Note: Descriptions are shown in the official language in which they were submitted.
CA 02835314 2013-11-28
MASS SPECTROMETER
The present invention relates to a method of mass spectrometry
and a mass spectrometer.
It has become common practice to analyse proteins by first
enzymatically or chemically digesting the protein and then analysing
the peptide products by mass spectrometry. The mass spectrometry
analysis of the peptide products normally entails measuring the mass
of the peptide products. This method is sometimes referred to as
"peptide mapping" or "peptide fingerprinting".
It is also known to induce parent or precursor peptide ions to
fragment and to then measure the mass of one or more fragment or
daughter ions as a way of seeking to identify the parent or precursor
peptide ion. The fragmentation pattern of a peptide ion has also
been shown to be a successful way of distinguishing isobaric peptide
ions. Thus the mass to charge ratio of one or more fragment or
daughter ions may be used to identify the parent or precursor peptide
ion and hence the protein from which the peptide was derived. In
some instances the partial sequence of the peptide can also be
determined from the fragment or daughter ion spectrum. This
information may be used to determine candidate proteins by searching
protein and genomic databases.
Alternatively, a candidate protein may be eliminated or
confirmed by comparing the masses of one or more observed fragment or
daughter ions with the masses of fragment or daughter ions which
might be expected to be observed based upon the peptide sequence of
the candidate protein in question. The confidence in the
identification increases as more peptide parent or precursor ions are
induced to fragment and their fragment masses are shown to match
those expected.
It is desired to provide an improved method of mass
spectrometry and mass spectrometer.
According to an aspect of the present invention there is
provided a method of mass spectrometry comprising:
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passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device comprising an Electron
Capture Dissociation fragmentation device;
repeatedly switching, altering or varying the Electron Capture
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented upon interacting with electrons to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device comprising an Electron
Capture Dissociation fragmentation device;
repeatedly switching, altering or varying the Electron Capture
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented upon interacting with electrons to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
recognising first parent or precursor ions of interest from the
first sample;
automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio; and
comparing the intensity of .the first parent or precursor ions
of interest with the intensity of the second parent or precursor
ions.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
According to the preferred embodiment the Electron Capture
Dissociation fragmentation device is preferably repeatedly switched
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between the first and second modes during a single experimental run
or during a single analysis of a sample.
In the first mode of operation the electrons preferably have an
energy selected from the group consisting of: (i) < 1 eV; (ii) 1-2
eV; (iii) 2-3 eV; (iv) 3-4 eV; and (v) 4-5 eV.
In the first mode of operation the relatively low energy
electrons are preferably confined by a relatively strong magnetic
field.
The ions to be fragmented are preferably confined within an ion
guide. An AC or RF voltage is preferably applied to the electrodes
of the ion guide in order to create a radial pseudo-potential filed
or well which preferably acts to confine ions radially within the ion
guide.
The relatively low energy electrons are preferably confined by
a magnetic field which preferably overlaps or superimposes the ion
guiding region of the ion guide so that multiply charged analyte ions
are caused to interact with the relatively low energy electrons.
Fragmentation of ions by Electron Capture Dissociation preferably
does not involve causing internal vibrational energy to be introduced
to the ions.
The method preferably further comprises providing an electron
source. In the first mode of operation the electron source
preferably generates a plurality of electrons which are arranged to
interact with the parent or precursor ions.
In the second mode of operation the electron source is
preferably switched OFF so that analyte ions preferably do not
interact with any electrons and hence preferably are not caused to
fragment.
According to an aspect of the present invention there is
provided method of mass spectrometry comprising:
passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device comprising an Electron
Transfer Dissociation fragmentation device;
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repeatedly switching, altering or varying the Electron Transfer
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented upon interacting with reagent ions to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device comprising an Electron
Transfer Dissociation fragmentation device;
repeatedly switching, altering or varying the Electron Transfer
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented upon interacting with reagent ions to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
recognising first parent or precursor ions of interest from the
first sample;
automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio; and
comparing the intensity of the first parent or precursor ions
of interest with the intensity of the second parent or precursor
ions.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
According to the preferred embodiment the Electron Transfer
Dissociation fragmentation device is preferably repeatedly switched
between the first and second modes during a single experimental run
or during a single analysis of a sample.
=
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According to an aspect of the present invention there is
provided method of mass spectrometry comprising:
passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device comprising a Surface
Induced Dissociation fragmentation device;
repeatedly switching, altering or varying the Surface Induced
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented upon impinging upon a surface or target plate to produce
fragment or daughter ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device comprising a Surface
Induced Dissociation fragmentation device;
repeatedly switching, altering or varying the Surface Induced
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented upon impinging upon a surface or target plate to produce
fragment or daughter ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented;
recognising first parent or precursor ions of interest from the
first sample;
automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio; and
comparing the intensity of the first parent or precursor ions
of interest with the intensity of the second parent or precursor
ions.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
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According to the preferred embodiment the Surface Induced
Dissociation fragmentation device is preferably repeatedly switched
between the first and second modes during a single experimental run
or during a single analysis of a sample.
In the first mode of operation the parent or precursor ions are
preferably directed, diverted or deflected on to the surface or
target plate. In the second mode of operation the parent or
precursor ions preferably are not directed, diverted or deflected on
to the surface or target plate.
The surface or target plate preferably comprises a self-
assembled monolayer. The surface or target plate preferably
comprises a fluorocarbon or hydrocarbon monolayer.
The surface or target plane is preferably arranged in a plane
which is substantially parallel to the direction of travel of the
parent or precursor ions in the second mode of operation i.e. when
ions are preferably transmitted past the surface or target plate
without being directed on to the surface or target plate.
According to an aspect of the present invention there is
provided method of mass spectrometry comprising:
passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented or reacted to produce fragment, daughter, product or
adduct ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented or reacted;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device;
repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented or reacted to produce fragment, daughter, product or
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adduct ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented or reacted;
recognising first parent or precursor ions of interest from the
first sample;
automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio; and
comparing the intensity of the first parent or precursor ions
of interest with the intensity of the second parent or precursor
ions;
wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) an Electron Collision or
Impact Dissociation fragmentation device; (ii) a Photo Induced
Dissociation ("PID") fragmentation device; (iii) a Laser Induced
Dissociation fragmentation device; (iv) an infrared radiation induced
dissociation device; (v) an ultraviolet radiation induced
dissociation device; (vi) a nozzle-skimmer interface fragmentation
device; (vii) an in-source fragmentation device; (viii) an ion-source
Collision Induced Dissociation fragmentation device; (ix) a thermal
or temperature source fragmentation device; (x) an electric field
induced fragmentation device; (xi) a magnetic field induced
fragmentation device; (xii) an enzyme digestion or enzyme degradation
fragmentation device; (xiii) an ion-ion reaction fragmentation
device; (xiv) an ion-molecule reaction fragmentation device; (xv) an
ion-atom reaction fragmentation device; (xvi) an ion-metastable ion
reaction fragmentation device; (xvii) an ion-metastable molecule
reaction fragmentation device; (xviii) an ion-metastable atom
reaction fragmentation device; (xix) an ion-ion reaction device for
reacting ions to form adduct or product ions; (xx) an ion-molecule
reaction device for reacting ions to form adduct or product ions;
(xxi) an ion-atom reaction device for reacting ions to form adduct or
product ions; (xxii) an ion-metastable ion reaction device for
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reacting ions to form adduct or product ions; (xxiii) an ion-
metastable molecule reaction device for reacting ions to form adduct
or product ions; and (xxiv) an ion-metastable atom reaction device
for reacting ions to form adduct or product ions.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
According to the preferred embodiment the collision,
fragmentation or reaction device is preferably repeatedly switched
between the first and second modes during a single experimental run
or during a single analysis of a sample.
According to an aspect of the present invention there is
provided method of mass spectrometry comprising:
passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device comprising an Electron
Capture Dissociation fragmentation device;
repeatedly switching, altering or varying the Electron Capture
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented upon interacting with electrons to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device comprising an Electron
Capture Dissociation fragmentation device;
repeatedly switching, altering or varying the Electron Capture
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented upon interacting with electrons to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
recognising first parent or precursor ions of interest from the
first sample; =
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automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio;
determining a first ratio of the intensity of the first parent
or precursor ions of interest to the intensity of other parent or
precursor ions in the first sample;
determining a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
comparing the first ratio with the second ratio.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
According to the preferred embodiment the Electron Capture
Dissociation fragmentation device is preferably repeatedly switched
between the first and second modes during a single experimental run
or during a single analysis of a sample.
In the first mode of operation the electrons preferably have an
energy selected from the group consisting of: (i) < 1 eV; (ii) 1-2
eV; (iii) 2-3 eV; (iv) 3-4 eV; and (v) 4-5 eV.
In the first mode of operation the electrons are preferably
confined by a magnetic field.
The method preferably further comprises providing an electron
source. In the first mode of operation the electron source
preferably generates a plurality of electrons which are arranged to
interact with the parent or precursor ions. In the second mode of
operation the electron source is preferably switched OFF.
According to another aspect of the present invention there is
provided a method of mass spectrometry comprising:
passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device comprising an Electron
Transfer Dissociation fragmentation device;
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repeatedly switching, altering or varying the Electron Transfer
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented upon interacting with reagent ions to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device comprising an Electron
Transfer Dissociation fragmentation device;
repeatedly switching, altering or varying the Electron Transfer
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented upon interacting with reagent ions to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
recognising first parent or precursor ions of interest from the
first sample;
automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio;
determining a first ratio of the intensity of the first parent
or precursor ions of interest to the intensity of other parent or
precursor ions in the first sample;
determining a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
comparing the first ratio with the second ratio.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
According to the preferred embodiment the Electron Transfer
Dissociation fragmentation device is preferably repeatedly switched
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between the first and second modes during a single experimental run
or during a single analysis of a sample.
According to an aspect of the present invention there is
provided method of mass spectrometry comprising:
passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device comprising a Surface
Induced Dissociation fragmentation device;
repeatedly switching, altering or varying the Surface Induced
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented upon impinging upon a surface or target plate to produce
fragment or daughter ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device comprising a Surface
Induced Dissociation fragmentation device;
repeatedly switching, altering or varying the Surface Induced
Dissociation fragmentation device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented upon impinging upon a surface or target plate to produce
fragment or daughter ions and a second mode wherein substantially
fewer parent or precursor ions are fragmented;
recognising first parent or precursor ions of interest from the
first sample;
automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio;
determining a first ratio of the intensity of the first parent
or precursor ions of interest to the intensity of other parent or
precursor ions in the first sample;
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determining a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
comparing the first ratio with the second ratio.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
According to the preferred embodiment the Surface Induced
Dissociation fragmentation device is preferably repeatedly switched
between the first and second modes during a single experimental run
or during a single analysis of a sample.
In the first mode of operation the parent or precursor ions are
preferably directed, diverted or deflected on to the surface or
target plate.
In the second mode of operation the parent or precursor ions
are preferably not directed, diverted or deflected on to the surface
or target plate.
The surface or target plate preferably comprises a self-
assembled monolayer. The surface or target plate preferably
comprises a fluorocarbon or hydrocarbon monolayer.
The surface or target plane is preferably arranged in a plane
which is substantially parallel to the direction of travel of the
parent or precursor ions in the second mode of operation.
According to an aspect of the present invention there is
provided a method of mass spectrometry comprising:
passing parent or precursor ions from a first sample to a
collision, fragmentation or reaction device;
repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the first sample are
fragmented or reacted to produce fragment, daughter, product or
adduct ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented or reacted;
passing parent or precursor ions from a second sample to a
collision, fragmentation or reaction device;
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repeatedly switching, altering or varying the collision,
fragmentation or reaction device between a first mode wherein at
least some of the parent or precursor ions from the second sample are
fragmented or reacted to produce fragment or daughter ions and a
second mode wherein substantially fewer parent or precursor ions are
fragmented or reacted;
recognising first parent or precursor ions of interest from the
first sample;
automatically determining the intensity of the first parent or
precursor ions of interest, the first parent or precursor ions of
interest having a first mass to charge ratio;
automatically determining the intensity of second parent or
precursor ions from the second sample which have the same first mass
to charge ratio;
determining a first ratio of the intensity of the first parent
or precursor ions of interest to the intensity of other parent or
precursor ions in the first sample;
determining a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
comparing the first ratio with the second ratio;
wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) an Electron Collision or
Impact Dissociation fragmentation device; (ii) a Photo Induced
Dissociation ("PID") fragmentation device; (iii) a Laser Induced
Dissociation fragmentation device; (iv) an infrared radiation induced
dissociation device; (v) an ultraviolet radiation induced
dissociation device; (vi) a nozzle-skimmer interface fragmentation
device; (vii) an in-source fragmentation device; (viii) an ion-source
Collision Induced Dissociation fragmentation device; (ix) a thermal
or temperature source fragmentation device; (x) an electric field
induced fragmentation device; (xi) a magnetic field induced
fragmentation device; (xii) an enzyme digestion or enzyme degradation
fragmentation device; (xiii) an ion-ion reaction fragmentation
device; (xiv) an ion-molecule reaction fragmentation device; (xv) an
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ion-atom reaction fragmentation device; (xvi) an ion-metastable ion
reaction fragmentation device; (xvii) an ion-metastable molecule
reaction fragmentation device; (xviii) an ion-metastable atom
reaction fragmentation device; (xix) an ion-ion reaction device for
reacting ions to form adduct or product ions; (xx) an ion-molecule
reaction device for reacting ions to form adduct or product ions;
(xxi) an ion-atom reaction device for reacting ions to form adduct or
product ions; (xxii) an ion-metastable ion reaction device for
reacting ions to form adduct or product ions; (xxiii) an ion-
metastable molecule reaction device for reacting ions to form adduct
or product ions; and (xxiv) an ion-metastable atom reaction device
for reacting ions to form adduct or product ions.
According to the preferred embodiment the parent or precursor
ions comprise doubly, triply, quadruply charged ions or ions having
five or more charges.
According to the preferred embodiment the collision,
fragmentation or reaction device is preferably repeatedly switched
between the first and second modes during a single experimental run
or during a single analysis of a sample.
A reaction device should be understood as comprising a device
wherein ions, atoms or molecules are rearranged or reacted so as to
form a new species of ion, atom or molecule. An X-Y reaction
fragmentation device should be understood as meaning a device wherein
X and Y combine to form a product which then fragments. This is
different to a fragmentation device per se wherein ions may be caused
to fragment without first forming a product. An X-Y reaction device
should be understood as meaning a device wherein X and Y combine to
form a product and wherein the product does not necessarily then
fragment.
According to the present invention ions are collided,
fragmented or reacted in a device other than a Collision Induced
Dissociation fragmentation device. According to a particularly
preferred embodiment an Electron Capture Dissociation ("ECD") or an
Electron Transfer Dissociation ("ETD") fragmentation device may be
used to fragment analyte ions.
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Polypeptide chains are made up of amino acid residues which
have certain masses. There are three different bonds along a peptide
backbone and when a bond is broken the charge may remain either at
the N-terminal part of the structure or the C-terminal part of the
structure. When a polypeptide is fragmented there are six possible
= fragmentation series which are commonly referred to as: a, b, c and
x, y, z.
With Collision Induced Dissociation the most common
fragmentation route is for fragmentation to occur through the amide
bond (II). If the charge remains on the N-terminal then the ion is
referred to as a b series ion. If the charge remains on the C-
terminal then the ion is referred to as a y series ion.
Subscripts may be used to indicate how many amino acids
residues are contained in the fragment. For example, b3 is the
fragment ion resulting from cleavage of the amide bond (II) such that
charge remains on the N-terminal and wherein there are 3 amino acid
residues in the fragment.
According to an embodiment of the present invention when an
Electron Capture Dissociation ("ECD") or an Electron Transfer
Dissociation ("ETD") fragmentation device is used to fragment ions
then the polypeptide chain can be fragmented at different positions
to those positions where fragmentation would be expected to occur if
the polypeptide were fragmented by Collision Induced Dissociation.
In particular, an Electron Capture Dissociation ("ECD") or an
Electron Transfer Dissociation ("ETD") device enable x and c series
fragment ions predominantly to be produced. In certain circumstances
it is particularly advantageous to cause ions to fragment into x and
c series fragment ions rather than b and y series fragment ions (as
would be the case by Collision Induced Dissociation). In some
situations a more complete sequence is possible using ECD or ETD and
there can also be less ambiguity in identifying fragment ions. This
can make the process of sequencing the peptide easier.
Polypeptides may also be modified by Post Translational
Modifications such as phosphorylation. The use of an ECD or ETD
fragmentation device and the resulting fragmentation series which are
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produced enables Post Translational Modifications such as
phosphorylation to be more easily observed. It is also possible to
make a determination as to where the modification occurs along the
length of the polypeptide.
According to another embodiment the collision, fragmentation or
reaction device may comprise a Surface Induced Dissociation
fragmentation device. Collision Induced Dissociation can be viewed
as being a relatively slow process in that fragmentation is often the
result of multiple collisions between ions and gas molecules. As a
result fragmentation tends to be averaged out and a relatively broad
range of fragmentation products are typically observed. In contrast,
Surface Induced Dissociation can be viewed as being a relatively
sudden or instantaneous process. As a result a polypeptide may
fragment in a very specific manner. In certain situations this can
be particularly useful since it can reveal certain useful information
about the structure of the polypeptide.
It will therefore be appreciated that the present invention is
particularly advantageous in that parent or precursor ions are
preferably fragmented via different fragmentation routes to those
that may be obtained by Collision Induced Dissociation. Furthermore,
the present invention also enables Post Translational Modifications
of peptides to be observed and a determination to be made as to where
the modification sits in the peptide. The present invention is also
particularly advantageous compared to conventional approaches to
fragmenting analyte ions and attempting to elucidate structural
information relating to the analyte ions by analysing the
corresponding fragment ions.
It will therefore be appreciated that the present invention is
particularly advantageous in that parent or precursor ions are
preferably fragmented via different fragmentation routes to those
that may be obtained by Collision Induced Dissociation. Furthermore,
the present invention also enables Post Translational Modifications
of peptides to be observed and a determination to be made as to where
the modification sits in the peptide.
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The present invention is therefore particularly advantageous
compared to conventional arrangements.
Other arrangements are also contemplated wherein instead of
determining a first ratio of first parent or precursor ions to other
parent or precursor ions, a first ratio of first parent or precursor
ions to certain fragment, product, daughter or adduct ions may be
determined. Similarly, a second ratio of second parent or precursor
ions to certain fragment, product, daughter or adduct ions may be
determined and the first and second ratios compared.
The method preferably comprises automatically switching,
altering or varying the collision, fragmentation or reaction device
between at least the first mode and the second mode at least once
every 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 seconds.
The other parent or precursor ions present in the first sample
and/or the other parent or precursor ions present in the second
sample may either be endogenous or exogenous to the sample. The
other parent or precursor ions present in the first sample and/or the
other parent or precursor ions present in the second sample may
additionally used as a chromatographic retention time standard.
According to one embodiment parent or precursor ions,
preferably peptide ions, from two different samples are analysed in
separate experimental runs. In each experimental run parent or
precursor ions are passed to a collision, fragmentation or reaction
device. The collision, fragmentation or reaction device is
preferably repeatedly switched, altered or varied between a
fragmentation or reaction mode and a substantially non-fragmentation
or reaction mode. The ions emerging from the collision,
fragmentation or reaction device or which have been transmitted
through the collision, fragmentation or reaction device are then
preferably mass analysed. The intensity of parent or precursor ions
having a certain mass to charge ratio in one sample are then compared
with the intensity of parent or precursor ions having the same
certain mass to charge ratio in the other sample. A direct
comparison of the parent or precursor ion expression level may be
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made or the intensity of parent or precursor ions in a sample may
first be compared with an internal standard. An indirect comparison
may therefore be made between the ratio of parent or precursor ions
in one sample relative to the intensity of parent or precursor ions
relating to an internal standard and the ratio of parent or precursor
ions in the other sample relative to the intensity of parent or
precursor ions relating to preferably the same internal standard. A
comparison of the two ratios may then be made. Although the
preferred embodiment is described as relating to comparing the parent
or precursor ion expression level in two samples, it is apparent that
the expression level of parent or precursor ions in three or more
samples may be compared.
Parent or precursor ions may be considered to be expressed
significantly differently in two samples if their expression level
preferably differs by more than 1%, 10%, 50%, 100%, 150%, 200%, 250%,
300%, 350%, 400%, 450%, 500%, 1000%, 5000% or 10000%.
The collision, fragmentation or reaction device is preferably
maintained at a pressure selected from the group consisting of: (i)
greater than or equal to 0.0001 mbar; (ii) greater than or equal to
0.001 mbar; (iii) greater than or equal to 0.005 mbar; (iv) greater
. than or equal to 0.01 mbar; (v) between 0.0001 and 100 mbar; and (vi)
between 0.001 and 10 mbar. Preferably, the collision, fragmentation
or reaction device is maintained at a pressure selected from the
group consisting of: (i) greater than or equal to 0.0001 mbar; (ii)
greater than or equal to 0.0005 mbar; (iii) greater than or equal to
0.001 mbar; (iv) greater than or equal to 0.005 mbar; (v) greater
than or equal to 0.01 mbar; (vi) greater than or equal to 0.05 mbar;
(vii) greater than or equal to 0.1 mbar; (viii) greater than or equal
to 0.5 mbar; (ix) greater than or equal to 1 mbar; (x) greater than
or equal to 5 mbar; and (xi) greater than or equal to 10 mbar.
Preferably, the collision, fragmentation or reaction device is
maintained at a pressure selected from the group consisting of: (i)
less than or equal to 10 mbar; (ii) less than or equal to 5 mbar;
(iii) less than or equal to 1 mbar; (iv) less than or equal to 0.5
mbar; (v) less than or equal to 0.1 mbar; (vi) less than or equal to
CA 02835314 2013-11-28
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0.05 mbar; (vii) less than or equal to 0.01 mbar; (viii) less than or
equal to 0.005 mbar; (ix) less than or equal to 0.001 mbar; (x) less
than or equal to 0.0005 mbar; and (xi) less than or equal to 0.0001
mbar.
According to a less preferred embodiment, gas in the collision,
fragmentation or reaction device may be maintained at a first
pressure when the collision, fragmentation or reaction device is in
the high fragmentation or reaction mode and at a second lower
pressure when the collision, fragmentation or reaction device is in
the low fragmentation or reaction mode. According to another less
preferred embodiment, gas in the collision, fragmentation or reaction
device may comprise a first gas or a first mixture of gases when the
collision, fragmentation or reaction device is in the high
fragmentation or reaction mode and a second different gas or a second
different mixture of gases when the collision, fragmentation or
reaction device is in the low fragmentation or reaction mode.
Parent ions which are considered to be parent or precursor ions
of interest are preferably identified. This may comprise determining
the mass to charge ratio of the parent or precursor ions of interest,
preferably accurately to less than or equal to 20 ppm, 15 ppm, 10 ppm
or 5 ppm. The determined mass to charge ratio of the parent or
precursor ions of interest may then be compared with a database of
ions and their corresponding mass to charge ratios and hence the
identity of the parent or precursor ions of interest can be
established.
According to the preferred embodiment the step of identifying
the parent or precursor ions of interest comprises identifying one or
more fragment, product, daughter or adduct ions which are determined
to result from fragmentation of the parent or precursor ions of
interest. Preferably, the step of identifying one or more fragment,
product, daughter or adduct ions further comprises determining the
mass to charge ratio of the one or more fragment, product, daughter
or adduct ions to less than or equal to 20 ppm, 15 ppm, 10 ppm or 5
Rom.
CA 02835314 2013-11-28
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The step of identifying first parent or precursor ions of
interest may comprise determining whether parent or precursor ions
are observed in a mass spectrum obtained when the collision,
fragmentation or reaction device is in a low fragmentation or
reaction mode for a certain time period and the first fragment,
product, daughter or adduct ions are observed in a mass spectrum
obtained either immediately before the certain time period, when the
collision, fragmentation or reaction device is in a high
fragmentation or reaction mode, or immediately after the certain time
period, when the collision, fragmentation or reaction device is in a
high fragmentation or reaction mode.
The step of identifying first parent or precursor ions of
interest may comprise comparing the elution times of parent or
precursor ions with the pseudo-elution time of first fragment,
product, daughter or adduct ions. The fragment, product, daughter or
adduct ions are referred to as having a pseudo-elution time since
fragment, product, daughter or adduct ions do not actually physically
elute from a chromatography column. However, since at least some of
the fragment, product, daughter or adduct ions are fairly unique to
particular parent or precursor ions, and the parent or precursor ions
may elute from the chromatography column only at particular times,
then the corresponding fragment, product, daughter or adduct ions may
similarly only be observed at substantially the same elution time as
their related parent or precursor ions. Similarly, the step of
identifying first parent or precursor ions of interest may comprise
comparing the elution profiles of parent or precursor ions with the
pseudo-elution profile of first fragment, product, daughter or adduct
ions. Again, although fragment, product, daughter or adduct ions do
not actually physically elute from a chromatography column, they can
be considered to have an effective elution profile since they will
tend to be observed only when specific parent or precursor ions elute
from the column and as the intensity of the eluting parent or
precursor ions varies over a few seconds so similarly the intensity
of characteristic fragment, product, daughter or adduct ions will
also vary in a similar manner.
CA 02835314 2013-11-28
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Ions may be determined to be parent or precursor ions by
comparing two mass spectra obtained one after the other, a first mass
spectrum being obtained when the collision, fragmentation or reaction
device was in a high fragmentation or reaction mode and a second mass
spectrum obtained when the collision, fragmentation or reaction
device was in a low fragmentation or reaction mode, wherein ions are
determined to be parent or precursor ions if a peak corresponding to
the ions in the second mass spectrum is more intense than a peak
corresponding to the ions in the first mass spectrum. Similarly,
ions may be determined to be fragment, product, daughter or adduct
ions if a peak corresponding to the ions in the first mass spectrum
is more intense than a peak corresponding to the ions in the second
mass spectrum. According to another embodiment, a mass filter may be
provided upstream of the collision, fragmentation or reaction device
wherein the mass filter is arranged to transmit ions having mass to
charge ratios within a first range but to substantially attenuate
ions having mass to charge ratios within a second range and wherein
ions are determined to be fragment, product, daughter or adduct ions
if they are determined to have a mass to charge ratio falling within
the second range.
The first parent or precursor ions and the second parent or
precursor ions are preferably determined to have mass to charge
ratios which differ by less than or equal to 40 ppm, 35 ppm, 30 ppm,
ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm. The first parent or
25 precursor ions and the second parent or precursor ions may have been
determined to have eluted from a chromatography column after
substantially the same elution time. The first parent or precursor
ions may also have been determined to have given rise to one or more
first fragment, product, daughter or adduct ions and the second
parent or precursor ions may have been determined to have given rise
to one or more second fragment, product, daughter or adduct ions,
wherein the one or more first fragment, product, daughter or adduct
ions and the one or more second fragment, product, daughter or adduct
ions have substantially the same mass to charge ratio. The mass to
charge ratio of the one or more first fragment, product, daughter or
CA 02835314 2013-11-28
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adduct ions and the one or more second fragment, product, daughter or
adduct ions may be determined to differ by less than or equal to 40
ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm.
The first parent or precursor ions may also be determined to
have given rise to one or more first fragment, product, daughter or
adduct ions and the second parent or precursor ions may have been
determined to have given rise to one or more second fragment,
product, daughter or adduct ions and wherein the first parent or
precursor ions and the second parent or precursor ions are observed
in mass spectra relating to data obtained in the low fragmentation or
reaction mode at a certain point in time and the one or more first
and second fragment, product, daughter or adduct ions are observed in
mass spectra relating to data obtained either immediately before the
certain point in time, when the collision, fragmentation or reaction
device is in the high fragmentation or reaction mode, or immediately
after the certain point in time, when the collision, fragmentation or
reaction device is in the high fragmentation or reaction mode.
The first parent or precursor ions may be determined to have
given rise to one or more first fragment, product, daughter or adduct
ions and the second parent or precursor ions may be determined to
have given rise to one or more second fragment, product, daughter or
adduct ions if the first fragment, product, daughter or adduct ions
have substantially the same pseudo-elution time as the second
fragment, product, daughter or adduct ions.
The first parent or precursor ions may be determined to have
given rise to one or more first fragment, product, daughter or adduct
ions and the second parent or precursor ions may be determined to
have given rise to one or more second fragment, product, daughter or
adduct ions and wherein the first parent or precursor ions are
0
determined to have an elution profile which correlates with a pseudo-
elution profile of a first fragment, product, daughter or adduct ion
and wherein the corresponding second parent or precursor ions are
determined to have an elution profile which correlates with a pseudo-
elution profile of a second fragment, product, daughter or adduct
ion.
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According to another embodiment the first parent or precursor
ions and the second parent or precursor ions which are being compared
may be determined to be multiply charged. This may rule out a number
of fragment, product, daughter or adduct ions which quite often tend
to be singly charged. The first parent or precursor ions and the
second parent or precursor ions may according to a more preferred
embodiment be determined to have the same charge state. According to
another embodiment, the parent or precursor ions being compared in
the two different samples may be determined to give rise to fragment,
product, daughter or adduct ions which have the same charge state.
The first sample and/or the second sample may comprise a
plurality of different biopolymers, proteins, peptides, polypeptides,
oligionucleotides, oligionucleosides, amino acids, carbohydrates,
sugars, lipids, fatty acids, vitamins, hormones, portions or
fragments of DNA, portions or fragments of cDNA, portions or
fragments of RNA, portions or fragments of mRNA, portions or
fragments of tRNA, polyclonal antibodies, monoclonal antibodies,
ribonucleases, enzymes, metabolites, polysaccharides, phosphorylated
peptides, phosphorylated proteins, glycopeptides, glycoproteins or
steroids. The first sample and/or the second sample may also
comprise at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,
300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,
3500, 4000, 4500, or 5000 molecules having different identities.
The first sample may be taken from a diseased organism and the
second sample may be taken from a non-diseased organism.
Alternatively, the first sample may be taken from a treated organism
and the second sample may be taken from a non-treated organism.
According to another embodiment the first sample may be taken from a
mutant organism and the second sample may be taken from a wild type
organism.
Molecules from the first and/or second samples are preferably
separated from a mixture of other molecules prior to being ionised by
High Performance Liquid Chromatography ("HPLC"), anion exchange,
anion exchange chromatography, cation exchange, cation exchange
chromatography, ion pair reversed-phase chromatography,
CA 02835314 2013-11-28
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chromatography, single dimensional electrophoresis, multi-dimensional
electrophoresis, size exclusion, affinity, reverse phase
chromatography, Capillary Electrophoresis Chromatography ('CEC"),
electrophoresis, ion mobility separation, Field Asymmetric Ion
Mobility Separation ("FAIMS") or capillary electrophoresis.
According to a particularly preferred embodiment the first and
second sample ions comprise peptide ions. The peptide ions
preferably comprise the digest products of one or more proteins. An
attempt may be made to identify a protein which correlates with
parent peptide ions of interest. Preferably, a determination is made
as to which peptide products are predicted to be formed when a
protein is digested and it is then determined whether any predicted
peptide product(s) correlate with parent or precursor ions of
interest. A determination may also be made as to whether the parent
or precursor ions of interest correlate with one or more proteins.
The first and second samples may be taken from the same
organism or from different organisms.
A check may be made to confirm that the first and second parent
or precursor ions being compared really are parent or precursor ions
rather than fragment, product, daughter or adduct ions. A high
fragmentation or reaction mass spectrum relating to data obtained in
the high fragmentation or reaction mode may be compared with a low
fragmentation or reaction mass spectrum relating to data obtained in
the low fragmentation or reaction mode wherein the mass spectra were
obtained at substantially the same time. A determination may be made
that the first and/or the second parent or precursor ions are not
fragment, product, daughter or adduct ions if the first and/or the
second parent or precursor ions have a greater intensity in the low
fragmentation or reaction mass spectrum relative to the high
fragmentation or reaction mass spectrum. Similarly, fragment,
product, daughter or adduct ions may be recognised by noting ions
having a greater intensity in the high fragmentation or reaction mass
spectrum relative to the low fragmentation or reaction mass spectrum.
Parent ions from the first sample and parent or precursor ions
from the second sample are preferably passed to the same collision,
CA 02835314 2013-11-28
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fragmentation or reaction device. However, according to a less
preferred embodiment, parent or precursor ions from the first sample
and parent or precursor ions from the second sample may be passed to
different collision, fragmentation or reaction devices.
According to an aspect of the present invention there is
provided a mass spectrometer comprising:
an Electron Capture Dissociation fragmentation device which is
arranged and adapted to be repeatedly switched in use between a first
mode wherein at least some parent or precursor ions are fragmented
upon interacting with electrons to produce fragment or daughter ions
and a second mode wherein substantially fewer parent or precursor
ions are fragmented;
a mass analyser; and
a control system which in use:
(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio; and
(iv) compares the intensity of the first parent or precursor
ions of interest with the intensity of the second parent or precursor
ions.
According to an aspect of the present invention there is
provided a mass spectrometer comprising:
an Electron Transfer Dissociation fragmentation device which is
arranged and adapted to be repeatedly switched in use between a first
mode wherein at least some parent or precursor ions are fragmented
upon interacting with reagent ions to produce fragment or daughter
ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
a mass analyser; and
a control system which in use:
CA 02835314 2013-11-28
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(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio; and
(iv) compares the intensity of the first parent or precursor
ions of interest with the intensity of the second parent or precursor
ions.
According to an aspect of the present invention there is
provided a mass spectrometer comprising:
a Surface Induced Dissociation fragmentation device which is
arranged and adapted to be repeatedly switched in use between a first
mode wherein at least some parent or precursor ions are fragmented
upon impinging upon a surface or target plate to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
a mass analyser; and
a control system which in use:
(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio; and
(iv) compares the intensity of the first parent or precursor
ions of interest with the intensity of the second parent or precursor
ions.
According to an aspect of the present invention there is
provided a mass spectrometer comprising:
=
CA 02835314 2013-11-28
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a collision, fragmentation or reaction device which is arranged
and adapted to be repeatedly switched in use between a first mode
wherein at least some parent or precursor ions are fragmented or
reacted to produce fragment, daughter, product or adduct ions and a
second mode wherein substantially fewer parent or precursor ions are
fragmented or reacted;
a mass analyser; and
a control system which in use:
(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio; and
(iv) compares the intensity of the first parent or precursor
ions of interest with the intensity of the second parent or precursor
ions;
wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) an Electron Collision or
Impact Dissociation fragmentation device; (ii) a Photo Induced
Dissociation ("PID") fragmentation device; (iii) a Laser Induced
Dissociation fragmentation device; (iv) an infrared radiation induced
dissociation device; (v) an ultraviolet radiation induced
dissociation device; (vi) a nozzle-skimmer interface fragmentation
device; (vii) an in-source fragmentation device; (viii) an ion-source
Collision Induced Dissociation fragmentation device; (ix) a thermal
or temperature source fragmentation device; (x) an electric field
induced fragmentation device; (xi) a magnetic field induced
fragmentation device; (xii) an enzyme digestion or enzyme degradation
fragmentation device; (xiii) an ion-ion reaction fragmentation
device; (xiv) an ion-molecule reaction fragmentation device; (xi) an
ion-atom reaction fragmentation device; (xvi) an ion-metastable ion
reaction fragmentation device; (xvii) an ion-metastable molecule
CA 02835314 2013-11-28
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reaction fragmentation device; (xviii) an ion-metastable atom
reaction fragmentation device; (xix) an ion-ion reaction device for
reacting ions to form adduct or product ions; (xx) an ion-molecule
reaction device for reacting ions to form adduct or product ions;
(xxi) an ion-atom reaction device for reacting ions to form adduct or
product ions; (xxii) an ion-metastable ion reaction device for
reacting ions to form adduct or product ions; (xxiii) an ion-
metastable molecule reaction device for reacting ions to form adduct
or product ions; and (xxiv) an ion-metastable atom reaction device
for reacting ions to form adduct or product ions.
According to an aspect of the present invention there is
provided a mass spectrometer comprising:
an Electron Capture Dissociation fragmentation device which is
arranged and adapted to be repeatedly switched in use between a first
mode wherein at least some parent or precursor ions are fragmented
upon interacting with electrons to produce fragment or daughter ions
and a second mode wherein substantially fewer parent or precursor
ions are fragmented;
a mass analyser; and
a control system which in use:
(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio;
(iv) determines a first ratio of the intensity of the first
parent or precursor ions of interest to the intensity of other parent
or precursor ions in the first sample;
(v) determines a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
(vi) compares the first ratio with the second ratio.
CA 02835314 2013-11-28
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According to an aspect of the present invention there is
provided a mass spectrometer comprising:
an Electron Transfer Dissociation fragmentation device which is
arranged and adapted to be repeatedly switched in use between a first
mode wherein at least some parent or precursor ions are fragmented
upon interacting with reagent ions to produce fragment or daughter
ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
a mass analyser; and
a control system which in use:
(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio;
(iv) determines a first ratio of the intensity of the first
parent or precursor ions of interest to the intensity of other parent
or precursor ions in the first sample;
(v) determines a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
(vi) compares the first ratio with the second ratio.
According to an aspect of the present invention there is
provided a mass spectrometer comprising:
a Surface Induced Dissociation fragmentation device which is
arranged and adapted to be repeatedly switched in use between a first
mode wherein at least some parent or precursor ions are fragmented
upon impinging upon a surface or target plate to produce fragment or
daughter ions and a second mode wherein substantially fewer parent or
precursor ions are fragmented;
a mass analyser; and
a control system which in use:
CA 02835314 2013-11-28
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(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio;
(iv) determines a first ratio of the intensity of the first
parent or precursor ions of interest to the intensity of other parent
or precursor ions in the first sample;
(v) determines a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
(vi) compares the first ratio with the second ratio.
According to an aspect of the present invention there is
provided a mass spectrometer comprising:
a collision, fragmentation or reaction device which is arranged
and adapted to be repeatedly switched in use between a first mode
wherein at least some parent or precursor ions are fragmented or
reacted to produce fragment, daughter, product or adduct ions and a
second mode wherein substantially fewer parent or precursor ions are
fragmented or reacted;
a mass analyser; and
a control system which in use:
(i) recognises first parent or precursor ions of interest from
a first sample, the first parent or precursor ions of interest having
a first mass to charge ratio;
(ii) determines the intensity of the first parent or precursor
ions of interest;
(iii) determines the intensity of second parent or precursor
ions from a second sample which have the same first mass to charge
ratio;
CA 02835314 2013-11-28
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(iv) determines a first ratio of the intensity of the first
parent or precursor ions of interest to the intensity of other parent
or precursor ions in the first sample;
(v) determines a second ratio of the intensity of the second
parent or precursor ions to the intensity of other parent or
precursor ions in the second sample; and
(vi) compares the first ratio with the second ratio;
wherein the collision, fragmentation or reaction device is
selected from the group consisting of: (i) an Electron Collision or
Impact Dissociation fragmentation device; (ii) a Photo Induced
Dissociation ("PID") fragmentation device; (iii) a Laser Induced
Dissociation fragmentation device; (iv) an infrared radiation induced
dissociation device; (v) an ultraviolet radiation induced
dissociation device; (vi) a nozzle-skimmer interface fragmentation
device; (vii) an in-source fragmentation device; (viii) an ion-source
Collision Induced Dissociation fragmentation device; (ix) a thermal
or temperature source fragmentation device; (x) an electric field
induced fragmentation device; (xi) a magnetic field induced
fragmentation device; (xii) an enzyme digestion or enzyme degradation
fragmentation device; (xiii) an ion-ion reaction fragmentation
device; (xiv) an ion-molecule reaction fragmentation device; (xv) an
ion-atom reaction fragmentation device; (xvi) an ion-metastable ion
reaction fragmentation device; (xvii) an ion-metastable molecule
reaction fragmentation device; (xviii) an ion-metastable atom
reaction fragmentation device; (xix) an ion-ion reaction device for
reacting ions to form adduct or product ions; (xx) an ion-molecule
reaction device for reacting ions to form adduct or product ions;
(xxi) an ion-atom reaction device for reacting ions to form adduct or
product ions; (xxii) an ion-metastable ion reaction device for
reacting ions to form adduct or product ions; (xxiii) an ion-
metastable molecule reaction device for reacting ions to form adduct
or product ions; and (xxiv) an ion-metastable atom reaction device
for reacting ions to form adduct or product ions.
The mass spectrometer preferably further comprises an ion
source. The ion source is preferably selected from the group
CA 02835314 2013-11-28
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consisting of: (i) an Electrospray ionisation ("ESI") ion source;
(ii) an Atmospheric Pressure Photo Ionisation ("APPI") ion source;
(iii) an Atmospheric Pressure Chemical Ionisation ("APCI") ion
source; (iv) a Matrix Assisted Laser Desorption Ionisation ("MALDIn)
ion source; (v) a Laser Desorption Ionisation ("LDI") ion source;
(vi) an Atmospheric Pressure Ionisation ("API") ion source; (vii) a
Desorption Ionisation on Silicon ("DIOS") ion source; (viii) an
Electron Impact ("El") ion source; (ix) a Chemical Ionisation ("CI")
ion source; (x) a Field Ionisation ("Fl") ion source; (xi) a Field
Desorption ("FD") ion source; (xii) an Inductively Coupled Plasma
("ICP") ion source; (xiii) a Fast Atom Bombardment ("FAB") ion
source; (xiv) a Liquid Secondary Ion Mass Spectrometry (nLSIMS") ion
source; (xv) a Desorption Electrospray Ionisation ("DESI") ion
source; (xvi) a Nickel-63 radioactive ion source; (xvii) an
Atmospheric Pressure Matrix Assisted Laser Desorption Ionisation ion
source; and (xviii) a Thermospray ion source.
The ion source may comprise a pulsed or a continuous ion
source.
According to a particularly preferred embodiment the ion source
may comprise an Electrospray, Atmospheric Pressure Chemical
Ionisation ("APCI"), Atmospheric Pressure Photo Ionisation ("APPI"),
Matrix Assisted Laser Desorption Ionisation (nMALDI"), Laser
Desorption Ionisation ("LDI"), Inductively Coupled Plasma ("ICP"),
Fast Atom Bombardment ("FAB") or Liquid Secondary Ions Mass
Spectrometry ("LSIMP) ion source. Such ion sources may be provided
with an eluent over a period of time, the eluent having been
separated from a mixture by means of liquid chromatography or
capillary electrophoresis.
Alternatively, the ion source may comprise an Electron Impact
("El"), Chemical Ionisation ("CI") or Field Ionisation ("Fab) ion
source. Such ion sources may be provided with an eluent over a
period of time, the eluent having been separated from a mixture by
means of gas chromatography.
The mass analyser may comprise a quadrupole mass filter, a Time
of Flight ("TOF") mass analyser (an orthogonal acceleration Time of
CA 02835314 2013-11-28
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Flight mass analyser is particularly preferred), a 2D (linear) or 3D
(doughnut shaped electrode with two endcap electrodes) ion trap, a
magnetic sector analyser or a Fourier Transform Ion Cyclotron
Resonance ("FTICR") mass analyser.
The mass analyser is preferably selected from the group
consisting of: (i) a quadrupole mass analyser; (ii) a 2D or linear
quadrupole mass analyser; (iii) a Paul or 3D quadrupole mass
analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass
analyser; (vi) a magnetic sector mass analyser; (vii) Ion Cyclotron
Resonance ("ICR") mass analyser; (viii) a Fourier Transform Ion
Cyclotron Resonance ("FTICR") mass analyser; (ix) an electrostatic or
orbitrap mass analyser; (x) a Fourier Transform electrostatic or
orbitrap mass analyser; and (xi) a Fourier Transform mass analyser;
(xii) a Time of Flight mass analyser; (xiii) an orthogonal
acceleration Time of Flight mass analyser; (xiv) an axial
acceleration Time of Flight mass analyser; and (xv) a quadrupole rod
set mass filter or mass analyser.
The mass spectrometer may further comprise an ion trap or ion
guide arranged upstream and/or downstream of the collision,
fragmentation or reaction device. The ion trap or ion guide is
preferably selected from the group consisting of:
(i) a multipole rod set or a segmented multipole rod set ion
trap or ion guide comprising a quadrupole rod set, a hexapole rod
set, an octapole rod set or a rod set comprising more than eight
rods;
(ii) an ion tunnel or ion funnel ion trap or ion guide
comprising a plurality of electrodes or at least 2, 5, 10, 20, 30,
40, 50, 60, 70, 80, 90 or 100 electrodes having apertures through
which ions are transmitted in use, wherein at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 100% of the electrodes have apertures which are of
substantially the same size or area or which have apertures which
become progressively larger and/or smaller in size or in area;
(iii) a stack or array of planar, plate or mesh electrodes,
wherein the stack or array of planar, plate or mesh electrodes
CA 02835314 2013-11-28
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comprises a plurality or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20 planar, plate or mesh electrodes and
wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the planar,
plate or mesh electrodes are arranged generally in the plane in which
ions travel in use; and
(iv) an ion trap or ion guide comprising a plurality of groups
of electrodes arranged axially along the length of the ion trap or
ion guide, wherein each group of electrodes comprises: (a) a first
and a second electrode and means for applying a DC voltage or
potential to the first and second electrodes in order to confine ions
in a first radial direction within the ion guide; and (b) a third and
a fourth electrode and means for applying an AC or RF voltage to the
third and fourth electrodes in order to confine ions in a second
radial direction within the ion guide.
The ion trap or ion guide preferably comprises an ion tunnel or
ion funnel ion trap or ion guide wherein at least 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 100% of the electrodes have internal diameters or dimensions
selected from the group consisting of: (i) 5 1.0 mm; (ii) 5 2.0 mm;
(iii) 5 3.0 mm; (iv) 5 4.0 mm; (v) 5 5.0 mm; (vi) 5 6.0 mm; (vii) 5
7.0 mm; (viii) 5 8.0 mm; (ix) 5 9.0 mm; (x) 5 10.0 mm; and (xi) >
10.0 mm.
The ion trap or ion guide preferably further comprises first AC
or RF voltage means arranged and adapted to apply an AC or RF voltage
to at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the plurality of
electrodes of the ion trap or ion guide in order to confine ions
radially within the ion trap or ion guide.
The first AC or RF voltage means is preferably arranged and
adapted to apply an AC or RF voltage having an amplitude selected
from the group consisting of: (i) < 50 V peak to peak; (ii) 50-100 V
peak to peak; (iii) 100-150 V peak to peak; (iv) 150-200 V peak to
peak; (v) 200-250 V peak to peak; (vi) 250-300 V peak to peak; (vii)
300-350 V peak to peak; (viii) 350-400 V peak to peak; (ix) 400-450 V
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peak to peak; (x) 450-500 V peak to peak; and (xi) > 500 V peak to
peak.
The first AC or RF voltage means is preferably arranged and
adapted to apply an AC or RP voltage having a frequency selected from
8,5 MHz; (xxii) 8.5-9.0 MHz; (xxiii) 9.0-9.5 MHz; (xxiv) 9.5-10.0
MHz; and (xxv) > 10.0 MHz.
The ion trap or ion guide is preferably arranged and adapted to
receive a beam or group of ions and to convert or partition the beam
15 or group of ions such that a plurality or at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate
packets of ions are confined and/or isolated in the ion trap or ion
guide at any particular time, and wherein each packet of ions is
separately confined and/or isolated in a separate axial potential
20 well formed within the ion trap or ion guide.
The mass spectrometer preferably further comprises means
arranged and adapted to urge at least some ions upstream and/or
downstream through or along at least 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
25 100% of the axial length of the ion trap or ion guide in a mode of
operation.
The mass spectrometer preferably further comprises first
transient DC voltage means arranged and adapted to apply one or more
transient DC voltages or potentials or one or more transient DC
30 voltage or potential waveforms to the electrodes forming the ion trap
or ion guide in order to urge at least some ions upstream and/or
downstream along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial
length of the ion trap or ion guide.
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The mass spectrometer preferably further comprises AC or RF
voltage means arranged and adapted to apply two or more phase-shifted
AC or RF voltages to electrodes forming the ion trap or ion guide in
order to urge at least some ions upstream and/or downstream along at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the ion
trap or ion guide.
The collision, fragmentation or reaction device may comprise a
quadrupole rod set, an hexapole rod set, an octopole or higher order
rod set or an ion tunnel comprising a plurality of electrodes having
apertures through which ions are transmitted. The apertures are
preferably substantially the same size. The collision, fragmentation
or reaction device may, more generally, comprise a plurality of
electrodes connected to an AC or RF voltage supply for radially
confining ions within the collision, fragmentation or reaction
device. An axial DC voltage gradient may or may not be applied along
at least a portion of the length of the ion tunnel collision,
fragmentation or reaction device. The collision, fragmentation or
reaction device may be housed in a housing or otherwise arranged so
that a substantially gas-tight enclosure is formed around the
collision, fragmentation or reaction device apart from an aperture to
admit ions and an aperture for ions to exit from and optionally a
port for introducing a gas. A gas such as helium, argon, nitrogen,
air or methane may be introduced into the collision, fragmentation or
reaction device.
Other arrangements are also contemplated wherein the collision,
fragmentation or reaction device is not repeatedly switched, altered
or varied between a high fragmentation or reaction mode and a low
fragmentation or reaction mode. For example, the collision,
fragmentation or reaction device may be left permanently ON and
arranged to fragment or react ions received within the collision,
fragmentation or reaction device. An electrode or other device may
be provided upstream of the collision, fragmentation or reaction
device. A high fragmentation or reaction mode of operation would
occur when the electrode or other device allowed ions to pass to the
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collision, fragmentation or reaction device. A low fragmentation or
reaction mode of operation would occur when the electrode or other
device caused ions to by-pass the collision, fragmentation or
reaction device and hence not be fragmented therein.
Other embodiments are also contemplated which would be useful
where particular parent or precursor ions could not be easily
observed since they co-eluted with other commonly observed peptide
ions. In such circumstances the expression level of fragment,
product, daughter or adduct ions is compared between two samples.
According to the preferred embodiment instead of comparing the
expression levels of parent or precursor ions in two different
samples and seeing whether the expression levels are significantly
different so as to warrant further investigation, an initial
recognition is preferably made that parent or precursor ions of
interest are present in a sample.
According to a preferred embodiment, the step of recognising
first parent or precursor ions of interest comprises recognising
first fragment, product, daughter or adduct ions of interest.
The first fragment, product, daughter or adduct ions of
interest may be optionally identified by, for example, determining
their mass to charge ratio preferably to less than or equal to 20
ppm, 15 ppm, 10 ppm or 5 ppm.
Having recognised and optionally identified fragment, product,
daughter or adduct ions of interest, it is then necessary to
determine which parent or precursor ion gave rise to that fragment,
product, daughter or adduct ion.
The step of recognising first parent or precursor ions of
interest may comprise determining whether parent or precursor ions
are observed in a mass spectrum obtained when the collision,
fragmentation or reaction device is in a low fragmentation or
reaction mode for a certain time period and first fragment, product,
daughter or adduct ions of interest are observed in a mass spectrum
obtained either immediately before the certain time period, when the
collision, fragmentation or reaction device is in a high
fragmentation or reaction mode, or immediately after the certain time
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period, when the collision, fragmentation or reaction device is in a
high fragmentation or reaction mode.
The step of recognising first parent or precursor ions of
interest may comprise comparing the elution times of parent or
precursor ions with the pseudo-elution time of first fragment,
product, daughter or adduct ions of interest. The step of
recognising first parent or precursor ions of interest may also
comprise comparing the elution profiles of parent or precursor ions
with the pseudo-elution profile of first fragment, product, daughter
or adduct ions of interest.
According to another less preferred embodiment, parent or
precursor ions of interest may be recognised immediately by virtue of
their mass to charge ratio without it being necessary to recognise
and identify fragment, product, daughter or adduct ions of interest.
According to this embodiment the step of recognising first parent or
precursor ions of interest preferably comprises determining the mass
to charge ratio of the parent or precursor ions preferably to less
than or equal to 20 ppm, 15 ppm, 10 ppm or 5 ppm. The determined
mass to charge ratio of the parent or precursor ions may then be
compared with a database of ions and their corresponding mass to
charge ratios.
According to another embodiment, the step of recognising first
parent or precursor ions of interest comprises determining whether
parent or precursor ions give rise to fragment, product, daughter or
adduct ions as a result of the loss of a predetermined ion or a
predetermined neutral particle.
Parent ions of interest may be identified in a similar manner
to the preferred embodiment.
The other preferred features of the preferred embodiment apply
equally to the other arrangement.
It will be apparent that the above described embodiments which
relate to recognising parent or precursor ions of interest and
comparing the expression level of parent or precursor ions of
interest in one sample with corresponding parent or precursor ions in
another sample may employ the method and apparatus relating to the
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preferred embodiment. Therefore, the same preferred features which
are recited with respect to the preferred embodiment may also be used
with the embodiments which relate to recognising parent or precursor
ions of interest and then comparing the expression level of the
parent or precursor ions of interest in one sample with corresponding
parent or precursor ions in another sample.
If parent or precursor ions having a particular mass to charge
ratio are expressed differently in two different samples, then
according to the preferred embodiment further investigation of the
parent or precursor ions of interest then occurs. This further
investigation may comprise seeking to identify the parent or
precursor ions of interest which are expressed differently in the two
different samples. In order to verify that the parent or precursor
ions whose expression levels are being compared in the two different
samples really are the same ions, a number of checks may be made.
Measurements of changes in the abundance of proteins in complex
protein mixtures can be extremely informative. For example, changes
to the abundance of proteins in cells, often referred to as the
protein expression level, could be due to different cellular
stresses, the effect of stimuli, the effect of disease or the effect
of drugs. Such proteins may provide relevant targets for study,
screening or intervention. The identification of such proteins will
normally be of interest. Such proteins may be identified by the
method of the preferred embodiment.
Therefore, according to the preferred embodiment a new
criterion for the discovery of parent or precursor ions of interest
is based on the quantification of proteins in two different samples.
This requires the determination of the relative abundances of their
peptide products in two or more samples. However, the determination
of relative abundance requires that the same peptide ions must be
compared in the two (or more) different samples and ensuring that
this happens is a non-trivial problem. Hence, it is necessary to be
able to recognise and preferably identify the peptide ion to the
extent that it can at least be uniquely recognised within the sample.
Such peptide ions may be adequately recognised by measurement of the
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mass of the parent or precursor ion and by measurement of the mass to
charge ratio of one or more fragment, product, daughter or adduct
ions derived from that parent or precursor ion. The specificity with
which the peptides may be recognised may be increased by the
determination of the accurate mass of the parent or precursor ion
and/or the accurate mass of one or more fragment, product, daughter
or adduct ions.
The same method of recognising parent or precursor ions in one
sample is also preferably used to recognise the same parent or
precursor ions in another sample and this enables the relative
abundances of the parent or precursor ions in the two different
samples to be measured.
Measurement of relative abundances allows discovery of proteins
with a significant change or difference in expression level of that
protein. The same data allows identification of that protein by the
method already described in which several or all fragment, product,
daughter or adduct ions associated with each such peptide product ion
is discovered by closeness of fit of their respective elution times.
Again, the accurate measurement of the masses of the parent or
precursor ion and associated fragment, product, daughter or adduct
ions substantially improves the specificity and confidence with which
the protein may be identified.
The specificity with which the peptides may be recognised may
also be increased by comparison of retention times. For example, the
HPLC or CE retention or elution times will be measured as part of the
procedure for associating fragment, product, daughter or adduct ions
with parent or precursor ions, and these elution times may also be
compared for the two or more samples. The elution times may be used
to reject measurements where they do not fall within a pre-defined
time difference of each other. Alternatively, retention times may be
used to confirm recognition of the same peptide when they do fall
within a predefined window of each other. Commonly there may be some
redundancy if the parent or precursor ion accurate mass, one or more
fragment, product, daughter or adduct ion accurate masses, and the
retention times are all measured and compared. In many instances
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just two of these measurements will be adequate to recognise the same
peptide parent or precursor ion in the two or more samples. For
example, measurement of just the accurate parent or precursor ion
mass to charge ratio and a fragment, product, daughter or adduct ion
mass to charge ratio, or the accurate parent or precursor ion mass to
charge ratio and the retention time, may well be adequate.
Nevertheless, the additional measurements may be used to confirm the
recognition of the same parent peptide ion.
The relative expression levels of the matched parent peptide
ions may be quantified by measuring the peak areas relative to an
internal standard.
The preferred embodiment does not require any interruption to
the acquisition of data and hence is particularly suitable for
quantitative applications. According to an embodiment one or more
endogenous peptides common to both mixtures which are not changed by
the experimental state of the samples may used as an internal
standard or standards for the relative peak area measurements.
According to another embodiment an internal standard may be added to
each sample where no such internal standard is present or can be
relied upon. The internal standard, whether naturally present or
added, may also serve as a chromatographic retention time standard as
well as a mass accuracy standard.
Ideally more than one peptide parent or precursor ion may be
measured for each protein to be quantified. For each peptide the
same means of recognition is preferably used when comparing
intensities in each of the different samples. The measurements of
different peptides serves to validate the relative abundance
measurements. Furthermore, the measurements from several peptides
provides a means of determining the average relative abundance, and
of determining the relative significance of the measurements.
According to one embodiment all parent or precursor ions may be
identified and their relative abundances determined by comparison of
their intensities to those of the same identity in one or more other
samples.
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In another embodiment the relative abundance of all parent or
precursor ions of interest, discovered on the basis of their
relationship to a predetermined fragment, product, daughter or adduct
ion, may be determined by comparison of their intensities to those of
the same identity in one or more other samples.
In another embodiment the relative abundance of all parent or
precursor ions of interest, discovered on the basis of their giving
rise to a predetermined mass loss, may be determined by comparison of
their intensities to those of the same identity in one or more other
samples.
In another embodiment it may be merely required to quantify a
protein already identified. The protein may be in a complex mixture,
and the same means for separation and recognition may be used as that
already described. Here it is only necessary to recognise the
relevant peptide product or products and measure their intensities in
one or more samples. The basis for recognition may be that of the
peptide parent or precursor ion mass or accurate mass, and that of
one or more fragment, product, daughter or adduct ion masses, or
accurate masses. Their retention times may also be compared thereby
providing a means of confirming the recognition of the same peptide
or of rejecting unmatched peptides.
The preferred embodiment is applicable to the study of
proteomics. However, the same methods of identification and
quantification may be used in other areas of analysis such as the
study of metabolomics.
The method is appropriate for the analysis of mixtures where
different components of the mixture are first separated or partially
separated by a means such as chromatography that causes components to
elute sequentially.
The source of ions may preferably yield mainly molecular ions
or pseudo-molecular ions and relatively few (if any) fragment,
product, daughter or adduct ions. Examples of such sources include
atmospheric pressure ionisation sources (e.g. Electrospray and APCI)
and Matrix Assisted Laser Desorption Ionisation (MALDI).
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If the two main operating modes of the collision, fragmentation
or reaction device are suitably set, then parent or precursor ions
can be recognised by virtue of the fact that they will be relatively
more intense in the mass spectrum without substantial fragmentation
or reaction. Similarly, fragment, product, daughter or adduct ions
can be recognised by virtue of the fact that they will be relatively
more intense in the mass spectrum with substantial fragmentation or
reaction.
The mass analyser may comprise a quadrupole, Time of Flight,
ion trap, magnetic sector or FT-ICR mass analyser. According to a
preferred embodiment the mass analyser should be capable of
determining the exact or accurate mass to charge value for ions.
This is to maximise selectivity for detection of characteristic
fragment, product, daughter or adduct ions or mass losses, and to
maximise specificity for identification of proteins.
The mass analyser preferably samples or records the whole
spectrum simultaneously. This ensures that the elution times
observed for all the masses are not modified or distorted by the mass
analyser, and in turn would allow accurate matching of the elution
times of different masses, such as parent or precursor and fragment,
product, daughter or adduct ions. It also helps to ensure that the
quantitative measurements are not compromised by the need to measure
abundances of transient signals.
A mass filter, preferably a quadrupole mass filter, may be
provided upstream of the collision, fragmentation or reaction device.
The mass filter may have a highpass filter characteristic and, for
example, be arranged to transmit ions having a mass to charge ratio
greater than or equal to 100, 150, 200, 250, 300, 350, 400, 450 or
500. Alternatively, the mass filter may have a lowpass or bandpass
filter characteristic.
An ion guide may be provided upstream of the collision,
fragmentation or reaction device. The ion guide may comprise either
a hexapole, quadrupole, octopole or higher order multipole rod set.
In another embodiment the ion guide may comprise an ion tunnel ion
guide comprising a plurality of electrodes having apertures through
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which ions are transmitted in use. Preferably, at least 90% of the
electrodes have apertures which are substantially the same size.
Alternatively, the ion guide may comprise a plurality of ring
electrodes having substantially tapering internal diameters ("ion
funnel").
Parent ions that belong to a particular class of parent or
precursor ions, and which are recognisable by a characteristic
fragment, product, daughter or adduct ion or characteristic neutral
loss are traditionally discovered by the methods of parent or
precursor ion scanning or constant neutral loss scanning. Previous
methods for recording parent or precursor ion scans or constant
neutral loss scans involve scanning one or both quadrupoles in a
triple quadrupole mass spectrometer, or scanning the quadrupole in a
tandem quadrupole orthogonal TOF mass spectrometer, or scanning at
least one element in other types of tandem mass spectrometers. As a
consequence, these methods suffer from the low duty cycle associated
with scanning instruments. As a further consequence, information may
be discarded and lost whilst the mass spectrometer is occupied
recording a parent or precursor ion scan or a constant neutral loss
scan. As a further consequence these methods are not appropriate for
use where the mass spectrometer is required to analyse substances
eluting directly from gas or liquid chromatography equipment.
According to the preferred embodiment, a tandem quadrupole
orthogonal Time of Flight mass spectrometer in used in a way in which
parent or precursor ions of interest are discovered using a method in
which sequential low and high collision energy mass spectra are
recorded. The switching, altering or varying back and forth is
preferably not interrupted. Instead a complete set of data is
acquired, and this is then processed afterwards. Fragment, product,
daughter or adduct ions may be associated with parent or precursor
ions by closeness of fit of their respective elution times. In this
way parent or precursor ions of interest may be confirmed or
otherwise without interrupting the acquisition of data, and
information need not be lost.
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According to one embodiment, possible parent or precursor ions
of interest may be selected on the basis of their relationship to a
predetermined fragment, product, daughter or adduct ion. The
predetermined fragment, product, daughter or adduct ion may comprise,
for example, immonium ions from peptides, functional groups including
phosphate group P03- ions from phosphorylated peptides or mass tags
which are intended to cleave from a specific molecule or class of
molecule and to be subsequently identified thus reporting the
presence of the specific molecule or class of molecule. A parent or
precursor ion may be short listed as a possible parent or precursor
ion of interest by generating a mass chromatogram for the
predetermined fragment, product, daughter or adduct ion using high
fragmentation or reaction mass spectra. The centre of each peak in
the mass chromatogram is then determined together with the
corresponding predetermined fragment, product, daughter or adduct ion
elution time(s). Then for each peak in the predetermined fragment,
product, daughter or adduct ion mass chromatogram both the low
fragmentation or reaction mass spectrum obtained immediately before
the predetermined fragment, product, daughter or adduct ion elution
time and the low fragmentation or reaction mass spectrum obtained
immediately after the predetermined fragment, product, daughter or
adduct ion elution time are interrogated for the presence of
previously recognised parent or precursor ions. A mass chromatogram
for any previously recognised parent or precursor ion found to be
present in both the low fragmentation or reaction mass spectrum
obtained immediately before the predetermined fragment, product,
daughter or adduct ion elution time and the low fragmentation or
reaction mass spectrum obtained immediately after the predetermined
fragment, product, daughter or adduct ion elution time is then
generated and the centre of each peak in each mass chromatogram is
determined together with the corresponding possible parent or
precursor ion of interest elution time(s). The possible parent or
precursor ions of interest may then be ranked according to the
closeness of fit of their elution time with the predetermined
fragment, product, daughter or adduct ion elution time, and a list of
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final possible parent or precursor ions of interest may be formed by
rejecting possible parent or precursor ions of interest if their
elution time precedes or exceeds the predetermined fragment, product,
daughter or adduct ion elution time by more than a predetermined
amount.
According to an alternative embodiment, a parent or precursor
ion may be shortlisted as a possible parent or precursor ion of
interest on the basis of it giving rise to a predetermined mass loss.
For each low fragmentation or reaction mass spectrum, a list of
target fragment, product, daughter or adduct ion mass to charge
values that would result from the loss of a predetermined ion or
neutral particle from each previously recognised parent or precursor
ion present in the low fragmentation or reaction mass spectrum is
generated. Then both the high fragmentation or reaction mass
spectrum obtained immediately before the low fragmentation or
reaction mass spectrum and the high fragmentation or reaction mass
spectrum obtained immediately after the low fragmentation or reaction
mass spectrum are interrogated for the presence of fragment, product,
daughter or adduct ions having a mass to charge value corresponding
with a target fragment, product, daughter or adduct ion mass to
charge value. A list of possible parent or precursor ions of
interest (optionally including their corresponding fragment, product,
daughter or adduct ions) is then formed by including in the list a
parent or precursor ion if a fragment, product, daughter or adduct
ion having a mass to charge value corresponding with a target
fragment, product, daughter or adduct ion mass to charge value is
found to be present in both the high fragmentation or reaction mass
spectrum immediately before the low fragmentation or reaction mass
spectrum and the high fragmentation or reaction mass spectrum
immediately after the low fragmentation or reaction mass spectrum. A
mass loss chromatogram may then be generated based upon possible
candidate parent or precursor ions and their corresponding fragment,
product, daughter or adduct ions. The centre of each peak in the
mass loss chromatogram is determined together with the corresponding
mass loss elution time(s). Then for each possible candidate parent
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or precursor ion a mass chromatogram is generated using the low
fragmentation or reaction mass spectra. A corresponding fragment,
product, daughter or adduct ion mass chromatogram is also generated
for the corresponding fragment, product, daughter or adduct ion. The
centre of each peak in the possible candidate parent or precursor ion
mass chromatogram and the corresponding fragment, product, daughter
or adduct ion mass chromatogram are then determined together with the
corresponding possible candidate parent or precursor ion elution
time(s) and corresponding fragment, product, daughter or adduct ion
elution time(s). A list of final candidate parent or precursor ions
may then be formed by rejecting possible candidate parent or
precursor ions if the elution time of a possible candidate parent or
precursor ion precedes or exceeds the corresponding fragment,
product, daughter or adduct ion elution time by more than a
predetermined amount.
Once a list of parent or precursor ions of interest has been
formed (which preferably comprises only some of the originally
recognised parent or precursor ions and possible parent or precursor
ions of interest) then each parent or precursor ion of interest can
then be identified.
Identification of parent or precursor ions may be achieved by
making use of a combination of information. This may include the
accurately determined mass or mass to charge ratio of the parent or
precursor ion. It may also include the masses or mass to charge
ratios of the fragment, product, daughter or adduct ions. In some
instances the accurately determined masses or mass to charge ratios
of the fragment, product, daughter or adduct ions may be preferred.
It is known that a protein may be identified from the masses or mass
to charge ratios, preferably the exact masses or mass to charge
ratios, of the peptide products from proteins that have been
enzymatically digested. These may be compared to those expected from
a library of known proteins. It is also known that when the results
of this comparison suggest more than one possible protein then the
ambiguity can be resolved by analysis of the fragments of one or more
of the peptides. The preferred embodiment allows a mixture of
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proteins, which have been enzymatically digested, to be identified in
a single analysis. The masses or mass to charge ratios, or exact
masses or mass to charge ratios, of all the peptides and 'their
associated fragment, product, daughter or adduct ions may be searched
against a library of known proteins. Alternatively, the peptide
masses or mass to charge ratios, or exact masses or mass to charge
ratios, may be searched against the library of known proteins, and
where more than one protein is suggested the correct protein may be
confirmed by searching for fragment, product, daughter or adduct ions
which match those to be expected from the relevant peptides from each
candidate protein.
The step of identifying each parent or precursor ion of
interest preferably comprises recalling the elution time of the
parent or precursor ion of interest, generating a list of possible
fragment, product, daughter or adduct ions which comprises previously
recognised fragment, product, daughter or adduct ions which are
present in both the low fragmentation or reaction mass spectrum
obtained immediately before the elution time of the parent or
precursor ion of interest and the low fragmentation or reaction mass
spectrum obtained immediately after the elution time of the parent or
precursor ion of interest, generating a mass chromatogram of each
possible fragment, product, daughter or adduct ion, determining the
centre of each peak in each possible fragment, product, daughter or
adduct ion mass chromatogram, and determining the corresponding
possible fragment, product, daughter or adduct ion elution time(s).
The possible fragment, product, daughter or adduct ions may then be
ranked according to the closeness of fit of their elution time with
the elution time of the parent or precursor ion of interest. A list
of fragment, product, daughter or adduct ions may then be formed by
rejecting fragment, product, daughter or adduct ions if the elution
time of the fragment, product, daughter or adduct ion precedes or
exceeds the elution time of the parent or precursor ion of interest
by more than a predetermined amount.
The list of fragment, product, daughter or adduct ions may be
yet further refined or reduced by generating a list of neighbouring
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parent or precursor ions which are present in the low fragmentation
or reaction mass spectrum obtained nearest in time to the elution
time of the final candidate parent or precursor ion. A mass
chromatogram of each parent or precursor ion contained in the list is
then generated and the centre of each mass chromatogram is determined
along with the corresponding neighbouring parent or precursor ion
elution time(s). Any fragment, product, daughter or adduct ion
having an elution time which corresponds more closely with a
neighbouring parent or precursor ion elution time than with the
elution time of a parent or precursor ion of interest may then be
rejected from the list of fragment, product, daughter or adduct ions.
Fragment, daughter, product or adduct ions may be assigned to a
parent or precursor ion according to the closeness of fit of their
elution times, and all fragment, product, daughter or adduct ions
which have been associated with the parent or precursor ion may be
listed.
An alternative embodiment which involves a greater amount of
data processing but yet which is intrinsically simpler is also
contemplated. Once parent and fragment, product, daughter or adduct
ions have been identified, then a parent or precursor ion mass
chromatogram for each recognised parent or precursor ion is
generated. The centre of each peak in the parent or precursor ion
mass chromatogram and the corresponding parent or precursor ion
elution time(s) are then determined. Similarly, a fragment, product,
daughter or adduct ion mass chromatogram for each recognised
fragment, product, daughter or adduct ion is generated, and the
centre of each peak in the fragment, product, daughter or adduct ion
mass chromatogram and the corresponding fragment, product, daughter
or adduct ion elution time(s) are then determined. Rather than then
identifying only a sub-set of the recognised parent or precursor
ions, all (or nearly all) of the recognised parent or precursor ions
are then identified. Fragment ions are assigned to parent or
precursor ions according to the closeness of fit of their respective
elution times and all fragment, product, daughter or adduct ions
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which have been associated with a parent or precursor ion may then be
listed.
Passing ions through a mass filter, preferably a quadrupole
mass filter, prior to being passed to the collision, fragmentation or
reaction device presents an alternative or an additional method of
recognising a fragment, product, daughter or adduct ion. A fragment,
product, daughter or adduct ion may be recognised by recognising ions
in a high fragmentation or reaction mass spectrum which have a mass
to charge ratio which is not transmitted by the collision,
fragmentation or reaction device i.e. fragment, product, daughter or
adduct ions are recognised by virtue of their having a mass to charge
ratio falling outside of the transmission window of the mass filter.
If the ions would not be transmitted by the mass filter then they
must have been produced in the collision, fragmentation or reaction
device.
Various embodiments of the present invention will now be described,
by way of example only, and with reference to the accompanying
drawings in which:
Fig. 1 is a schematic drawing of a preferred mass spectrometer;
Fig. 2 shows a schematic of a valve switching arrangement
during sample loading and desalting and the inset shows desorption of
a sample from an analytical column;
Fig. 3A shows a fragment or daughter ion mass spectrum and Fig.
3B shows the corresponding parent or precursor ion mass spectrum
obtained when a mass filter upstream of a collision cell was arranged
so as to transmit ions having a mass to charge ratio > 350 to the
collision cell;
Fig. 4A shows a mass chromatogram of a parent or precursor ion,
Fig. 4B shows a mass chromatogram of a parent or precursor ion, Fig.
4C shows a mass chromatogram of a parent or precursor ion, Fig. 4D
shows a mass chromatogram of a fragment or daughter ion and Fig. 4E
shows a mass chromatogram of a fragment or daughter;
Fig. 5 shows the mass chromatograms of Figs. 4A-E superimposed
upon one another;
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Fig. 6 shows a mass chromatogram of the Asparagine immonium ion
which has a mass to charge ratio of 87.04;
Fig. 7 shows a mass spectrum of the peptide ion T5 derived from
ADH which has the sequence ANELLINVK and a molecular weight of
1012.59;
Fig. 8 shows a mass spectrum of a tryptic digest of 13-Casein
obtained when a collision cell was in a low fragmentation mode;
Fig. 9 shows a mass spectrum of a tryptic digest of 13-Casein
obtained when a collision cell was in a high fragmentation mode;
Fig. 10 shows a processed and expanded view of the mass
spectrum shown in Fig. 9;
Fig. 11A shows a mass chromatogram of an ion from a first
sample having a mass to charge ratio of 880.4, Fig. 11B shows a
similar mass chromatogram of the same ion from a second sample, Fig.
11C shows a mass chromatogram of an ion from a first sample having a
mass to charge ratio of 5,82.3 and Fig. 11D shows a similar mass
chromatogram of the same ion from a second sample;
Fig. 12A shows a mass spectrum recorded from a first sample and
Fig. 12B shows a corresponding mass spectrum recorded from a second
sample which is similar to the first sample except that it contains a
higher concentration of the digest products of the protein Casein
which is common to both samples;
Fig. 13 shows the mass spectrum shown in Fig. 12A in more
detail and the insert shows an expanded part of the mass spectrum
showing isotope peaks at mass to charge ratio 880.4; and
Fig. 14 shows the mass spectrum shown in Fig. 12B in more
detail and the insert shows an expanded part of the mass spectrum
showing isotope peaks at mass to charge ratio 880.4.
A preferred embodiment will now be described with reference to
Fig. 1. A mass spectrometer 6 is shown which comprises an ion source
1, preferably an Electrospray Ionisation source, an ion guide 2
arranged downstream of the ion source 1, a quadrupole mass filter 3,
a collision, fragmentation or reaction device 4 and an orthogonal
acceleration Time of Flight mass analyser 5 incorporating a
reflectron. The ion guide 2 and mass filter 3 may be omitted if
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necessary. The mass spectrometer 6 is preferably interfaced with a
chromatograph, such as a liquid chromatograph (not shown) so that the
sample entering the ion source 1 may be taken from the eluent of the
liquid chromatograph.
The quadrupole mass filter 3 is preferably disposed in an
evacuated chamber which is maintained at a relatively low pressure
e.g. less than 10-5 mbar. The rod electrodes comprising the mass
filter .3 are preferably connected to a power supply which generates
both RF and DC potentials which determine the mass to charge value
transmission window of the mass filter 3.
The collision, fragmentation or reaction device 4 preferably
comprises a Surface Induced Dissociation ("SID") fragmentation
device, an Electron Transfer Dissociation fragmentation device or an
Electron Capture Dissociation fragmentation device.
According to an embodiment the collision, fragmentation or
reaction device 4 may comprise an Electron Capture Dissociation
fragmentation device. According to this embodiment multiply charged
analyte ions are preferably caused to interact with relatively low
energy electrons. The electrons preferably have energies of < 1 eV
or 1-2 eV. The electrons are preferably confined by a relatively
strong magnetic field and are directed so that the electrons collide
with the analyte ions which are preferably confined within an RF ion
guide which is preferably arranged within the fragmentation device 4.
An AC or RF voltage is preferably applied to the electrodes of the RF
ion guide so that a radial pseudo-potential well is preferably
created which preferably acts to confine ions radially within the ion
guide so that the ions can interact with the low energy electrons.
According to another embodiment the collision, fragmentation or
reaction device 4 may comprise an Electron Transfer Dissociation
fragmentation device. According to this embodiment positively
charged analyte ions are preferably caused to interact with
negatively charged reagent ions. The negatively charged reagent ions
are preferably injected into an RF ion guide or ion trap located
within the collision, fragmentation or reaction device 4. An AC or
RF voltage is preferably applied to the electrodes of the RF ion
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guide so that a radial pseudo-potential well is preferably created
which preferably acts to confine ions radially within the ion guide
so that the ions can interact with the negatively charged reagent
ions. According to a less preferred embodiment negatively charged
analyte ions may alternatively be arranged to interact with
positively charged reagent ions.
According to another embodiment the collision, fragmentation or
reaction device 4 may comprise a Surface Induced Dissociation
fragmentation device. According to this embodiment ions are
preferably directed towards a surface or target plate with a
relatively low energy. The ions may, for example, be arranged to
have an energy of 1-10 eV. The surface or target plate may comprise
stainless steel or more preferably the surface or target plate may
comprise a metallic plate coated with a monolayer of fluorocarbon or
hydrocarbon. The monolayer preferably comprises a self-assembled
monolayer. The surface or target plate may be arranged in a plane
which is substantially parallel with the direction of travel of ions
through the Surface Induced Dissociation fragmentation device in a
mode of operation wherein ions are not fragmented. In a mode of
operation wherein it is desired to fragment ions, the ions may be
deflected onto or towards the surface or target plate so that the
ions impinge the surface or target plate at a relatively shallow
angle with respect to the surface of target plate. Fragment ions are
preferably produced as a result of the analyte ions colliding with
the surface or target plate. The fragment ions are preferably
directed off or away from the surface or target plate at a relatively
shallow angle with respect to the surface or target plate. The
fragment ions are then preferably arranged to assume a trajectory
which preferably corresponds with the trajectory of ions which are
transmitted through or past the Surface Induced Dissociation
fragmentation device in a mode of operation wherein ions are not
substantially fragmented.
The collision, fragmentation or reaction device 4 may comprise
an Electron Collision or Impact Dissociation fragmentation device
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wherein ions are fragmented upon collisions with relatively energetic
electrons e.g. wherein the electrons have > 5ev.
According to other embodiments the collision, fragmentation or
reaction device 4 may comprise a Photo Induced Dissociation ("PID")
fragmentation device, a Laser Induced Dissociation fragmentation
device, an infrared radiation induced dissociation device, an
ultraviolet radiation induced dissociation device, a thermal or
temperature source fragmentation device, an electric field induced
fragmentation device, a magnetic field induced fragmentation device,
an enzyme digestion or enzyme degradation fragmentation device, an
ion-ion reaction fragmentation device, an ion-molecule reaction
fragmentation device, an ion-atom reaction fragmentation device, an
ion-metastable ion reaction fragmentation device, an ion-metastable
molecule reaction fragmentation device, an ion-metastable atom
reaction fragmentation device, an ion-ion reaction device for
reacting ions to form adduct or product ions, an ion-molecule
reaction device for reacting ions to form adduct or product ions, an
ion-atom reaction device for reacting ions to form adduct or product
ions, an ion-metastable ion reaction device for reacting ions to form
adduct or product ions, an ion-metastable molecule reaction device
for reacting ions to form adduct or product ions or an ion-metastable
atom reaction device for reacting ions to form adduct or product
ions.
According to an embodiment the collision, fragmentation or
reaction device may form part of the ion source 1. For example, the
collision, fragmentation or reaction device may comprise a nozzle-
skimmer interface fragmentation device, an in-source fragmentation
device or an ion-source Collision Induced Dissociation fragmentation
device.
The collision, fragmentation or reaction device 4 may comprise
either a quadrupole or hexapole rod set which may be enclosed in a
substantially gas-tight casing (other than having a small ion
entrance and exit orifice) into which a gas such as helium, argon,
nitrogen, air or methane may be introduced at a pressure of between
10-4 and 10-1 mbar, further preferably 10-1 mbar to 10-2 mbar.
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Suitable AC or RF potentials for the electrodes comprising the
collision, fragmentation or reaction device 4 are provided by a power
supply (not shown).
Ions generated by the ion source 1 are transmitted by the ion
guide 2 and pass via an interchamber orifice 7 into vacuum chamber 8.
Ion guide 2 is preferably maintained at a pressure intermediate that
of the ion source 1 and the vacuum chamber 8. In the embodiment
shown, ions may be mass filtered by mass filter 3 before entering the
preferred collision, fragmentation or reaction device 4. However,
the mass filter 3 is an optional feature of this embodiment. Ions
exiting from the collision, fragmentation or reaction device 4 or
which have been transmitted through the collision, fragmentation or
reaction device 4 preferably pass to a mass analyser which preferably
comprises a Time of Flight mass analyser 5. Other ion optical
components, such as further ion guides and/or electrostatic lenses,
may be provided which are not shown in the figures or described
herein. Such components may be used to maximise ion transmission
between various parts or stages of the mass spectrometer. Various
vacuum pumps (not shown) may be provided for maintaining optimal
vacuum conditions. The Time of Flight mass analyser 5 incorporating
a reflectron operates in a known way by measuring the transit time of
the ions comprised in a packet of ions so that their mass to charge
ratios can be determined.
A control means (not shown) preferably provides control signals
for the various power supplies (not shown) which respectively provide
the necessary operating potentials for the ion source 1, the ion
guide 2, the quadrupole mass filter 3, the collision, fragmentation
or reaction device 4 and the Time of Flight mass analyser 5. These
control signals determine the operating parameters of the mass
spectrometer, for example the mass to charge ratios transmitted
through the mass filter 3 and the operation of the analyser 5. The
control means comprise a computer (not shown) which may also be used
to process the mass spectral data acquired. The computer may also
display and store mass spectra produced by the mass analyser 5 and
receive and process commands from an operator. The control means may
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be set to perform automatically various methods and make various
determinations without operator intervention, or may optionally
require operator input at various stages.
The control means is preferably arranged to switch, vary or
alter the collision, fragmentation or reaction device 4 back and
forth between at least two different modes. If the collision,
fragmentation or reaction device 4 comprises an Electron Capture
Dissociation fragmentation device then the electron source or beam
may be switched ON in a first mode of operation and may be switched
OFF in a second mode of operation. If the collision, fragmentation
or reaction device 4 comprises an Electron Transfer Dissociation
fragmentation device 4 then reagent ions may be injected into an ion
guide or ion trap comprising analyte ions in a first mode of
operation and substantially no reagent ions may be injected into the
ion guide or ion trap in a second mode of operation. If the
collision, fragmentation or reaction device 4 comprises a Surface
Induced Dissociation fragmentation device then the analyte ions may
be directed so that they collide or impinge upon the surface or
target plate in a first mode of operation and the analyte ions may be
directed straight past the surface or target plate in a second mode
of operation so that the analyte ions do not collide or impinge upon
the surface of target plate.
In one embodiment the control means may switch, alter or vary
between modes approximately every second. When the mass spectrometer
6 is used in conjunction with an ion source 1 being provided with an
eluent separated from a mixture by means of liquid or gas
chromatography, the mass spectrometer 6 may be run for several tens
of minutes over which period of time several hundred high and low
fragmentation or reaction mass spectra may be obtained.
At the end of the experimental run the data which has been
obtained is preferably analysed and parent or precursor ions and
fragment, product, daughter or adduct ions can be recognised on the
basis of the relative intensity of a peak in a mass spectrum obtained
when the collision, fragmentation or reaction device 4 was in one
mode compared with the intensity of the same peak in a mass spectrum
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obtained approximately a second later in time when the collision,
fragmentation or reaction device 4 was in the second mode.
According to an embodiment, mass chromatograms for each parent
and fragment, product, daughter or adduct ion are generated and
fragment, product, daughter or adduct ions are assigned to parent or
precursor ions on the basis of their relative elution times.
An advantage of this method is that since all the data is
acquired and subsequently processed then all fragment, product,
daughter or adduct ions may be associated with a parent or precursor
ion by closeness of fit of their respective elution times. This
allows all the parent or precursor ions to be identified from their
fragment, product, daughter or adduct ions, irrespective of whether
or not they have been discovered by the presence of a characteristic
fragment, product, daughter or adduct ion or characteristic "neutral
loss".
According to another embodiment an attempt maybe made to reduce
the number of parent or precursor ions of interest. A list of
possible (i.e. not yet finalised) parent or precursor ions of
interest may be formed by looking for parent or precursor ions which
may have given rise to a predetermined fragment, product, daughter or
adduct ion of interest e.g. an immonium ion from a peptide.
Alternatively, a search may be made for parent and fragment, product,
daughter or adduct ions wherein the parent or precursor ion could
have fragmented or reacted into a first component comprising a
predetermined ion or neutral particle and a second component
comprising a fragment, product, daughter or adduct ion. various
steps may then be taken to further reduce/refine the list of possible
parent or precursor ions of interest to leave a number of parent or
precursor ions of interest which are then preferably subsequently
identified by comparing elution times of the parent or precursor ions
of interest and fragment, product, daughter or adduct ions. As will
be appreciated, two ions could have similar mass to charge ratios but
different chemical structures and hence would most likely fragment
differently enabling a parent or precursor ion to be identified on
the basis of a fragment, product, daughter or adduct ion.
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A sample introduction system is shown in more detail in Fig. 2.
Samples may be introduced into the mass spectrometer 6 by means of a
Micromass (RTM) modular CapLC system. For example, samples may be
loaded onto a C18 cartridge (0.3 mm x 5 mm) and desalted with 0.1%
HCOOH for 3 minutes at a flow rate of 30pL per minute. A ten port
valve may then switched such that the peptides are eluted onto the
analytical column for separation, see inset of Fig. 2. Flow from two
pumps A and B may be split to produce a flow rate through the column
of approximately 200n1/min.
A preferred analytical column is a PicoFrit (RTM) column packed
with Waters (RTM) Symmetry C18 set up to spray directly into the mass
spectrometer 6. An Electrospray potential (ca. 3kV) may be applied
to the liquid via a low dead volume stainless steel union. A small
amount e.g. 5 psi (34.48 kPa) of nebulising gas may be introduced
around the spray tip to aid the Electrospray process.
Data may be acquired using a mass spectrometer 6 fitted with a
Z-spray (RTM) nanof low Electrospray ion source. The mass
spectrometer may be operated in the positive ion mode with a source
temperature of 80 C and a cone gas flow rate of 401/hr.
The instrument may be calibrated with a multi-point calibration
using selected fragment, product, daughter or adduct ions that
result, for example, from the fragmentation of Glu-fibrinopeptide b.
Data may be processed using the MassLynx (RTM) suite of software.
Switching a Collision Induced Decomposition fragmentation cell
between two different modes of operation is not intended to fall
within the scope of the present invention. However, experimental
results which were obtained according to this method will nonetheless
be presented since they serve to illustrate aspects of the present
invention.
Figs. 3A and 3B show respectively fragment or daughter and
parent or precursor ion spectra of a tryptic digest of alcohol
dehydrogenase (ADH). The fragment or daughter ion spectrum shown in
Fig. 3A was obtained by maintaining a gas collision cell at a
relatively high potential around 30V which resulted in significant
fragmentation of ions passing therethrough. The parent or precursor
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ion spectrum shown in Fig. 3B was obtained at low collision energy
e.g. less than or equal to 5V. The data presented in Fig. 3B was
obtained using a mass filter 3 arranged upstream of the collision
cell and set to transmit ions having a mass to charge value greater
than 350. The mass spectra in this particular example were obtained
from a sample eluting from a liquid chromatograph, and the spectra
were obtained sufficiently rapidly and close together in time so that
they essentially correspond to the same component or components
eluting from the liquid chromatograph.
The mass spectrum shown in Fig. 3A was obtained using a
collision cell to fragment ions by Collision Induced Dissociation.
Such an approach is not intended to fall within the scope of the
present invention. However, the mass spectra which were obtained and
the following description relating to the processing of the mass
spectral data illustrate various aspects of the present invention.
In Fig. 33, there are several high intensity peaks in the
parent or precursor ion spectrum, e.g. the peaks at 418.7724 and
568.7813, which are substantially less intense in the corresponding
fragment or daughter ion spectrum shown in Fig. 3A. These peaks may
therefore be recognised as being parent or precursor ions. Likewise,
ions which are more intense in the fragment or daughter ion spectrum
shown in Fig. 3A than in the parent or precursor ion spectrum shown
in Fig. 3B may be recognised as being fragment or daughter ions. As
will also be apparent, all the ions having a mass to charge value
less than 350 in the high fragmentation mass spectrum shown in Fig.
3A can be readily recognised as being fragment or daughter ions on
the basis that they have a mass to charge value less than 350 and the
fact that only parent or precursor ions having a mass to charge value
greater than 350 were transmitted by the mass filter 5 to the
collision cell.
Figs. 4A-E show respectively mass chromatograms for three
parent or precursor ions and two fragment or daughter ions. The
parent or precursor ions were determined to have mass to charge
ratios of 406.2 (peak "MC1"), 418.7 (peak "MC2") and 568.8 (peak
"MC3") and the two fragment or daughter ions were determined to have
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mass to charge ratios of 136.1 (peaks "MC4" and "MC5") and 120.1
(peak "MC6").
It can be seen that parent or precursor ion peak MC1 (mass to
charge ratio 406.2) correlates well with fragment or daughter ion
peak MC5 (mass to charge ratio 136.1) i.e. a parent or precursor ion
with a mass to charge ratio of 406.2 seems to have fragmented to
produce a fragment or daughter ion with a mass to charge ratio of
136.1. Similarly, parent or precursor ion peaks MC2 and MC3
correlate well with fragment or daughter ion peaks MC4 and MC6, but
it is difficult to determine which parent or precursor ion
corresponds with which fragment or daughter ion.
Fig. 5 shows the peaks of Figs. 4-E overlaid on top of one
other and redrawn at a different scale. By careful comparison of the
peaks of MC2, MC3, MC4 and MC6 it can be seen that in fact parent or
precursor ion MC2 and fragment or daughter ion MC4 correlate well
whereas parent or precursor ion MC3 correlates well with fragment or
daughter ion MCG. This suggests that parent or precursor ions with a
mass to charge ratio of 418.7 fragmented to produce fragment or
daughter ions with a mass to charge ratio of 136.1 and that parent or
precursor ions with mass to charge ratio 568.8 fragmented to produce
fragment or daughter ions with a mass to charge ratio of 120.1.
This cross-correlation of mass chromatograms may be carried out
using automatic peak comparison means such as a suitable peak
comparison software program running on a suitable computer.
Fig. 6 show the mass chromatogram for the fragment or daughter
ion having a mass to charge ratio of 87.04 extracted from a HPLC
separation and mass analysis obtained using mass spectrometer 6. It
is known that the immonium ion for the amino acid Asparagine has a
mass to charge value of 87.04. This chromatogram was extracted from
all the high energy spectra recorded on the mass spectrometer 6.
Fig. 7 shows the full mass spectrum corresponding to scan number 604.
This was a low energy mass spectrum recorded on the mass spectrometer
6, and is the low energy spectrum next to the high energy spectrum at
scan 605 that corresponds to the largest peak in the mass
chromatogram of mass to charge ratio 87.04. This shows that the
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parent or precursor ion for the Asparagine immonium ion at mass to
charge ratio 87.04 has a mass of 1012.54 since it shows the singly
charged (M+H) ion at mass to charge ratio 1013.54, and the doubly
charged (M+2H)+4 ion at mass to charge ratio 507.27.
Fig. 8 shows a mass spectrum from a low energy spectra recorded
on a mass spectrometer 6 of a tryptic digest of the protein 13-Casein.
The protein digest products were separated by HPLC and mass analysed.
The mass spectra were recorded on a mass spectrometer 6 operating in
a MS mode and alternating between low and high collision energy in a
gas collision cell for successive spectra. Fig. 9 shows a mass
spectrum from the high energy spectra recorded at substantially the
same time that the low energy mass spectrum shown in Fig. 8 relates
to. Fig. 10 shows a processed and expanded view of the mass spectrum
shown in Fig. 9 above. For this spectrum, the continuum data has
been processed so as to identify peaks and display them as lines with
heights proportional to the peak area, and annotated with masses
corresponding to their centroided masses. The peak at mass to charge
ratio 1031.4395 is the doubly charged (M+211)." ion of a peptide, and
the peak at mass to charge ratio 982.4515 is a doubly charged
fragment or daughter ion. It has to be a fragment or daughter ion
since it is not present in the low energy spectrum. The mass
difference between these ions is 48.9880. The theoretical mass for
H3PO4 is 97.9769, and the mass to charge value for the doubly charged
H3PO4' ion is 48.9884, a difference of only 8 ppm from that observed.
It is therefore assumed that the peak having a mass to charge ratio
of 982.4515 relates to a fragment or daughter ion resulting from a
peptide ion having a mass to charge of 1031.4395 losing a H3P041+ ion.
Some experimental data is now presented which illustrates the
ability of the preferred embodiment to quantify the relative
abundance of two proteins contained in two different samples which
comprise a mixture of proteins.
A first sample contained the tryptic digest products of three
proteins BSA, Glycogen Phosphorylase B and Casein. These three
proteins were initially present in the ratio 1:1:1. Each of the
three proteins had a concentration of 330 fmol/pl. A second sample
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contained the tryptic digest products of the same three proteins BSA,
Glycogen Phosphorylase B and Casein. However, the proteins were
initially present in the ratio 1:1:X. X was uncertain but believed
to be in the range 2-3. The concentration of the proteins BSA and
Glycogen Phosphorylase B in the second sample mixture was the same as
in the first sample, namely 330 fmol/pl.
The experimental protocol which was followed was that 1 pl of
sample was loaded for separation on to a HPLC column at a flow rate
of 4 pl/min. The liquid flow was then split such that the flow rate
to the nano-electrospray ionisation source was approximately 200
nl/min.
mass spectra were recorded on the mass spectrometer 6. Mass
spectra were recorded at alternating low and high collision energy
using nitrogen collision gas. The low-collision energy mass spectra
were recorded at a collision voltage of 10V and the high-collision
energy mass spectra were recorded at a collision voltage of 33V. The
mass spectrometer was fitted with a Nano-Lock-Spray device which
delivered a separate liquid flow to the source which may be
occasionally sampled to provide a reference mass from which the mass
calibration may be periodically validated. This ensured that the
mass measurements were accurate to within an RMS accuracy of 5 ppm.
Data were recorded and processed using the MassLynx (RTM) data
system.
The first sample was initially analysed and the data was used
as a reference. The first sample was then analysed a further two
times. The second sample was analysed twice. The data from these
analyses were used to attempt to quantify the (unknown) relative
abundance of Casein in the second sample.
All data files were processed automatically generating a list
of ions with associated areas and high-collision energy spectra for
each experiment. This list was then searched against the Swiss-Prot
protein database using the ProteinLynx (RTM) search engine.
Chromatographic peak areas were obtained using the Waters (RTM) Apex
Peak Tracking algorithm. Chromatograms for each charge state found
to be present were summed prior to integration.
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The experimentally determined relative expression level of
various peptide ions normalised with respect to the reference data
for the two samples are given in the following tables.
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BSA peptide ions Sample 1 Sample 1 Sample 2 Sample 2
Run 1 Run 2 Run 1 Run 2
FKDLGEEHFK 0.652 0.433 0.914 0.661
HLVDEPQNLIK 0.905 0.829 0.641 0.519
KVPQVSTPTLVEVSR 1.162 0.787 0.629 0.635
LVNELTEFAK 1.049 0.795 0.705 0.813
LGEYGFQNALIVR 1.278 0.818 0.753 0.753
AEFVEVTK 1.120 0.821 0.834 0.711
Average 1.028 0.747 0.746 0.682
Glycogen Sample 1 Sample 1 Sample 2 Sample 2
Phophorylase B Run 1 Run 2 Run 1 Run 2
peptide ions
VLVDLER 1.279 0.751 n/a 0.701
TNFDAFPDK 0.798 0.972 0.691 0.699
EIWGVEPSR 0.734 0.984 1.053 1.054
LITAIGDVVNHDPVVGDR 1.043 0.704 0.833 0.833
VLPNDNFFEGK 0.969 0.864 0.933 0.808
QIIEQLSSGFFSPK 0.691 n/a 1.428 1.428
VAAAFPGDVDR 1.140 0.739 0.631 0.641
Average . 0.951 0.836 0.928 0.881
CASEIN Sample 1 Sample 1 Sample 2 Sample 2
Peptide sequence Run 1 Run 2 Run 1 Run 2
EDVPSER 0.962 0.941 2.198 1.962
HQGLPQEVLNENLLR 0.828 0.701 1.736 2.090
FFVAPFPEVFGK 1.231 0.849 2.175 1.596
Average 1.007 0.830 2.036 1.883
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Peptides whose sequences were confirmed by high-collision
energy data are underlined in the above tables. Confirmation means
that the probability of this peptide, given its accurate mass and the
corresponding high-collision energy data, is larger than that of any
other peptide in the database given the current fragmentation or
reaction model. The remaining peptides are believed to be correct
based on their retention time and mass compared to those for
confirmed peptides. It was expected that there would be some
experimental error in the results due to injection volume errors and
other effects.
When using BSA as an internal reference, the relative abundance
of Glycogen Phosphorylase B in the first sample was determined to be
0.925 (first analysis) and 1.119 (second analysis) giving an average
of 1Ø The relative abundance of Glycogen Phosphorylase B in the
second sample was determined to be 1.244 (first analysis) and 1.292
(second analysis) giving an average of 1.3. These results compare
favourably with the expected value of 1.
Similarly, the relative abundance of Casein in the first sample
was determined to be 0.980 (first analysis) and 1.111 (second
analysis) giving an average of 1Ø The relative abundance of Casein
in the second sample was determined to be 2.729 (first analysis) and
2.761 (second analysis) giving an average of 2.7. These results
compare favourably with the expected values of 1 and 2-3.
The following data relates to chromatograms and mass spectra
obtained from the first and second samples. One peptide having the
sequence HQGLPQEVLNENLLR and derived from Casein elutes at almost
exactly the same time as the peptide having the sequence LVNELTEFAK
derived from BSA. Although this is an unusual occurrence, it
provided an opportunity to compare the abundance of Casein in the two
different samples.
Figs. 11A-D show four mass chromatograms, two relating to the
first sample and two relating to the second sample. Fig. 11A shows a
mass chromatogram relating to the first sample for ions having a mass
to charge ratio of 880.4 which corresponds with the peptide ion
(M+2H)" having the sequence 11(2GLPQEVLNENLLR and which is derived
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from Casein. Fig. 11B shows a mass chromatogram relating to the
second sample which corresponds with the same peptide ion having the
sequence HQGLPQEVLNENLLR which is derived from Casein.
Fig. 11C shows a mass chromatogram relating to the first sample
for ions having a mass to charge ratio of 582.3 which corresponds
with the peptide ion (M+2H)++ having the sequence LVNELTEFAK and
which is derived from BSA. Fig. 11D shows a mass chromatogram
relating to the, second sample which corresponds with the same peptide
ion having the sequence LVNELTEFAK and which is derived from BSA.
The mass chromatograms show that the peptide ions having a mass to
charge ratio of mass to charge ratio 582.3 derived from BSA are
present in both samples in roughly equal amounts whereas there is
approximately a 100% difference in the intensity of peptide ion
having a mass to charge ratio of 880.4 derived from Casein.
Fig. 12A show a parent or precursor ion mass spectrum recorded
after around 20 minutes from the first sample and Fig. 12B shows a
parent or precursor ion mass spectrum recorded after around
substantially the same time from the second sample. The mass spectra
show that the ions having a mass to charge ratio of 582.3 (derived
from BSA) are approximately the same intensity in both mass spectra
whereas ions having a mass to charge ratio of 880.4 which relate to a
peptide ion from Casein are approximately twice the intensity in the
second sample compared with the first sample. This is consistent
with expectations.
Fig. 13 shows the parent or precursor ion mass spectrum shown
in Fig. 12A in more detail. Peaks corresponding with BSA peptide
ions having a mass to charge of 582.3 and peaks corresponding with
the Casein peptide ions having a mass to charge ratio of 880.4 can be
clearly seen. The insert shows the expanded part of the spectrum
showing the isotope peaks of the peptide ion having a mass to charge
ratio of 880.4. Similarly, Fig. 14 shows the parent or precursor ion
mass spectrum shown in Fig. 12B in more detail. Again, peaks
corresponding with BSA peptide ions having a mass to charge ratio of
582.3 and peaks corresponding with the Casein peptide ions having a
mass to charge ratio of 880.4 can be clearly seen. The insert shows
CA 02835314 2013-11-28
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the expanded part of the spectrum showing the isotope peaks of the
peptide ion having a mass to charge ratio of 880.4. It is apparent
from Figs. 12-14 and from comparing the inserts of Figs. 13 and 14
that the abundance of the peptide ion derived from Casein which has a
mass spectral peak of mass to charge ratio 880.4 is approximately
twice the abundance in the second sample compared with the first
sample.
Although the present invention has been described with
reference to preferred embodiments, it will be understood by those
skilled in the art that various changes in form and detail may be
made without departing from the scope of the invention as set forth
in the accompanying claims.
