U.S. patent application number 10/390031 was filed with the patent office on 2004-01-29 for catalytically generated mass labels.
This patent application is currently assigned to XZILLION GMBH & CO.. Invention is credited to Schmidt, Gunter, Thompson, Andrew Hugin.
Application Number | 20040018565 10/390031 |
Document ID | / |
Family ID | 10818648 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018565 |
Kind Code |
A1 |
Schmidt, Gunter ; et
al. |
January 29, 2004 |
Catalytically generated mass labels
Abstract
Provided is a method for assaying a substance, which method
comprises contacting the substance with an assay agent comprising a
catalytic agent to associate the substance with the catalytic
agent, contacting the resulting associated substance with a label
precursor, and detecting a label, wherein the label precursor is
capable of reacting catalytically with the catalytic agent to
release the label, and wherein the label is a mass label. Also
provided is a kit for assaying a substance, which kit comprises an
assay agent comprising a catalytic agent, and a label precursor
capable of reacting catalytically with the catalytic agent to
release a label, wherein the label is a mass label.
Inventors: |
Schmidt, Gunter; (Houghton,
GB) ; Thompson, Andrew Hugin; (Alloway, GB) |
Correspondence
Address: |
Samuel C. Miller, III
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Assignee: |
XZILLION GMBH & CO.
65926
Frankfurt AM Main
DE
|
Family ID: |
10818648 |
Appl. No.: |
10/390031 |
Filed: |
July 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10390031 |
Jul 16, 2003 |
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09508049 |
May 3, 2000 |
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6649354 |
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09508049 |
May 3, 2000 |
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PCT/GB98/02690 |
Sep 7, 1998 |
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Current U.S.
Class: |
435/7.5 ;
435/7.92 |
Current CPC
Class: |
C12Q 1/6872 20130101;
Y10S 436/824 20130101; G01N 33/531 20130101; Y10S 436/823 20130101;
Y10S 435/814 20130101; Y10S 530/81 20130101; Y10S 436/805 20130101;
Y10S 530/811 20130101; G01N 33/58 20130101 |
Class at
Publication: |
435/7.5 ;
435/7.92 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 1997 |
GB |
9718921.1 |
Claims
1. A method for assaying a substance, which method comprises
contacting the substance with an assay agent comprising a catalytic
agent to associate the substance with the catalytic agent,
contacting the resulting associated substance with a label
precursor, and detecting a label, wherein the label precursor is
capable of reacting catalytically with the catalytic agent to
release the label, and wherein the label is a mass label.
2. A method according to claim 1, wherein the catalytic agent is
capable of cleaving a specific chemical bond, or a bond in a
specific molecular environment.
3. A method according to claim 1 or claim 2, wherein prior to
contacting the associated substance with the label precursor, the
associated substance is separated from unassociated assay
agent.
4. A method according to any preceding claim, in which the label
precursor comprises a label attached to a carrier moiety via a
linker, the label being specific to the linker, and wherein the
linker is capable of being cleaved by a specific catalytic agent to
release the label.
5. A method according to claim 4, wherein the carrier moiety is
selected such that it allows the label precursor to be captured on
a solid phase.
6. A method according to claim 5, wherein the carrier moiety
comprises biotin for attaching the label precursor to an avidinated
solid phase.
7. A method according to any preceding claim, wherein the assay
agent comprises a recognition moiety which is capable of
associating with a specific substance.
8. A method according to claim 7, wherein the recognition moiety
comprises an antibody, an oligonucleotide, a protein, a peptide, an
enzyme, an RNA aptamer and/or a dextran.
9. A method according to any preceding claim, wherein the catalytic
agent comprises an organic catalyst, a protein enzyme and/or a
catalytic RNA molecule.
10. A method according to claim 9, wherein the catalytic agent
comprises a hydrolytic enzyme, such as lysozyme, trypsin,
chymotrypsin, an elastase, an alkaline phosphitase or a
peroxidase.
11. A method according to any preceding claim, wherein the
catalytic agent comprises an endonuclease and the label precursor
comprises an oligonucleotide linker capable of being cleaved
catalytically by the endonuclease to release the label.
12. A method according to any preceding claim for assaying a
mixture comprising a plurality of substances, which method
comprises contacting the mixture with an assay agent comprising an
array of catalytic agents to associate each substance in the
mixture with a catalytic agent specific to that substance,
contacting the resulting associated substances with an array of
label precursors, and detecting a plurality of labels, wherein each
catalytic agent in the array is specific to one substance in the
mixture, each label precursor is capable of reacting catalytically
with a catalytic agent specific to that precursor to release a
label, and each label is specific to its precursor and is a mass
label.
13. A method according to claim 12, wherein each label precursor
comprises a label attached to a carrier moiety via a linker and the
carrier moiety of each label precursor is selected such that it has
a different mass to each of the mass labels in the array of label
precursors.
14. A method for gene profiling comprising detecting one or more
mRNA moieties by means of a method as defined in any preceding
claim.
15. A kit for assaying a substance, which kit comprises an assay
agent comprising a catalytic agent, and a label precursor capable
of reacting catalytically with the catalytic agent to release a
label, wherein the label is a mass label.
16. A kit according to claim 15 for assaying a mixture of
substances, which kit comprises an assay agent comprising an array
of catalytic agents and an array of label precursors, wherein each
catalytic agent in the array is specific to one substance in the
mixture, each label precursor is capable of reacting catalytically
with a catalytic agent specific to that precursor to release a
label, and each label is specific to its precursor and is a mass
label.
17. A kit according to claim 15 or claim 16, wherein the catalytic
agent is capable of cleaving a specific chemical bond, or a bond in
a specific molecular environment.
18. A kit according to any of claims 15-17, in which the label
precursor comprises a label attached to a carrier moiety via a
linker, the label being specific to the linker, and wherein the
linker is capable of being cleaved by a specific catalytic agent to
release the label.
19. A kit according to any of claims 18, wherein the carrier moiety
is selected such that it allows the label precursor to be captured
on a solid phase.
20. A kit according to claim 19, wherein the carrier moiety
comprises biotin for attaching the label precursor to an avidinated
solid phase.
21. A kit according to any of claims 15-20, wherein the assay agent
comprises a recognition moiety which is capable of associating with
a specific substance.
22. A kit according to any of claims 15-21, wherein the assay agent
comprises an antibody, an oligonucleotide, a protein, a peptide, an
enzyme, an RNA aptamer and/or a dextran.
23. A kit according to any of claims 15-22, wherein the catalytic
agent comprises an organic catalyst, a protein enzyme and/or a
catalytic RNA molecule.
24. A kit according to claim 23, wherein the catalytic agent
comprises a hydrolytic enzyme, such as lysozyme, trypsin,
chymotrypsin, an elastase, an alkaline phosphitase or a
peroxidase.
25. A kit according to any of claims 15-24, wherein the catalytic
agent comprises an endonuclease and the label precursor comprises
an oligonucleotide linker capable of being cleaved catalytically by
the endonuclease to release the label.
26. A kit according to any of claims 16-25, wherein each label
precursor comprises a label attached to a carrier moiety via a
linker and the carrier moiety of each label precursor is selected
such that it has a different mass to each of the mass labels in the
array of label precursors.
Description
[0001] This invention relates to a method for assaying a substance
which is present in a very low concentration. Specifically, this
invention concerns a method which employs catalysis to amplify a
signal. The method is especially suited to biological assays. The
invention has the further advantage that it allows the simultaneous
assay of a mixture comprising a plurality of substances, even when
these substances are present in very low quantities.
[0002] Biological assays must often achieve a sensitivity to target
molecules in the low femotomolar to attomolar range. Assays based
on ligand binding can achieve this but often require signal
amplification to indicate the success of the ligand binding in a
detectable manner. Radioisotope labelled ligands can achieve this
but there are safety implications in using radio-active agents
which makes them dangerous and expensive to use. Furthermore, since
there is no trivial or cheap way to differentiate between different
kinds of radiation, only one radiolabelled ligand can be used in
any one assay.
[0003] Colorimetric, chemoluminescent assays and fluorescence based
systems permit signal amplification in a variety of systems,
however the number of labels that can be used simultaneously is
limited due to spectral overlap effects of emission based
systems.
[0004] An object of the present invention is to solve the above
problems. Accordingly, the present invention provides a method for
assaying a substance, which method comprises contacting the
substance with an assay agent comprising a catalytic agent to
associate the substance with the catalytic agent, contacting the
resulting associated substance with a label precursor, and
detecting a label, wherein the label precursor is capable of
reacting catalytically with the catalytic agent to release the
label, and wherein the label is a mass label.
[0005] The present invention also provides a kit for assaying a
substance, which kit comprises an assay agent comprising a
catalytic agent, and a label precursor capable of reacting
catalytically with the catalytic agent to release a label, wherein
the label is a mass label.
[0006] The invention will now be described in further detail by way
of example only, with reference to the accompanying drawings, in
which:
[0007] FIG. 1 shows a label precursor (secondary signal generating
agent) which can be used in the present invention;
[0008] FIG. 2 shows a label precursor in further detail; and
[0009] FIG. 3 shows an arrangement for carrying out orthogonal
time-of-flight mass spectrometry, which can be used in conjunction
with the present invention.
[0010] A technology that will permit amplification of a signal in a
non-radioactive manner and which permits very many labels to be
used simultaneously is described here. Such a system requires
probes to be labelled with catalytic agents that will cleave a
specific chemical bond, or a bond in a specific molecular
environment. A signal is generated by the cleavage of a secondary
molecule which comprises a mass label, bonded by a specifically
cleavable linker to a carrier molecule. The mass label identifies
the linker that is cleaved. In an array of probes labelled with
catalysts, only one catalytic cleavage agent should cleave any
single linker. Thus the mass label identifies the corresponding
probe. An array of secondary molecules is thus required, one for
each probe with a unique linker and a unique mass label identifying
each secondary molecule.
[0011] Typical assays involving probes of this form would be
similar in principal to those used in colorimetric or fluorescence
based systems. Labelled probe is added to target molecules in a
sample where binding takes place. Bound probe is separated from
unbound probe and then the presence of bound probe is assayed.
[0012] Numerous systems exist to separate labelled probe from
unlabelled probe. The method used will depend largely on the nature
of the probe target interactions. Some systems will be discussed
here by way of example but these are by no means limiting.
[0013] Assaying the presence of bound probe is effected with this
system by adding a known quantity of the secondary carrier molecule
linked to its corresponding mass label. Any given bound probe will
carry a catalytic cleavage agent suitable to cleave a specific
linker releasing a corresponding mass label. The catalyst can
liberate a massive quantity of label in a given time and can thus
generate a significant amplification of signal.
[0014] Probe Molecules
[0015] Probe molecules include but are not limited to antibodies,
oligonucleotides, peptides and proteins, including enzymes, RNA
aptamers, dextrans.
[0016] Catalytic Agents
[0017] Catalytic agents include but are not limited to protein
enzymes, catalytic RNA molecules, organic catalysts.
[0018] Favourable enzyme systems include hydrolytic enzymes such as
lysozyme, trypsin, chymotrypsin, elastase, alkaline phosphatase,
peroxidases. Restriction endonucleases would be an excellent class
of enzymes to exploit for non-DNA target molecules as
oligonucleotide linkers bearing the recognition sequence for a
specific endonuclease would allow a significant number of carrier
molecules to be generated.
[0019] `Secondary` Signal Generating Agents
[0020] Secondary agents comprise a mass label, cleavably linked to
a carrier molecule via a linker that is cleaved specifically by a
catalytic agent. The mass label identifies the linker used. This in
turn should be specific to only one catalyst in any given assay
thus allowing liberation of mass label from its carrier to be
related back to the presence of a single probe molecule.
[0021] Carrier Molecules
[0022] Carrier molecules must be capable of being readily linked to
cleavable linker groups. A single carrier should be able to be
linked to all or as many as possible of available linkers to allow
combinatorial reuse of carrier molecules. Furthermore a single
carrier could be derivitised with multiple linker groups coupled to
mass labels to give a high density of secondary agent from which to
generate a large signal.
[0023] One could for example derivitise all the exposed amine
residues of a small protein, or one could target carboxyl and
hydroxyl groups with a linker group as these are all groups that
can be fairly readily derivitised.
[0024] Carrier molecules should not inhibit cleavage reactions and
should be easily separable from cleaved mass labels. Mass
spectrometry facilitates this separation. In a simple case the
carrier molecule need only be chosen to fall outside the mass range
of mass labels. For example, if the mass range in which mass labels
are designed to occur is up to a mass charge ratio of 2000, then
the carrier molecule would be chosen to have a mass/charge ratio of
greater than 2000. Furthermore, it should be resistant to
fragmentation to ensure that fragmentation products do not coincide
with mass label peaks.
[0025] A potentially simpler alternative supported by mass
spectrometry is to choose a carrier molecule that takes a different
charge from the mass labels. Thus if mass labels that become
negatively charged on ionisation are used then a carrier that
becomes positively charged is appropriate. However, carrier that is
still linked to uncleaved mass label, should preferably also take a
net positive charge to facilitate separation of labels from
uncleaved carrier.
[0026] Separation outside the mass spectrometer is also possible.
Carrier molecules could be as simple as biotin cleavably linked to
a mass label. This would permit uncleaved carrier to be easily
separated from cleaved mass labels by an avidin affinity column.
Clearly any immobilisation system could be used, including but not
limited to poly-histidine metal coordination systems,
photocross-linking agents, antibodies, RNA aptamers,
oligonucleotides, etc.
[0027] Linkers
[0028] Linker groups should permit specific cleavage by a single
catalytic agent from a family of such agents used in a multiplexed
probing reaction.
[0029] Saccharides of N-acetyl glucosamine (GlcNAc) are an
effective substrate for lysozyrne as are alternating
oligosaccharides of N-acetyl muramic acid and GlcNAc, particularly
hexasaccharides of each.
[0030] The serine proteases are a class of enzymes with fairly
distinct specificities. Trypsin will cleave peptide bonds only
after lysine or arginine residues. Chymotrypsin will cleave only
after large hydrophobic residues. Thus peptide linkers with these
distinct features would be appropriate to use with their
corresponding enzyme.
[0031] Restriction endonucleases have very high specificity for
their substrate, have a very diverse array of substrates and are
manufactured in quantity for reasonable prices. Eco RI cuts at the
sequence 5'-GAATTC-3', while Hind III cuts at 5'-AAGCTT-3' and Hpa
I cuts at 5'-GTTAAC-3'. Thus with a group of restriction enzymes as
catalytic markers on a probe molecule, one could use
oligonucleotides as linkers. An oligonucleotide biotinylated at one
terminus and bearing a mass label at the other terminus would be a
relatively simple reagent to produce. This sort of system would not
be appropriate for use with DNA targets.
[0032] Gene Profiling Using Catalytically Generated Mass Labels
[0033] Gene expression profiling techniques often fail to detect
mRNAs expressed at very low levels. Most gene expression profiling
techniques require conversion of mRNA into cDNA before assaying for
particular sequences. Certain mRNAs are missed in the reverse
transcription process. Low level mRNAs that are reverse transcribed
into cDNAs are still at very low levels and may be missed by the
assaying technique. Differential display techniques and arbitrarily
primed reverse transcription techniques use sets of primers,
usually fluorescently or radiolabelled primers to generate
fragments of cDNA of specific lengths. The primer sets are designed
statistically to prime as many mRNAs as possible with as few
primers as possible. To detect the fragments generated by each
primer in a mixed reaction is not really possible in conventional
techniques so primers must be spatially resolved which means a
separate reaction vessel for each primer or set of primer to be
followed independently. If single assays could be performed where
each primer can labelled independently then significant savings in
reagents, manpower and time would be achieved. This invention
permits individually labelled primers to be used.
[0034] For assaying specifically for low levels of specific mRNAs
for which specific primer pairs are available this technology is
well suited. One could label one primer for each pair with an
immobilisation agent such as biotin. The second primer could be
labelled with a unique catalyst. mRNA or single stranded cDNA could
be primed with the first primer and the primed template can be
extended by a reverse transcriptase or polymerase respectively. The
double stranded product can then be immobilised, if the primer is
not already immobilised. One can achieve linear amplification of
the template with further cycles of denaturation and priming with
the first primer. The double stranded products are then denatured
and the free strands, unused primer, etc. is washed away leaving
immobilised single stranded template. The second primer can be
added at this stage to prime the immobilised strand and can be
extended by a polymerase. Unincorporated primer is washed away
after synthesis of the second strand. Incorporation of the second
primer can then be assayed by addition of cleavable mass label
carriers. Clearly many mRNAs can be assayed for simultaneously in a
system like this.
[0035] Oligonucleotide Ligase Assays
[0036] The oligonucleotide ligase assay is a technique for fast
genotyping. The principle is simple for a given single nucleotide
polymorphism (SNP), two oligonucleotides are necessary. One
immediately complementary to a region 5' of the SNP and one
adjacent to the 5' oligonucleotide which overlaps the SNP. The 5'
oligonucleotide is typically biotin labelled, while the 3'
oligonucleotide is typically fluorescently labelled, 1 dye is used
to identify each of the 4 possible bases that the SNP might adopt.
An amplified clone of a gene or DNA fragment of interest is assayed
in a ligase reaction. The 5' and 3' oligonucleotides are allowed to
hybridise to their complementary sequences in the target DNA in the
presence of ligase. The 3' oligonucleotide complementary to the SNP
bearing sequence should be preferentially ligated to the 5'
oligonucleotide over the other 3 possible oligonucleotides. The
ligation can be assayed by capturing the 5' oligonucleotides onto
an avidinated substrate and determining the quantity and frequency
of fluorescence generated by the ligated 3' oligonucleotide.
[0037] The small number of commercially available fluorescent dyes
is a limit on this system being extended to determine the nature of
multiple SNPs in a large gene such as the cystic fibrosis gene.
Using catalytically cleavable mass labels and corresponding
catalyst labelled oligonucleotides would permit a multiplexed
reaction wherein numerous SNPs in a single gene could be assayed
for.
[0038] Features of Mass Labels
[0039] To achieve the required behaviour from a mass label, certain
chemical properties are desirable. These are represented in
particular molecular groups or moieties that can be incorporated
into mass labels in a number of ways.
[0040] For the purposes of generating mass labels, favoured labels
require a specifically cleavable bond in the linker and
fragmentation resistant bonds in the mass label.
[0041] For optimal performance using present techniques a
mass/charge ratio of up to 2000 to 3000 units is the optimal range
for such labels as this corresponds to the range over which singly
charged entities can be reliably detected with greatest
sensitivity, however labels of mass less than 100 to 200 daltons
are not ideal as the low mass end of the spectrum tends to be
populated by solvent molecules, small molecule impurities, multiple
ionisation peaks and fragmentation peaks.
[0042] To permit detection one requires labels that have a net
charge, but are preferably not multiply ionisable, i.e. they have a
fixed single charge. Furthermore they should be resistant to
fragmentation. This ensures that each peak in the mass/charge
spectrum corresponds to a single label and simplifies the analysis
of the data. Furthermore this reduces any ambiguity in the
determination of the quantity of the label, which is very important
for some of the applications for which this invention has been
developed.
[0043] Various functionalities exist which carry or can carry
positive charges for positive ion mass spectrometry. These include
but are not limited to amines particularly tertiary amines and
quaternary amines. Quaternary ammonium groups carry a single
positive charge and do not require ionisation. For positive ion
spectrometry these allow great sensitivity. Hence preferred
positive ion mass labels should carry at least one such group.
[0044] Various functionalities are available to carry a negative
charge for negative ion mass spectrometry which include but are not
limited to carboxylic acids, phosphonates, phosphates, phenolic
hydroxyls, sulphonic acids, sulphonilamides, sulphonyl urea,
tetrazole and perfluoro alcohol.
[0045] Ionisation Techniques
[0046] For many biological mass spectrometry applications so called
`soft` ionisation techniques are used. These allow large molecules
such as proteins and nucleic acids to be put into the mass
spectrometer in solutions with mild pH and at low concentrations.
Two such techniques are ideal for use with this invention;
electrospray ionisation and Matrix Assisted Laser Desorption
Ionisation (MALDI).
[0047] Electrospray Ionisation
[0048] Electrospray ionisation requires that the dilute solution of
biomolecule be `atomised` into the spectrometer, i.e. in a fine
spray. The solution is, for example, sprayed from the tip of a
needle across an electrostatic field gradient or into a stream of
dry nitrogen in an electrostatic field. The mechanism of ionisation
is not fully understood but is thought to work broadly as follows.
In a stream of nitrogen the solvent is evaporated. With a small
droplet, this results in concentration of the biomolecule. Given
that most biomolecules have a net charge this increases the
electrostatic repulsion of the dissolved protein. As evaporation
continues this repulsion ultimately becomes greater than the
surface tension of the droplet and the droplet `explodes` into
smaller droplets. The electrostatic field helps to further overcome
the surface tension of the droplets. The evaporation continues from
the smaller droplets which, in turn, explode iteratively until
essentially the biomolecules are in the vapour phase, as is all the
solvent. This technique is of particular importance in the use of
mass labels in that the technique imparts a relatively small amount
of energy to ions in the ionisation process and the energy
distribution within a population tends to fall in a narrower range
when compared with other techniques. The ions are accelerated out
of the ionisation chamber through a pair of electrodes. The
potential difference across these electrodes determines whether
positive or negative ions pass into the mass analyser and also the
energy with which these ions enter the mass spectrometer. This is
of significance when considering fragmentation of ions in the mass
spectrometer. The more energy imparted to a population of ions the
more likely it is that fragmentation will occur. By adjusting the
accelerating voltage used to accelerate ions from the ionisation
chamber one can control the fragmentation of ions.
[0049] Matrix Assisted Laser Desorption Ionisation (MALDI)
[0050] MALDI requires that the biomolecule solution be embedded in
a large molar excess of an photo-excitable `matrix`. The
application of laser light of the appropriate frequency (266 nm
beam for nicotinic acid) results in the excitation of the matrix
which in turn leads to excitation and ionisation of the embedded
biomolecule. This technique imparts a significant quantity of
translational energy to ions, but tends not to induce excessive
fragmentation despite this. Accelerating voltages can again be used
to control fragmentation with this technique though.
[0051] MALDI techniques can be supported in two ways. One can embed
mass labelled DNA in a MALDI matrix, where the labels themselves
are not specifically excitable by laser or one can construct labels
that contain the necessary groups to allow laser energisation. The
latter approach means the labels do not need to be embedded in a
matrix before performing mass spectrometry. Such groups include
nicotinic, sinapinic or cinnamic acid moieties. MALDI based
cleavage of labels would probably be most effective with a
photocleavable linker as this would avoid a cleavage step prior to
performing MALDI mass spectrometry. The various excitable
ionisation agents have different excitation frequencies so that a
different frequency can be chosen to trigger ionisation from that
used to cleave the photolysable linker. These excitable moieties
are easily derivitised using standard synthetic techniques in
organic chemistry so labels with multiple masses can be constructed
in a combinatorial manner.
[0052] Fragmentation Within the Mass Spectrometer
[0053] Fragmentation is a highly significant feature of mass
spectrometry. With respect to this invention it is important to
consider how one intends to identify a mass label. At the two
extremes one can either design molecules that are highly resistant
to fragmentation and identify a label by the appearance of the
label's molecular ion in the mass spectrum. One would thus design
families of labels to have unique molecular ions. At the other
extreme one can design a molecule with a highly characteristic
fragmentation pattern that would identify it. In this case one must
design families of labels with non-overlapping patterns or with at
least one unique fragmentation species for each label by which to
identify each label unambiguously. Fragmentation is, however, a
property of the molecule and of the ionisation technique used to
generate the ion. Different techniques impart differing amounts of
energy to the ion and the chemical environment of the ions will
vary considerably, thus labels that are appropriate for one mass
spectrometry technique may be inappropriate in others. The
preferred approach is to design fragmentation resistant molecules,
although some fragmentation is inevitable. This means one aims to
identify molecules with a single major species, either the
molecular ion or a single very highly populated fragment ion.
[0054] Determining Bond Stability in the Mass Spectrometer
[0055] In neutral molecules it is reasonably simple to determine
whether a molecule is resistant to fragmentation, by consideration
of bond strengths. However, when the molecule is ionised, the bond
strength may increase or decrease in ways that are difficult to
determine a priori. For example, given a bond, X-Y, we can
write:
[0056] un-ionised:
X-Y.fwdarw.X'+Y'
.thrfore.D(X-Y)=.DELTA.H(X')+.DELTA.H(Y')-.DELTA.H(X-Y)
[0057] But
D(X-Y).sup.+=.DELTA.H(X.sup.+)+.DELTA.H(Y')-.DELTA.H(X-Y.sup.+)
.thrfore.D(X-Y)-D(X-Y).sup.+=.DELTA.H(X.sup.-)-.DELTA.H(X.sup.+)
-.DELTA.H(X-Y)-.DELTA.H(X-Y.sup.+)
[0058] and
I(X.sup.-)=.DELTA.H(X.sup.+)-.DELTA.H(X.sup.-)
I(X-Y)=.DELTA.H(X-Y.sup.+)-.DELTA.H(X-Y)
.thrfore.D(X-Y)-D(X-Y).sup.+=I(X-Y)-I(X.sup.-)
[0059] This means D(X-Y)-D(X-Y).sup.+>0 if
I(X-Y)>I(X.sup.-)
[0060] Similarly D(X-Y)-D(X-Y).sup.+<0 if
I(X-Y)<I(X.sup.-)
[0061] since both I(X-Y) and I(X.sup.-) are positive.
[0062] .thrfore.a stronger bond results if
I(X-Y)<I(X.sup.-).
[0063] In the equations above, D(A-B) refers to bond energy of the
species in parentheses, I(N) refers to the ionisation energy of the
species in parentheses and delta-H is the free energy of formation
of the species in parentheses. The upshot of the equations above is
that in order to predict whether a bond is likely to be stable
under a given set of ionisation conditions we need to know the
energy of ionisation of the molecule and the energy of ionisation
of the neutral fragment that results from fragmentation at the bond
in question.
[0064] For example, consider the C-N bond in aniline:
I(NH.sub.2.sup.-)=11.14 electronvolts (ev) and I(C.sub.6H.sub.5
NH.sub.2)=7.7 ev
.thrfore.I(C.sub.6H.sub.5NH.sub.2)<I(NH.sub.2.sup.-) by 3.44
ev
[0065] The alternative cleavage at this bond is:
I(C.sub.6H.sub.5)=9.35 electronvolts(ev) and
I(C.sub.6H.sub.5NH.sub.2)=7.7 ev
.thrfore.I(C.sub.6H.sub.5NH.sub.2)<I(C.sub.6H.sub.5)
[0066] This bond is thus not easily broken in the ion. Aniline, if
it has sufficient initial energy to fragment, is generally observed
to cleave releasing HCN, rather than by cleavage of a C--N bond.
Similarly considerations apply to phenol:
I(OH.sup.-)=13 ev and I(C.sub.6H.sub.5OH)=8.47 ev
.thrfore.I(C.sub.6H.sub.5OH)<I(OH.sup.-)
[0067] The alternative cleavage at this bond is
I(C.sub.6H.sub.5.sup.-)=9.35 ev and I(C.sub.6H.sub.5OH)=8.47 ev
.thrfore.I(C.sub.6H.sub.5OH)<O(C.sub.6H.sub.5.sup.-)
[0068] Thus C--O cleavage is not observed.
[0069] Determining the differences in ionisation energies of
molecule and neutral fragments is a general working principle which
can be used to predict likely ion bond strengths. If the energy
added during ionisation is less than the ionic bond strength then
ionisation will not be observed. Typical ionic bonds that have good
strength include, aryl-O, aryl-N, aryl-S bonds. Generally,
aliphatic type bonds become less stable in ionic form. Thus single
C--C bonds are weak in the ion but C.dbd.C is still strong.
Aryl-C.dbd.C tends to be strong too for the same reasons as aryl-O,
etc. Aryl or Aryl-F bonds are also strong in ionic form which is
appealing as fluorocarbons are cheap to manufacture and are
chemically inert.
[0070] Similar considerations apply to negative ions, except one
must use electron affinities in the equations above rather than
ionisation energies.
[0071] Mass Label Chemistries
[0072] For any practically or commercially useful system it is
important that construction of labels be as simple as possible
using as few reagents and processing steps as possible. A
combinatorial approach in a which a series of monomeric molecular
units are available to be used in multiple combinations with each
other would be ideal.
[0073] One can synthesise mass labels using standard organic
chemistry techniques. Such labels ought to carry a single charge
bearing group and should be resistant to fragmentation in the mass
spectrometry technique used. Amine derivatives, quaternary ammonium
ions or positive sulphur centres are good charge carriers if
positive ions mass spectrometry is used. These have extremely good
detection properties that generate clean sharp signals. Similarly,
negatively charged ions can be used, so molecules with carboxylic
acid, sulphonic acid and other moieties are appropriate for
negative ion spectrometry. Labels for MALDI mass spectrometry can
be generated by derivitising known molecules that are excitable by
UV laser light, such as sinapinnic acid or cinnamic acid, of which
a number of derivatives are already commercially available.
Fragmentation resistant groups are discussed above. For a text on
organic chemistry see:
[0074] Vogel's "Textbook of Organic Chemistry" 4th Edition, Revised
by B. S. Fumiss, A. J. Hannaford, V. Rogers, P. W. G. Smith &
A. R. Tatchell, Longman, 1978.
[0075] Amino Acids
[0076] With a small number of amino acids such as glycine, alanine
and leucine, a large number of small peptides with different masses
can be generated using standard peptide synthesis techniques well
known in the art. With more amino acids many more labels can be
synthesised.
[0077] E. Atherton and R. C. Sheppard, editors, `Solid Phase
Peptide Synthesis: A Practical Approach`, IRL Press, Oxford.
[0078] Carbohydrates
[0079] Similarly carbohydrate molecules are useful monomeric units
that can be synthesised into heteropolymers of differing masses but
these are not especially amenable to ESMS.
[0080] Gait, M. J. editor, `Oligonucleotide Synthesis: A Practical
Approach`, IRL Press, Oxford,
[0081] Eckstein, editor, `Oligonucleotides and Analogues: A
Practical Approach`, IRL Press, Oxford, 1991
[0082] Other Labelling Chemistries
[0083] Clearly almost any molecule can be tacked onto another as a
label. Obviously the properties of such labels in the mass
spectrometer will vary. In terms of analysing biomolecules it will
be important that the labels be inert, bear a single charge and be
resistant to fragmentation.
[0084] Quantification and Mass Spectrometry
[0085] For the most part biochemical and molecular biological
assays are quantitative. The mass spectrometer is not a simple
device for quantification but use of appropriate instrumentation
can lead to great sensitivity. It must always be remembered that
the ion count is not a direct measure of the source molecule
quantity, the relationship is a complex function of the molecule's
ionisation behaviour. Quantitation is effected by scanning the mass
spectrum and counting ions at each mass/charge ratio scanned. The
count is integrated to give the total count at each point in the
spectrum over a given time. These counts can be related back to the
original qunatities of source molecules in a sample. Methods for
relating the ion count or current back to the quantity of source
molecule vary. External standards are one approach in which the
behaviour of the sample molecules is determined prior to
measurement of unknown sample. A calibration curve for each sample
molecule can be determined by measuring the ion current for serial
dilutions of a sample molecule when fed into the instrument
configuration being used.
[0086] Internal standards are probably the more favoured approach
rather than external standards, since an internal standard is
subjected to the same experimental conditions as the sample so any
experimental vagaries will affect both internal control and sample
molecule. To determine the quantity of a sample molecule, an
internal standard of a known quantity is added to the sample. The
internal standard is chosen to have a similar ionisation behaviour
as the molecule being measured. Thus the ratio of sample ion count
to standard ion count can be used to determine the quantity of
sample as the ratio of qunatities should be the same. Choosing
appropriate standards is the main difficulty with this approach.
One must find a molecule that is similar but not identical in its
mass spectrum. A favourable approach is to synthesise the sample
molecule with appropriate isotopes to give a slightly different
mass spectrum, for a molecule with the same chemical behaviour.
This approach might be less desirable than external standards for
use with large numbers of mass labels due to the added expense of
finding or synthesising appropriate internal standards but will
give better qunatification than external standards. An alternative
to isotope labelling is to identify a molecule that has similar but
not identical chemical behaviour as the sample in the mass
spectrometer. Finding such analogues is difficult and is a
significant task for large families of mass labels.
[0087] A compromise approach might be appropriate though, since
large families of mass labels will ideally be synthesised
combinatorially, and will thus be related chemically. A small
number of internal controls might be used, where each individual
control determines the quantities of a number of mass labels. The
precise relationship between internal standard and each mass label
might be determined in external calibration experiments to
compensate for any differences between them.
[0088] The configuration of the instrument is critical to
determining the actual ion count itself, particularly the
ionisation method and the separation method used. Certain
separation methods act as mass filters like the quadrupole which
only permits ions with a particular mass charge ratio to pass
through at one time. This means that a considerable proportion of
sample never reaches the detector. Furthermore most mass
spectrometers only detect one part of the mass spectrum at a time.
Given that a large proportion of the mass spectrum may be empty or
irrelevant but is usually scanned anyway, this means a further
large proportion of the sample is wasted. These factors may be a
problem in detecting very low abundance ions but these problems can
in large part be overcome by correct configuration of the
instrumentation.
[0089] To ensure better quantification one could attempt to ensure
all ions that are generated are detected. Mattauch-Herzog geometry
sector instruments permit this but have a number of limitations.
Sector instruments are organised into distinct regions, `sectors`,
that perform certain functions. In general the ionisation chamber
feeds into a free sector which feeds into an `electric sector`. The
electric sector essentially `focuses` the ion beam which is
divergent after leaving the ion source. The electronic sector also
ensures the ion stream has the same energy. This step results in
the loss of a certain amount of sample. This focused ion beam then
passes through a second free area into a magnetic sector which
splits the beam on the basis of its mass charge ratio. The magnetic
sector behaves almost like a prism. A photographic plate can be
placed in front of the split beam to measure the intensities of the
spectrum at all positions. Unfortunately there is a limit on the
dynamic range of these sorts of detector and it is messy and
cumbersome. Better dynamic range is achievable with electron
multiplier arrays, but at a cost of loss in resolution which is
limited by how close together the elements of the array can be
constructed. With a family of well characterised mass labels one
would probably monitor only sufficient peaks to sample all the mass
labels unambiguously. In general array detectors would allow one to
simultaneously and continuously monitor a number of regions of the
mass spectrum simultaneously, which might be applicable for use
with well characterised mass label families. The limit on the
resolution of closely spaced regions of the spectrum might restrict
the number of labels one might use, though, if array detectors are
chosen. For `selected ion monitoring` (SIM) the quadropole has an
advantage over many configurations in that the fields that filter
ions can be changed with extreme rapidity allowing a very high
sampling rate over a small number of peaks of interest.
[0090] Orthogonal TOF Mass Spectrometry
[0091] An approach that is preferable to array geometries is the
orthogonal time of flight mass spectrometer. This geometry that
allows for very fast sampling of an ion stream followed by almost
instantaneous detection of all ion species. The ion current leaving
the source, probably an electrospray source for many biological
applications, passes an electrode plate perpendicular to the
current. This plate is essentially an electrical gate and is used
to generate a repulsive potential which deflects the ion current
`orthogonally` into a time of flight mass analyser that uses a
reflectron. The reflectron is essentially a series of circular
electrodes that generate an increasingly repulsive electromagnetic
field that normalises ion energies and reflects the ion stream into
a detector. The reflectron is a simple device that greatly
increases the resolution of TOF analysers. Ions leaving the ion
source will have different energies, faster ions will penetrate the
repulsive field further than ions with a lower energy and so will
be delayed slightly with respect to the lower energy ions but since
they will arrive slightly before the lower energy ions they will
enter the TOF at roughly the same time so all the ions of a given
mass charge ratio will arrive at the detector at roughly the same
time. When the electrical gate is `closed` to deflect ions into the
TOF analyser, the timer is triggered. The flight time of the
deflected ions is recorded and this is sufficient to determine
their mass/charge ratio. The gate generally only sends a short
pulse of ions into the TOF analyser at any one time. Since the
arrival of all ions is recorded and since the TOF separation is
extremely fast, the entire mass spectrum is measured effectively
simultaneously. Furthermore, the gate electrode can sample the ion
stream at extremely high frequencies so very little sample is
required. For these reasons this geometry is extremely sensitive,
to the order of a few femtomoles.
[0092] Mass labels that can be used in the present invention
include those disclosed in GB 9700746.2, GB 9718255.4, GB
9726953.4, PCT/GB98/00127 and the UK application having Page White
and Farrer file number 87820. The contents of these applications
are incorporated herein by reference.
[0093] Specific methods for gene profiling in which the method of
the present invention can be used are disclosed in PCT/GB98/01134
and the PCT application having Page White and Farrer file number
88061. The contents of these applications are incorporated herein
by reference.
* * * * *