116) by the gas
flow are inserted into conic part of injector (117) which is
connected with a cylindrically symmetric funnel entrance of the
injector (13) capillary. This injector (13) may be heated to
prevent droplet condensation and adsorption of the sample species
on the walls. The inside pressure can vary over a wide range from a
few mTorr up to near atmosphere which is adjusted by the sizes of
capillary (13) and (17) and the speed of the pumps (24). The length
of this injector should not be very short and would be chosen
experimentally to provide enough time for species from the sample
to come to charge state equilibrium (and, perhaps, for H-D exchange
too) with the solution inside the droplets. Heating and splitting
of droplets is provided as before by a microwave voltage modulated
by several sonic or ultrasonic frequency voltages applied to the
solenoid (10) shown in more details in FIG. 1B. Further
transformation of the flow and methods of measurements are the same
as described in the previous sections both with RF trapping
operations at low pressure and without RF trapping at higher
pressures near or above atmosphere. The configuration in FIG. 3 is
very versatile for surface analysis. For example, an energetic ion
source (such as a laser or a particle beam) could be combined to
irradiate the surface (116) during droplet impingement. This would
function to erode the surface either prior to, during or after
droplet impingement. The energetic source could also be used to
pre-form ions on the surface either by direct ionization or by
matrix assisted laser desorption. In another configuration, an on
demand droplet generator in place of the (110) could be used to
impinge either neutral or charged droplets. Laser light scattering
velocity tracking of the droplets could accurately predict when and
in what spatial region the droplet was going to impinge surface
(116). At the moment just as the droplet was impinging the surface
a laser could also be pulsed to irradiate the droplet and surface.
The droplet meniscus would act as a lens to micro-focus the portion
of the laser beam which had impinged the droplet into a high
fluence spot immediately below where the droplet was hitting the
surface (116). In this way a MALDI plume would be produced from an
area less than the size of the droplet diameter. The ions and
neutrals from the plume would evaporate from the surface into the
oncoming droplet and then be captured and borne into the injector
(117) entrance to the mobility cell array. The source may also be
used with the teachings of Schultz et. al. (see U.S. Pat. No.
6,989,528; pending application Ser. No. 11/231,448 filed Sep. 21,
2005; and pending application Ser. No. 10/861,970, filed Jun. 4,
2004 and incorporated by reference as though fully described
herein) to impinge droplets which are either pure solvent or which
contain nanoparticulates which can act as MALDI active matrices and
as taught in these applications the droplet can function both to
sputter the surface into the injector (117) while depositing the
matrix active material. Energetic particle irradiation of the
surface can be synchronized before, during, and after the droplet
arrival at the surface (116).
[0076] In the context of the present invention, four measuring
units (400) each including a multi-channel IM cell combined with a
multi-channel data recording TOFMS (FIG. 4 which is a view along
the cross-section A-A of FIG. 1B) are used to collect and detect
positive and negative ions (i) directly produced from different ion
sources and co-mixed with a gas flow (40) including ESI ions (or
laser ablated ions, or chemical ionization of neutrals or
post-ionization of neutrals or neutral molecule with adducts) and
(ii) produced from fragments of zwitterions. Ions of a given type
are accumulated in the orthogonal IM injection region (41) in
separate traps (42) for each ion beam as described in detail above
and in FIG. 1A, FIG. 1B and FIG. 4. In FIG. 1B ions are
pre-selected by a combination of electric fields and gas flows in
the trapping region and are directed to different traps. For
optimum conditions of ion trapping and further transport in
mobility cells the gas pressure inside orthogonal IM injection
region is maintained at about 100 Torr by pumping (49). After
accumulation, ions move under increasing electric field into the
funnel-shape IM channels (53), ions in the conical sections of the
channels undergo a small gas counterflow. The remaining transport
through each multichannel IM unit (400) has already been
described.
[0077] FIG. 6 gives details of the multichannel RF interface (70)
to prevent ions from diverging from the axis by the gas flow (72).
The main function of the CID tubes (48) is to collect ions coming
from corresponding IM channels and transport them to the
multi-channel RF interface (70). However, high electric field
inside CID (48) tubes may be applied to provide collision induced
dissociation of some chosen ions. To focus each ion beam (73), a
multi-channel RF-ion guide (58) is used. This interface (70) shown
in detail (section A-A) in the top part of FIG. 6 is comprised of
pairs of rods (58) and confining plates (57) between each pair.
RF-voltage of the same phase is applied to rods. DC voltages of
rods and confining plates are the same. The voltage difference
between the confining plates and the TOFMS (50) corresponds to the
energy that ions need to enter the TOFMS and to be detected
(determined by TOFMS geometry). These plates allow ion confinement
(73) between rods. Ions (73) entering the orthogonal TOFMS (50)
have some divergence and different velocities. Due to RF-focusing
they are entering the TOFMS through small orifices below 1 mm
diameter, thus a single pump (52) is sufficient for good operating
pressure. Before entering the RF ion guide, ions have traveled
through the IM cell and thus low m/z ions arrive first. The arrival
time is roughly linear to m/z values. The slope of the mobility
time versus m/z varies with the type of ions. As the focusing force
provided by RF-field is proportional to quadratic voltage/frequency
ratio and inversely proportional to the ion mass to charge ratio,
it is possible to increase the amplitude of RF-voltage (or decrease
the frequency) applied to rods proportionally to the square root of
ion arrival time with the coefficient being the square root of the
slope of the mobility time versus m/z. Such RF-field adjustment
allows one to record small ions without defocusing and losing them
due to possible instability of their motion for large RF-fields.
Also, it provides an opportunity to effectively focus large mass
ions and achieve similar width ion beams for ions of all masses. It
is true for the singly charged ions and multi-charged ions will be
focused better proportionally to their charge. Usually CID provides
structural information about ions. Most valuable information about
parent ions is usually obtained from daughter ions whose mass is
close to the parent ion mass. It is possible to increase the
RF-field proportional to the square root of the ion mass to charge
ratio which is emerging from the mobility cell and thus have
optimal transport of all ions through the RF interface.
[0078] FIG. 5 shows some results of computer simulation of ion
motion in short (about 2 cm) mobility cells under 150 Torr helium
pressure in the third chamber of the mobility cell (81). Two types
of singly charged ions are shown: "light" ions, 720 Da mass, 100
.ANG..sup.2 collision cross section, and "heavy" ions 1000 Da mass,
150 .ANG..sup.2 collision cross section. The top window of the
figure shows the moment when light ions (small dark grey (red)
crosses (82)) are stopped inside the TOFMS (83) (shown as a cone at
the right side). Heavy ions (small light grey (green) crosses (84))
are moving in the middle of mobility cell. The black small crosses
(85) show discharged ions after their collisions to the walls. The
voltages applied to electrodes are shown below (86). Gas pressures
in Torr are shown for various chambers (87) on the top of the
chambers (beginning of the forth chamber of mobility cell). The
diameter of orifices between these chambers and the length of them
is 1 mm. The diameter of the exit orifice (88) is 0.2 mm. Just
after exit orifice on the top of the window residual pressure in
mTorr is shown (89). Pumping rate (500 L/sec) is shown below (90).
The final picture for the simulation is shown in the middle of FIG.
5. The status bar at the bottom (91) of this picture gives
information about the numbers of ions of both types which have
reached the final position of their motion. Here about 50% of them
survived during this motion. Two status bars (92) at the left top
part of the picture give the drift time in .mu.s for each type of
ions, standard deviation of mobility peak in ns, average final
velocity of the ions, its standard deviation and average angle of
ion divergence in radian. At the bottom of the figure the same
final situation is shown for the case without special focusing
electrode for ions near exit of mobility cell. The transmission of
ions in this case is less (40% and 33%) but the resolving power
(more than 25) is better than for the previous case (about 20).
[0079] Ion beams entering the TOFMS will have a width of about 1 mm
and a divergence of about 0.02-0.04 radian (when special interface
electrode assembly like (70) is used). If the maximum length of ion
path in the initial direction to the detector plate (75) is about
10 cm, the standard deviation of the ion beam width in the plane of
recording will be about 3 mm. As the distance between ion beams is
about 5 mm, individual beams will overlap to some extent on the
detector plate. So if the detector plate has eight anodes and each
one is for recording the corresponding ion beam, it will actually
record its own beam and some signals from the adjacent beams as
well. This property seems to be a drawback but it may be turned
into an important advantage. The fact that a small fraction of a
given ion beam is recorded in an adjacent channel can be used to
increase the dynamic range if the signal in the main channel is
saturated. It is the same principle as that taught in U.S. Pat. No.
6,747,271 of Gonin et al., through the use of large and small
anodes. It is particularly useful if there is no interference from
the other signals on that adjacent channel. This can easily be
achieved with the mobility and mass resolutions of the present
instrumentation, and with multi-channel data recording. Since the
IM channels are not likely to be identical, the same ions (same
mass and formed from the same pulse) traveling through different
channels will appear at different times so their signals will not
overlap. The coefficients used to recover the signal in the main
channel may be obtained by comparing the signals on the tails of
mobility peaks, i.e. where the main signal is not (yet) saturated.
These coefficients for known location and sizes of recording anodes
could be easily converted into angle divergence of ion beams if the
velocities of ions in axial direction are known. At the end of
RF-ion guide, the velocity of ions will not be very high, but close
to that in IM channels (few hundred meters per second for ion of
about 1000 Da mass which corresponds to a kinetic energy of 0.1
eV). Accelerating voltage of several tens of eV applied between
RF-ion guide and the TOFMS gives these ions a velocity of several
thousand meters per second with relative standard deviation due to
initial energy far less than 1%. Known angle divergence of ion
beams allows estimation of the ion fraction impinging adjacent
anodes. Thus, when an ion flow saturates signal in the main anode
it may be recovered by the small unsaturated signal fraction
impinging adjacent anodes. Also, better mobility peak profiling may
be provided by multi-TDC channel detection. Several anodes are
linked to the same TDC channel. An example of anode arrangement
with their TDC channel links is shown in FIG. 7. In this case the
distribution of ion counts for each ion beam (73) over the TDC
channels (shown in FIG. 8 for fifth ion beam) will be used for
calculation of ion intensities coming to the left and the right
halves of the detector plate with correction of possible signal
saturation using also the mathematical procedure of TDC dead-time
correction.
[0080] FIG. 9 schematically shows the cross-section of the trapping
region for multi-beam profiling of a surface sample (120) located
on a convex cylindrical substrate. The view from the top of this
region is given in FIG. 10. Several (eight for the figures)
energetic pulsed beams (121) (for example laser or ion beams)
produce evaporated sample plumes near the surface. Gas flows (132)
from mobility cells (128) and (129) or the one created by pumping
(127) provide motion of the plumes from the surface to the top of
the figure. Any and all means known in the art to create, modify
and control gas flows in this and all other regions of the
apparatus may be used. Examples of means to create, modify and
control gas flows include, but are not limited to, mechanical
variable diameter iris type orifices, variable leak valves, or more
sophisticated gas flow controllers, all of which may be under
computer control. All other means known to those of ordinary skill
in the art are also applicable, as well as any yet to be developed.
The main factor here is the rate of pumping (127) which is provided
through the slit (158). The others are the gas pressures at the
ends of mobility cells (128) and (129). Electric fields (130)
between bottom pair of mobility cells move ions from the plume;
positive (122) to the left mobility cell and negative (123) to the
right mobility cell. After some delay time after initiation of the
desorption pulse, the neutral part of the plume (124), shown in
FIG. 10 as (156), will have moved to the region between the two top
mobility cells (128) and (129). At that time, a post-ionization
laser pulse (164), shown in FIG. 11, can be used to produce
positive and negative ions from these neutrals. Using an electric
field (130) between the top pair of mobility cells (128) and (129)
shown in FIG. 11 as (154) and (162) with collimating electrodes
(163), one can insert positive ions (125) into the left cell and
negative ions (126) are inserted into the right one. Thus, the
flows of positive ions (155) and negative ions (161) inside the
corresponding mobility cells are formed. The preferred means for
post-ionization of neutrals is laser irradiation of the flow or
plume containing the neutrals, however other means, such as, but
not limited to, electron attachment, chemical ionization, use of a
metastable atom beam, helium ion Auger neutralization, and other
means known to those of skill in the art are applicable.
[0081] This embodiment removes one of the main restrictions to
analysis by IM-oTOFMS of a sample surface. The drift time in the
mobility cell is often longer than the time between applications of
the energetic ion desorption pulse. If only one analysis channel is
used then the rate at which the desorption pulses are applied is
limited to the time necessary for the IM cell to clear on analyte
ions. Thus if multiple beams are used, we approach or exceed the
analysis time possible when one laser and an MS are used to
interrogate a surface. An additional advantage is that the sample
does not need to be translated as rapidly from one spot to the
other if multiple channels are used in lieu of a single channel.
This considerably reduces the complexity and improves the
positional accuracy of the mechanical means of translating the
sample to different spots in front of the immobile focal point of
the desorption source.
Analysis of Aerosol Particles
[0082] Another important possibility is to use the basic principles
of the electrospray ion source described above and to modify it for
the investigation of aerosol particles. The aerosol particles may
be natural aerosols such as atmospheric aerosols or they may be
generated aerosols. The proposed modification is illustrated in
FIG. 11, FIG. 12 and FIG. 13. The left and the right parts of this
source are the same as those previously described using the
electrospray ion source see FIGS. 1B and 2. These parts have the
same numerical identifiers as described for FIG. 2. The flow (40)
with ions and neutrals is directed to the trapping region of the
source shown in FIG. 1B.
[0083] Aerosol particles under the flow of ambient air by
compressor (169) are directed inside the chamber (170) containing
some layer of radioactive element (such as .sup.210Po), e.g., as
typically used in conventional instruments for aerosol analysis.
Alpha particles of about 5 MeV energy produced by .sup.210Po ionize
air in chamber (170), create large amounts of positive and negative
ions. These ions move in the chamber under influence of electric
field orthogonal to initial flow of aerosol particles and charge
these particles. Positively charged particles come to the right
part of the chamber (170), negatively charged particles are
concentrated at the left part of the chamber. The particles having
zero total charge are moved by the gas flow to the bottom of the
chamber (170) through the capillary (187) and are directed out of
the chamber. By a computer controlled valve (189), they are moved
away or mixed with nitrogen gas flow and enter separation chamber
(186). Alternatively, they travel through capillaries (171) and
(172) when computer controlled valve (189) is closed and valves
(188) are open together with the flows. Positively and negatively
charged particles travel to the top and bottom parts of the chamber
(186) which is used both for separation of aerosol particles and
for transporting of the nebulizer gas (being now a mixture of
nitrogen with air and chosen part of aerosol particles) for
producing droplets (12) of solvent from the capillary (185).
Charged (positive are coming through the capillary (197),
negative-through (199)) or neutral aerosol particles (together with
nitrogen flow (198)) are moving with the nebulizer gas and are
faster than solvent droplets so they can penetrate and accumulate
inside droplets--(196) and (206); FIG. 13. Under the influence of
solvent molecules and solvent ions, the organic substances adsorbed
on the surface of the particle would become neutrals or ions in
solution ready for further processing by the above-described
electrospray technique. Sound frequency voltage applied to the
solenoid (10), shown in more details in FIG. 1, provides energy
into the droplets liquid flow around the aerosol particles and thus
enhances removing of adsorbed substances from the surface of
aerosol particles.
[0084] The cases of separation of charged aerosol particles and
neutral ones are shown in FIG. 12 and FIG. 13. Separation of
charged particles is provided by some version of FAIMS (Field
Asymmetric Ion Mobility Spectrometry). Neutral particles are
separated by gas flows due to differences in diffusion
coefficients.
[0085] The cross-section of the chamber (186) for the case of
charged particles separation is shown in bottom-left part of the
FIG. 12. This chamber is divided into parts by insulator (195). The
top part provides separation of positively charged aerosol
particles, the bottom part separates negatively charged ones. An
example of an asymmetric potential wave form (FAIMS) applied to the
top part of the chamber (186) is shown (207) in FIG. 12. Reverse
polarity wave-form (-FAIMS)-(208) is applied to the bottom part of
the chamber (186). The position of the zero potential line may be
changed to provide focusing of desired particles (190) and (200).
Under the influence of an electric field provided by these wave
forms and gas flows (205) and (203) only particles with some
relation between their charge and size would be focused inside the
chamber (186) in crescent-like shaped regions (190) and (200).
Other particles would come out of the chamber (191) and (201) or
concentrate around solvent capillary (185)-(192) and (202). To
prevent loss of charge for these particles, a solvent capillary
(185) is coated by an insulator (195). The potential of the solvent
capillary (185) is usually maintained at around 0. To remove the
particles (192) and (202) from the separation chamber (186) the
potential wave forms applied to the right half of the chamber (186)
are inverted in comparison to the left half. Insulator (193)
separates these two parts. As a result the selected particles (190)
and (200) come close to the solvent capillary and the particles
concentrated there before (192) and (202) come out of the
separation region (186)-(194) and (204). Thus charging of the
droplets (196) and (206) by desired particles is provided and other
particles are removed from the separation region (186).
[0086] Transport of neutral aerosol particles is shown in FIG. 13.
These particles come into the separation chamber with the flow of
nitrogen (210) and (220). Small particles with large diffusion
coefficients (211) and (221) can quickly go out of the separation
chamber (186). Larger particles with less diffusion coefficients
would go further along the separation chamber and emerge from it
(194) and (204) at some distance after their entrance point. The
flow of relatively large particles (212) and (222) would come to
the end of separation region to be caught by solvent droplets near
the end of the capillary (185). By changing the pumping (174)-FIG.
12, it is possible to change the rate of separation of neutral
aerosol particles and provide different size distribution of
particles coming into the solvent droplets.
[0087] To simultaneously analyze the largest possible portion of
the deflected charged or neutral aerosol particles it is possible
to use two of the measuring units shown in FIG. 5. Their coupling
to the described ion source is shown in FIG. 11. One possibility
for producing ions from adsorbed organic substances is by using
laser ablation from the beams (173). These beams are reflected from
mirrors (168) to become parallel to the surface of the separation
chamber (186) from where the considered particles (177) and (181)
have appeared. Each output orifice for these particles is located
opposite to some input funnel of the corresponding measuring unit.
The volume between this surface and input funnels of the measuring
units (the top one for analysis of positive ions and the bottom
unit--for the negative ions) is pumped (174) to have in this region
the pressure around 100 Torr. Electric fields (175) and (176) are
applied to insert ions against gas flows (179) and (182) to ion
traps (178) and (183) at the entrances of corresponding funnels.
When the ion accumulation in traps is finished they are inserted
inside mobility channels by pulse of strong electric field inside
funnels to provide positive (180) and negative (184) mobility
separating ion flows directed to corresponding multichannel
orthogonal TOFMS.
[0088] It is possible to change the composition of the solution (1)
to be mixed with the separated aerosol to contain additives which
can enhance the ionization probability of the organics dissolved in
the droplet after the droplet solvent extracts the aerosols. For
example, the solution might contain MALDI matrix or could even be a
suspension of nanoparticulates which may adsorb some of the organic
analyte which had been on the surface of the aerosol.
Method of Combining Isotopic Labeling Followed by IM-TOFMS Analysis
to Identify Unknown Molecular Complexes in Complex Systems.
[0089] The apparatus could be used for analysis of isotopically
labeled drugs or other desired isotopically labeled analytes. For
example, a precisely controlled mixture having a precisely
determined composition comprising identical drug molecules (some
precise portion of which are unlabeled (e.g., H) and the other
portion of which are labeled on non-exchangeable sites with
isotopes (e.g., D)), is introduced into a viable biological
organism. Samples are later taken of the tissue, blood, serum,
saliva, or whole cells, and analyzed. The ionized isotopic drug
pairs appears in the plot of IM vs m/z as two ions separated in m/z
by the precise difference between the mass of labeled and
unlabelled drug but both types of ions have almost identical ion
mobilities. This nearly identical mobility cross-section of
isotopically labeled pairs of otherwise identical molecules can be
use to search for drugs bound to unknown biomolecules (e.g.
protein, lipid) by computer searching the plots of IM vs m/z from
such samples. The drug/biomolecule complex will also be revealed by
the nearly horizontal shift in m/z of the IM vs m/z plot due to the
mass difference of the labeled and unlabelled drug. The recognition
that the precise mass shift and the characteristic horizontal shift
allows us to create a new approach to the identification of labeled
molecules and their complexes with biomolecules. By creating an
algorithm, we can search IM vs m/z plots for free drug in the midst
of biological background, which might arise from direct analysis
from complex biological samples such as tissue, saliva, blood, etc.
Furthermore, the determination of metabolic products of the drug,
and the binding of these metabolites or the binding of free drug
with unknown biomolecules can also be identified by such a
procedure. A further use of this method allows simultaneously
relating the proteome, lipidome, and glycolipidome, to the
metabolic products (metabolome) of a given sample. Yet a further
use of the method is for measuring variations of the entire
metabolome on a cell to cell basis from a biological cell culture
by aerosolizing the cell from suspensions and measuring and
correlating the IM-MS plots from each cell individually with one
another. Such an approach, whether cell by cell or averaged over
many cells, can be used when a cell culture is split and one half
is grown with isotopically enriched nutrient such a specific
peptide (e.g., deuterated leucine).
[0090] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
* * * * *