U.S. patent application number 11/916445 was filed with the patent office on 2009-02-26 for method for sequencing peptides and proteins using metastable-activated dissociation mass spectrometry.
This patent application is currently assigned to Ohio University. Invention is credited to Olivier Collin, Glen P. Jackson.
Application Number | 20090050798 11/916445 |
Document ID | / |
Family ID | 37498967 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090050798 |
Kind Code |
A1 |
Jackson; Glen P. ; et
al. |
February 26, 2009 |
Method for Sequencing Peptides and Proteins Using
Metastable-Activated Dissociation Mass Spectrometry
Abstract
Methods for fragmentation of large molecular ions, including
proteins, nucleic acids, dendromers, and nanomaterials, compatible
with several mass spectrometric techniques. The methods involve
providing a gas-phase ion and allowing the gas phase ion to undergo
collisions with metastable states of noble gases or nitrogen
gas.
Inventors: |
Jackson; Glen P.; (Athens,
OH) ; Collin; Olivier; (Athens, OH) |
Correspondence
Address: |
CALFEE HALTER & GRISWOLD, LLP
800 SUPERIOR AVENUE, SUITE 1400
CLEVELAND
OH
44114
US
|
Assignee: |
Ohio University
Athens
OH
|
Family ID: |
37498967 |
Appl. No.: |
11/916445 |
Filed: |
June 5, 2006 |
PCT Filed: |
June 5, 2006 |
PCT NO: |
PCT/US2006/021620 |
371 Date: |
June 16, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60687584 |
Jun 3, 2005 |
|
|
|
Current U.S.
Class: |
250/282 ;
250/281 |
Current CPC
Class: |
G01N 33/6848
20130101 |
Class at
Publication: |
250/282 ;
250/281 |
International
Class: |
B01D 59/44 20060101
B01D059/44 |
Claims
1. A method for fragmenting macromolecules in the gas phase, the
method comprising the steps of providing metastable atoms and
colliding the macromolecular ions with the metastable atoms.
2. The method of claim 1 wherein the macromolecules are collided
with the metastable atoms in a mass spectrometer.
3. The method of claim 2 wherein the mass spectrometer is a
quadrupole ion trap mass spectrometer.
4. The method of any one of claims 1-3 wherein macromolecules are
selected from the group consisting of peptides, proteins, nucleic
acids, dendodromers, and nanomaterials.
5. The method of any one of claims 1-4 wherein the macromolecules
have a molecular weight greater than about 3 kD.
6. The method of any one of claims 1-5 wherein the metastable atoms
are selected from metastable nitrogen atoms and metastable noble
gases.
7. The method of claim 6 wherein the metastable noble gases are
selected from metastable helium, metastable neon, and metastable
argon.
8. The method of any one of claims 1-7 wherein the macromolecules
are neutral.
9. The method of any one of claims 1-7 wherein the macromolecules
are positively charged ions.
10. The method of any one of claims 1-7 wherein the macromolecules
are negatively charged ions.
11. A method for activating macromolecules in the gas phase
comprising the steps of providing metastable atoms and colliding
the macromolecular ions with the metastable atoms.
12. The method of claim 11 wherein the activation results in
dissociation of the macromolecule.
13. The method of claim 12 wherein the macromolecule is selected
from the group consisting of peptides, proteins, nucleic acids,
dendodromers, and nanomaterials.
14. The method of claim 13 wherein the macromolecules have a
molecular weight of greater than about 3 kDa.
15. A system capable of directly determining the amino acid
sequence of large peptides and proteins comprising a mass
spectrometer and a source of metastable atoms and a detector,
wherein the large proteins or peptides collide with the metastable
ions and fragment and the fragments are detected by the
detector.
16. The system of claim 15 wherein the mass spectrometer is a
quadrupole mass spectrometer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/687,584, filed Jun. 3, 2005, the entirety of
which is incorporated herein by reference. This application also
claims priority to International Application No. PCT/US2006/021620,
filed Jun. 5, 2006, the entirety of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Tandem mass spectrometry summarizes a broad range of
techniques whereby mass selected ions are subjected to a second (or
more) level or mass spectrometric analysis. Such is the social and
technological importance of tandem mass spectrometry that to date
more than a million blood and plasma samples from newborns have
been tested for various disorders using such devices. Tandem mass
spectrometry is also a central technology for proteomics and other
important areas of macromolecular identification, such as drug and
metabolite monitoring for forensics.
[0003] At present, there are essentially three physical processes
by which the internal energy of gas phase ions is raised above the
dissociation threshold: 1) collisions with atoms, molecules, or
surfaces 2) photodissociation, and 3) dissociative recombination of
positively and negatively charged species. Of these methods,
collision activated dissociation (CAD), also called collision
induced dissociation (CID), is the most widely practiced method.
Although collisional activation has many advantages over
alternative activation methods, the major limitation of collisional
activation is in its ineffectiveness at dissociating high mass ions
(such as biomolecules) and molecules with high barriers for
dissociation.
[0004] The ineffectiveness of CAD for high mass ions stems from a
number of factors including 1) inefficient conversion of kinetic to
internal energy and 2) increased number of degrees of freedom. In
addition, CAD of biological ions often results in the loss of one
or more small neutral losses such as water or ammonia with the
consequence of uninformative fragmentation patterns. Wide-band
excitation has been described recently to attempt to overcome these
difficulties, but other problems remain. Significant ion losses,
and subsequent decreases in sensitivity are notable in CAD devices
because reagent ions and fragment ions are often scattered during,
or inefficiently collected, after the collisional processes. Also,
as the size of the reagent ion increases, so does the number of
fragment ions over which the residual charges must be spread.
Fragmentation into a large number of channels leads to decreased
sensitivity and may prevent the ability to perform MS.sup.n
(n>2) fragmentation analyses.
[0005] Surface-induced dissociation (SID) has been applied to ion
beam, quadrupole, and ICR-type instruments and shows many
improvements over CAD for dissociating large biomolecules. However,
significant complications arise from surface sputtering, surface
reactions, ion losses and collision angle effects.
[0006] In the "top down" approach to proteomics, the dissociation
of biomolecules in the kDa-MDa mass range is necessary, and this
can only be achieved using CAD if it is used in combination with
significant proton attachments to effect coulombic repulsions
within the biomolecules. A more promising approach to fragmenting
large biomolecules has been through electron capture dissociation
(ECD) in FT-ICR instruments. This particular technique seems
restricted to ICR instruments, however, and may not be applicable
to more commonly available mass spectrometers such as quadrupole
based systems. There is also the inherent requirement for multiple
charges on the reagent ion, as neutralization by an electron
reduces the overall charge of the reagent with each capture. For
large mono-positive ions, such as dendromers or polymers, ECD may
not be applicable.
[0007] Moreover, commercial instruments available today typically
cannot directly determine the amino acid sequence of large peptides
and whole proteins (e.g. >3 kDa). This is primarily due to the
difficulty of breaking apart large ions within mass spectrometers,
but also due to the inability to control where fragmentation takes
place within the bio-ions. If these limitations regarding the
fragmentation of large biomolecules could be overcome, biomedical
research that depended on protein identification could be
considerably accelerated.
[0008] Accordingly, there exists a need for additional or
complementary methods for dissociating macromolecular ions in the
gas phase. This need is especially essential for large biomolecules
of interest to human health, national security and forensic
applications wherein existing techniques are ineffective for
providing conclusive and reproducible results.
SUMMARY OF THE INVENTION
[0009] Provided are methods for the fragmentation of large
molecular ions, such as proteins, nucleic acids, dendromers, and
nanomaterials that are compatible with several mass spectrometric
techniques, including quadrupole ion trap mass spectrometry. The
methods comprise the steps providing a gas-phase ion and allowing
the gas-phase ion to undergo collisions with metastable states of
noble gases or N.sub.2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows the production of metastable argon atoms in a
pulsed discharge source.
[0011] FIG. 2 shows a schematic for combining a metastable atom
source with a quadrupole ion trap mass spectrometer to perform a
metastable-activated dissociation mass spectrometry (MAD-MS)
experiment.
[0012] FIG. 3 shows the time sequence for a metastable-activated
dissociation mass spectrometry (MAD-MS) experiment.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Provided herein is a new paradigm for inducing tandem mass
spectrometry of gas-phase ions by allowing the ions to undergo
controlled collisions with metastable states of nobles gases or
N.sub.2. Reactions involving metastable atoms with charged
reagents, such as ions stored in ion trapping instruments, offer
significant advantages over existing methods of ion activation.
Table 1 compares the activation method described herein to several
practiced activation methods.
TABLE-US-00001 TABLE 1 Basic description of some practiced
activation methods and the comparison to the method of activation
described herein.* Activation Energy method range Instruments
Comments PSD Low ReTOF Metastable decay caused by excess internal
energy from ionization. CID (CAD) Low IT, FTICR, QqX
Controlled-energy (1-100 eV.sub.lab) collisions with inert gases.
High TOF/TOF, sectors Same, but keV energies. SID Low XqQ, IT,
FTICR 1-100 eV.sub.lab collisions between ions and a metal or SAM
surface. High TOF/TOF, sectors Same, but at keV energies. ECD Low
FTICR Capture of low energy electrons by positive ions; radical ion
chemistry follows. IRMPD Low IT, FTICR IR laser slowly raises
internal energy of ions above dissociation thresholds BIRD Low IT,
FTICR Heated systems used as IR source to achieve similar
consequences as IRMPD. MAD (this Low or High IT, FTICR, QqX
Metastable atoms with work) different energies used to excite or
ionize ions. Radical ion chemistry may follow ionization. PSD =
post-source decay; CID = collision-induced dissociation; CAD =
collision-activated dissociation; SID = Surface-induced
dissociation; ECD = electron capture dissociation; IRMPD = infrared
multiphoton dissociation; BIRD = blockbody infrared radiative
dissociation; MAD = metastable-activated dissociation; ReTOF =
reflectron time-of-flight; IT = linear or quadrupole ion trap;
FTICR = fourier transform ion cyclotron resonance; QqX = mass
selective quadrupole followed by rf-only multipole followed by any
mass analyzer; TOF/TOF tandem time of flight; XqQ = any mass
selective device followed by an rf-only multipole followed by a
mass selective quadrupole; SAM = self-assembled monolayer.
[0014] Of these activation methods, collision-activated
dissociation (CAD), also called collision-induced dissociation
(CID), is the most widely practiced method. CAD of peptides and
small proteins has been extensively studied and the major
fragmentation pathways are well known. Although CAD has many
advantages over alternative activation methods, its major weakness
is its ineffectiveness at dissociating molecules with high energy
barriers for dissociation and high mass ions such as proteins. The
methods described herein overcome these limitations and could open
the door to sequencing large, intact proteins.
[0015] The ineffectiveness of CAD for high mass ions stems from two
major factors: 1) inefficient conversion of kinetic to internal
energy, and 2) increased number of degrees of freedom. In addition,
CAD of biological ions, especially proteins, often results in the
loss of one or more small neutral groups such as water or ammonia.
In these cases, little or no structural information is obtained.
Wide band excitation has been described recently to attempt to
overcome these difficulties, but other challenges remain. For
example, in CAD the precursor ions must be accelerated to enable
higher-energy collisions to occur. In ion trap mass spectrometers,
this often limits the sensitivity because of scattering of
precursor ions and inefficient collection of product ions.
Furthermore, as the size of the precursor ion increases, so does
the number of fragment ions over which the residual charges must be
distributed. Fragmentation into a large number of channels leads to
decreased sensitivity and the inability to perform MS.sup.n
(n>2). Although ion trap mass spectrometers dominate the market
for sequencing peptides, there is room for additional or
complementary methods for performing tandem mass spectrometry.
[0016] In the `top down` approach to proteomics, the dissociation
of biomolecules in the kDa-MDa range mass is necessary and this is
oftentimes achieved using CAD in combination with significant
proton attachments to cause coulombic repulsions within the
biomolecules. It has been shown that the charge state of proteins
undergoing CAD plays a significant yet unpredictable role in
determining the fragmentation pathways. Work is progressing in the
area of CAD of whole proteins, but the secondary and tertiary
structures of large proteins seem to favor a select number of
fragmentation pathways. Although whole proteins can be identified
using CAD, complete sequence coverage is rarely obtained.
[0017] A recent approach to fragmenting large biomolecules has been
through electron capture dissociation (ECD) in FT-ICR instruments.
One advantage of ECD is the extensive fragmentation along the amide
backbone, which has shown to generate more complete amino-acid
sequence coverage of peptides. An exception is that ECD strongly
favors certain fragmentation pathways, such as on the C-terminal
side of Trp. Recently, ECD has been applied to radio frequency ion
traps but does not yet seem to offer such promising results as ECD
conducted in FT-ICR spectrometers. Because ECD seems to be most
suitable to FT-ICR spectrometers, such expensive instrumentation
will not be a financially viable option for most researchers. There
is also an inherent requirement for ions to be positively and
preferably multiply-charged because electron capture is more
favorable under these conditions. Obviously, ECD is not possible
for negatively charged ions, which would exclude certain analyses
such as DNA and proteins analyzed in negative-ion mode.
[0018] The above discussion shows that there is a clear and present
need for complementary methods for dissociating bio-ions in the gas
phase. This capability is especially needed for large biomolecules
where existing techniques can struggle to be effective at providing
conclusive and reproducible results. The ability to sequence large
proteins would circumvent lengthy digestion, separation and
purification procedures and would enable biomedical and clinical
researchers to identify important proteins more rapidly. This new
activation method described herein is compatible with both new and
existing mass spectrometers and separation technologies.
[0019] Metastable atoms of noble gases occupy well known energy
levels. Argon has two metastable states at 11.55 and 11.72 eV above
the neutral ground state. Table 2 shows the energies of the
metastable atoms of different noble gases. In the absence of
collisions, metastable atoms can exist for several seconds, giving
them time to find a collision partner. When a metastable atom
collides with a neutral atom or molecule with a lower ionization
potential than the metastable level, the metastable is capable of
ionizing the neutral in a process called Penning Ionization
(PI),
M*+AB.fwdarw.M+AB*+e.sup.-
Where M* is the metastable atom and AB is the neutral molecule.
When energetically feasible, more than one in three collisions
result in deexcitation in this manner. This is very efficient. If
the metastable atom has more energy than the minimum required to
ionize the neutral, the excess energy can be distributed between
the kinetic energy and the internal energy of the products. The
ejected electron tends to carry any significant kinetic energy and
the newly generated molecular ion carries any excess internal
energy.
[0020] Excess internal energy resulting from PI can lead to
extensive fragmentation of polyatomic ions,
M*+AB.fwdarw.M+AB*.sup.++e.sup.-.fwdarw.A.sup.++B
whereas smaller molecules tend to display informative photoemission
spectra,
M*+AB.fwdarw.M+AB*.sup.++e.sup.-.fwdarw.AB.sup.++hv
These basic phenomena have been known for decades, but until
recently have not been utilized for the deliberate fragmentation of
polyatomic species. By using different noble gases or diatomic
molecules (such as N.sub.2), it is possible to generate metastable
atoms in a selective range of energies and thus provide Penning
ionization reactions with tunable degrees of fragmentation. While
reactions between metastable atoms and large macro-ions cannot be
found in the literature, studies using the methods described herein
suggest that in a collision between a gas-phase macro-ion and a
metastable atom can yield two possible results, depending on the
ionization potential of the ion: First, macro-ion/metastable
collisions may result in subsequent ionization of the macro-ion.
Reactions of this kind could continue until the n.sup.th IP of the
macro-ion is greater than the energy level of the metastable in
question. Second, macro-ion/metastable collisions may result in the
internal excitation of the ion, with or without ionization.
Multiple collisions of this kind could be used to increase the
internal energy of the ion until the threshold energy for
fragmentation is reached.
TABLE-US-00002 TABLE 2 Characteristics of metastable atoms of noble
gases.* Electron Excitation Life- Polarizability Atom configuration
State energy (eV) time (s) (.ANG..sup.3) He 1s2s 2.sup.1S.sub.0
20.62 ~0.02 >9 2.sup.3S.sub.1 19.82 >7900 46.9 Ne 2p.sup.53s
.sup.3P.sub.0 16.72 430 .sup.3P.sub.2 16.62 24.4 27.8 Ar 3p.sup.54s
.sup.3P.sub.0 11.72 44.9 .sup.3P.sub.2 11.55 55.9 47.9 Kr
4p.sup.55s .sup.3P.sub.0 10.56 0.49 .sup.3P.sub.2 9.92 85 50.7 Xe
5p.sup.56s .sup.3P.sub.0 9.45 0.08 .sup.3P.sub.2 8.32 150 63.6
[0021] In the first scenario above, repeated ionization reactions
may benefit macro-ion analysis in several ways:
[0022] First, PI results in the ejection of a single electron and
the generation of a radical positive ion. Radical ions are known to
be considerably more reactive than proton-bound bio-ions, and the
phenomenon may lead to rearrangements or fragmentations of the ion
in question (such as McLafferty-type rearrangements). Because
Penning ionization reactions are known to be electrophilic,
ionization is likely to take place on a site such as a lone pair of
electrons on an oxygen molecule of a carbonyl group. Therefore,
when a protein or peptide ion is ionized by Penning ionization and
the ion subsequently rearranged according to McLafferty-like
rearrangement, amide cleavage would result and the products may
provide amino acid sequence information for the reagent ion.
[0023] Second, increasing the charge state of the bio-ion increases
the coulombic repulsions within the bio-ion. This facilitates
unfolding and fragmentation of the bio-ion.
[0024] Third, a reagent ion with only one charge can undergo
Penning ionization to provide two products with two charges
distributed between them. It is possible that each of the product
(fragment) ions could retain a charge, in which case each of the
fragment ions can be detected in a mass-selective manner. In this
case, one could recapture product ions and subject them to
subsequent Penning-type reactions. This could lead to the ability
to perform MS.sup.n experiments.
[0025] In the second scenario above, the energy deposited with each
Penning excitation collision could be as high as 20 eV for the case
of helium metastable atoms. For comparison, CAD of a 10 kDa bio-ion
with argon as the collision gas would require at least 5200 eV in
the lab frame, assuming a completely inelastic collision. Such
energies are barely obtainable in laboratory-scale ion-beam
apparatus, and certainly not in quadrupole or ICR based
instruments. Multiple Penning collisions could build significant
internal energy in the macro-ions depending, of course, on the
balance between the radiative loss rate of the macro-ion and the
collision rate with metastable.
[0026] A distinct advantage of Penning excitation and ionization
over CAD or SID is that the kinetic energy of the reagent ions is
virtually unaffected by Penning collisions. Scattering losses
should be minimal and collection efficiencies of resulting
fragments should be very high. Another potential advantage is that
neutral fragments released during fragmentation could be re-ionized
via PI reactions with new metastable atoms. This could provide
another mechanism for improving the sensitivity of MS.sup.n
experiments. An additional feature is that Penning-type reactions
can take place with neutral molecules or positive or negatively
charged ions. Ion-ion reactions and electron-capture reactions are
more limited in their scope.
[0027] Very recent experiments exposing self-assembled monolayers
(SAM) to metastable atoms in vacuum show that ionized fragments of
the monolayer species are desorbed from the surface, indicating
that the metastable collisions have very shallow surface
interactions. In these experiments, C--H and C--C bond cleavages
were made possible by collisions with metastable helium atoms and
significant but uncharacterized structural changes were possible
using metastable argon atoms.
[0028] Studies of collisions between metastable atoms with organic
surfaces show that interactions of metastable atoms with large
macro-ions may result in little more than ejection of H.sup.+ or
CH.sub.3.sup.+ ions from the outermost surface of the macro-ion. In
this "worst case scenario" the resulting surface modifications of
trapped macro-ions could be determined by an accompanying
activation technique (such as CAD). For peptides and proteins, this
information is almost certain to provide valuable information
regarding the exposed surfaces in the folded ions. The utility of
metastable activated reactions to elucidate the composition of
single stranded (ss) and double stranded (ds) DNA fragments is also
be of interest. Penning ionization of ss or ds DNA may also enable
sequencing information to be obtained. This would allow the
identification of PCR products, for example. This has the potential
to have a dramatic impact on DNA sequencing capabilities.
Experimental Approach
[0029] The first prototype instrument is currently being configured
on a commercial platform ion trap mass spectrometer. Quadrupole ion
traps (QITs) have demonstrated ability to obtain isolated charge
states of macro-ions in the gas phase, and to store macro-ions for
extended periods. QITs operate over a range of pressures compatible
with metastable sources. A metastable atom source has been built
and initial testing suggests that metastable atoms are successfully
transferred to the trapping region.
[0030] FIG. 1 shows the result of an initial test of the metastable
source, demonstrating the ability to form metastable atoms in the
pulses or .about.100 .mu.s in width. The red line in FIG. 1 shows
the voltage applied to tungsten wire in the discharge region. When
it pulses to -600 V, a plasma is produced and photons are emitted.
These are detected down-stream in the vacuum chamber by an electron
multiplier. When the voltage returns to zero at "0" seconds,
photons are emitted as a result of recombination and metastable
atoms are subsequently formed. Two electrodes were used to prevent
electrons and ions from reaching the detector, so only photons and
highly energetic compounds can be detected. A double optical
chopper is currently being installed to block the photons from
reaching the detector (or ion trap) and thus will only allow
ground-state and excited-state neutral atoms to reach the
detector.
[0031] In one embodiment, the source would be configured to the
vacuum chamber of a quadrupole ion trap mass spectrometer and
metastable ions would enter the trapping region through a small
hole drilled through the ring electrode. Initial testing of the ion
trap confirms that such a hole does not deleteriously affect the
performance of the ion trap. A schematic of one possible
configuration is shown in FIG. 2.
[0032] FIG. 3 shows a typical time sequence of different electrical
components pertinent to the experiment.
[0033] The time sequence shown in just one example or a way in
which metastable-activated dissociation can be competed. Time
sequences for ion-ion reactions, ion-molecule reactions, CID, IRMPD
(infrared multi-photon dissociation) may be added. The time
sequences are labeled as follows: 1) pre-ion accumulation period;
2) ion accumulation from ESI/APCI/MALDI ion source; 3)
pre-isolation; 4) isolation of reagent ion of interest; 5) pre-MAD;
6) metastable-activated dissociation (MAD) of reagent ion; 7)
pre-detection; 8) detection of product ions.
[0034] In step 6, a metastable-atom-beam of selectable
pulse-duration would be introduced to the center of the trapping
region to effect excitation/ionization of the trapped macro-ions.
In some cases, it may be necessary to send multiple pulses of
metastable atoms to effect dissociation or other reaction with the
trapped ions. The macro-ions would be formed using a commercial
interface, most probably an electrospray, nanospray, or (AP) MALDI
source.
Applications
[0035] Tandem mass spectrometry of macro-ions is just one example
of a highly beneficial application involving metastable atom
activation. Other applications involving bio-ions include `top
down` and `bottom up` proteomics, generic screening, and forensic
applications, such as bacterial and viral screening. Any current
application of tandem mass spectrometry using one of the activation
methods identified in Table 1 could in principle be achieved using
this new method of activation. To date, CAD of single and double
stranded DNA material has provided very challenging interpretation
because backbone cleavages are not as frequent or as predictable as
for peptide fragmentation. Metastable-activated dissociation of DNA
analogues could be possible using this approach and could lead to
the ability to sequence DNA. Other macro-ion applications include
the structural analyses of polymers, dendromers and nano-materials,
all of which could undergo surface modifications or
fragmentation/rearrangement upon absorbing energy via Penning-type
collisions.
[0036] In addition to Penning-type collisions the metastable source
could be easily configured as a source for positive ions or
electrons for ion-ion reactions in the trapping region. Multiple
ion, electron or metastable sources may be configured in a single
device to provide additional dimensions of flexibility for
macro-molecular studies, forensic and bio-ion applications. An
example would be to perform ion-ion reactions prior to
metastable-ion reactions in order to obtain the macro-ion in the
desired charge state in maximum abundance prior to MAD tandem mass
spectrometry.
[0037] The vast literature on metastable atom-neutral collisions
(from atoms to liquids and large surfaces) suggests that a
metastable atom-bio-ion collision will result in one of two
possible outcomes: 1) A collision will result in ionization of the
bio-ion (B),
[B+nH.sup.+].sup.n++M*.fwdarw.[B+nH.sup.+].sup.(n+1).+M
+e.sup.-
This would lead to the generation of a radical cation, which is
known to be more reactive than a protonated, even electron cation.
This type of process is often accompanied by internal excitation,
rearrangement and subsequent fragmentation; or 2) A collision will
lead to excitation of the bio-ion without ionization.
[B+nH.sup.30 ].sup.n++M*.fwdarw.[B+nH+].sup.*n++M
Multiple collisions of this kind could be used to increase the
internal energy of the macro-ions until the fragmentation threshold
energy is reached.
[0038] The reaction products will depend on the energy of the
metastable atom used, the IP of the target and the reaction
chemistry involved. Preliminary data on neutral amides shows that
metastables tend to ionize the carbonyl oxygen atoms of small
amides; we hypothesize that charged amides will react similarly. As
discussed above, because metastable atoms are neutral in charge,
the charge of a precursor will have little, if any, effect on the
collision frequency. A simple protonated peptide will be considered
below to illustrate the selective chemistry expected to follow
metastable-atom activation. Scheme 1 shows a McLafferty-type
rearrangement for protonated dialanine. This reaction occurs via
hydrogen transfer from the .gamma.-position followed by bond
cleavage of the .beta.-bond. The reaction is simplified as a
concerted mechanism below. All of the commonly occurring amino
acids--with the exception of glycine--have a hydrogen in the
.gamma.-position and could fragment through this common
pathway.
##STR00001##
Scheme 2 shows another favored pathway for the
rearrangement/fragmentation of radical cations. This fragmentation
occurs via 60 -cleavage.
##STR00002##
[0039] Schemes 1 and 2 show that the expected
rearrangement/fragmentation pathways of metastable activation will
result in cleavage of the amide backbone. Amide backbone cleavage
is the key to sequencing peptides and proteins using mass
spectrometry. The reaction products generated by the
rearrangement/fragmentation of the dipeptide above would provide
all the information necessary to sequence the precursor molecule.
The distribution of proton and electron-hole charges between
products obviously will depend on the pathway taken--McLafferty
rearrangement or .alpha.-cleavage--but will also depend heavily on
the location of the proton prior to metastable activation. One can
clearly see in Scheme 2 that in some cases it will be possible to
observe two charged products from a singly-charged precursor ion.
This is a significant possibility, and one that could dramatically
improve the signal intensities and limits of detection in tandem
mass spectra. Sequencing polypeptides using this technology relies
on the correct interpretation of fragmentation spectra. The spectra
obtained via the new fragmentation method of MAD-MS is expected to
follow the same principles and the same methods of nomenclature
that are used to interpret the product ion spectra of any other
fragmentation method. These methods rely on the fact that
fragmentation can, and often does, occur at quite random amino-acid
positions on each peptide ion that is fragmented. If enough ions
are fragmented, one will obtain a distribution of fragmentation
product ions containing all the possible fragmentation positions.
The differences in mass between certain peaks in the product-ion
mass spectra relates to the masses of the amino acid residues at
those points, and thus a `ladder` of amino acid residues can be
generated for the peptide ion in question. Product ion spectra
obtained via MAD-MS should be searchable in any of the databases
that are already established for the assignment of CAD product-ion
spectra. Therefore, the new technology is expected to fit
seamlessly into existing protocols for data analysis.
[0040] Similar to ECD, we expect the transfer of energy from
metastable atoms to larger peptides and proteins to be a nonergodic
reaction. That is, the excess energy deposited from a metastable
atom to a bio-ion during ionization will not have time to
equilibrate over all the internal energy modes in the bio-ion.
Instead, the energy will go into fragmenting the bio-ion close to
the site of `impact`. This hypothesis is supported by several
facts: 1) ECD of peptides and proteins has recently been shown to
be nonergodic; 2) Recent investigation have demonstrated that when
self assembled monolayers (SAMs) are exposed to metastable atoms
fragmentation takes place at the site of impact. Therefore,
collisions between large proteins and metastable atoms would be
expected to cause localized destruction of the protein surfaces, in
a nonergodic process, and especially through pathways such as those
described in Schemes 1 and 2.
[0041] Unlike current activation methods, the method of energy
transfer described herein is independent of the mass of the
precursor ion. In many tandem mass spectrometers, ions are
accelerated using static or dynamic electric fields to encourage
higher-energy collisions with inert targets. When a small ion
collides with a gas such as helium, the kinetic energy can be
converted to internal energy quite effectively. However, for large
molecules, the transfer of energy is much less efficient. This is
the main reason why large biomolecules have been so difficult to
fragment using collisional technologies. In ECD and
metastable-activated dissociation the mode of energy transfer is
independent of the mass of the precursor ion. This is one reason
why ECD has proven to be so beneficial for structural
characterization of larger peptides and proteins. We therefore
expect metastable activation to function as effectively for larger
peptides and proteins as it would for small molecules.
[0042] An additional benefit of metastable activation is
highlighted in Schemes 1 and 2. That is, each metastable impact may
generate a new charge on the precursor ion. Therefore, the more
exposure a precursor ion has to metastable atoms, the more product
ions can be generated from that precursor. This possibility could
dramatically enhance the ability to observe and interpret
post-fragmentation spectra of proteins and peptides. This
improvement could permit smaller sample sizes of proteins to be
sequenced in a top-down manner without the requirement for
enzymatic digestions. This would obviate the need for multiple
wet-chemistry steps and could make the identification of proteins
easier and faster than is currently possible.
[0043] Metastable-activated dissociation is expected to reveal
surface active residues and functional groups of proteins and
macromolecules with three-dimensional structure in the gas phase.
This could help elucidate the three-dimensional structure and
folding of biomolecules. For example, large proteins can have
complicated three-dimensional structure that can be thought of as a
tightly knotted rope. Due to the method of energy transfer from
metastable atoms to target compounds, the activation method
described herein may only activate molecular orbitals present at
the very outermost surface of the protein. Therefore, only those
amino acid residues exposed at the surface of the protein during
initial activation will be selectively fragmented. This is similar
to using scissors to cut only the exposed loops of the knotted
rope. In some cases, simply `cutting` the amide backbone at the
surface will not be enough for a mass spectrometer to determine
where the protein was cut: the protein will need to be `untied` so
that the m/z ratios of the products can be used to identify where
the original protein was cut. In this scenario, conventional
collision-activated dissociation could be used in concert with
metastable activation to facilitate the `untying` of the peptide
products. In addition to the conformational information elucidated
in this manner, exposing the peptide fragmentation products to
additional activation with metastable atoms could provide sequence
information and/or post translational modification information.
[0044] The examples described herein are for illustrative purposes
only and are not meant to limit the scope of the invention as
defined in the claims.
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