U.S. patent application number 14/697455 was filed with the patent office on 2015-08-27 for mass spectrometric quantitation.
This patent application is currently assigned to Electrophoretics Limited. The applicant listed for this patent is Electrophoretics Limited. Invention is credited to Karsten Kuhn, Ian Pike, Peter Schulzknappe.
Application Number | 20150241443 14/697455 |
Document ID | / |
Family ID | 37988840 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150241443 |
Kind Code |
A1 |
Schulzknappe; Peter ; et
al. |
August 27, 2015 |
MASS SPECTROMETRIC QUANTITATION
Abstract
Provided is a method of assaying for an analyte, including
combining a test sample having the analyte, with a calibration
sample having at least two different aliquots of the analyte, each
aliquot having a different known quantity of the analyte. The test
sample and each aliquot are differentially labeled with one or more
isobaric mass labels each with a mass spectrometrically distinct
mass marker group, such that the test sample and each aliquot of
the calibration sample can be distinguished by mass spectrometry.
The method further includes determining by mass spectrometry the
quantity of analyte in the test sample and in each aliquot, and
calibrating the quantity of analyte in the test sample against
known and determined quantities of analytes in the aliquots.
Inventors: |
Schulzknappe; Peter;
(Frankfurt-am-Main, DE) ; Pike; Ian; (Cobham
Surrey, GB) ; Kuhn; Karsten; (Hofheim Am Taunus,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Electrophoretics Limited |
Cobham Surrey |
|
GB |
|
|
Assignee: |
Electrophoretics Limited
Cobham Surrey
GB
|
Family ID: |
37988840 |
Appl. No.: |
14/697455 |
Filed: |
April 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12530747 |
Mar 8, 2010 |
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PCT/EP2008/052962 |
Mar 12, 2008 |
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14697455 |
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Current U.S.
Class: |
435/5 ; 435/29;
435/6.1; 435/7.92; 436/15 |
Current CPC
Class: |
G01N 2333/775 20130101;
G01N 33/6851 20130101; G01N 2560/00 20130101; H01J 49/0009
20130101; Y10T 436/105831 20150115; G01N 33/96 20130101; G01N 1/28
20130101; G01N 2333/765 20130101; G01N 33/58 20130101; G01N
2001/2893 20130101; G01N 33/6848 20130101; G01N 2458/15 20130101;
G01N 2570/00 20130101; G01N 2333/4703 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2007 |
GB |
0704764 |
Claims
1-46. (canceled)
47. A method of assaying for an analyte, comprising: a) combining a
test sample, comprising the analyte, with a calibration sample
comprising at least two different aliquots of the analyte, each
aliquot having a different known quantity of the analyte, wherein
the sample and each aliquot are differentially labeled with one or
more isobaric mass labels each with a mass spectrometrically
distinct mass marker group, such that the test sample and each
aliquot of the calibration sample can be distinguished by mass
spectrometry; b) determining by mass spectrometry the quantity of
the analyte in the test sample and the quantity of analyte in each
aliquot in the calibration sample, and calibrating the quantity of
the analyte in the test sample against the known and determined
quantities of the analytes in the aliquots in the calibration
sample.
48. A method according to claim 47, wherein the test sample
comprises a plurality of different analytes and a calibration
sample is provided for each different analyte, and wherein step (b)
is repeated for each different analyte, preferably wherein the
plurality of analytes are peptide fragments of a protein or
polypeptide which are produced by chemical or enzymatic processing
of the protein or polypeptide prior to step (a), and/or wherein the
different aliquots of each calibration sample are selected such
that each calibration sample provides a range of quantities of
analyte which are different to the range of quantities of analyte
in other calibration samples.
49. A method according to claim 47, wherein a plurality of test
samples are assayed for an analyte, preferably wherein each of the
plurality of test samples is assayed for the same analyte, and/or
wherein each of the test samples is differentially labelled with
one or more of the isobaric mass labels and combined with a single
calibration sample in step (a), and the quantity of the analyte in
each sample is determined simultaneously in step (b), or
alternatively wherein each test sample is labelled with the same
mass label, and steps (a) and (b) are repeated for each different
sample, preferably wherein the same calibration sample is used for
each test sample to be assayed.
50. A method according to claim 47, wherein the method comprises a
further step prior to step (a) of differentially labelling each
test sample or each aliquot of the calibration sample with one or
more isobaric mass labels, preferably wherein the method comprises
a further step of combining the differentially labelled aliquots to
produce a calibration sample prior to step (a).
51. A method according to claim 47, wherein step (b) comprises: i)
in a mass spectrometer selecting and fragmenting ions of a mass to
charge ratio corresponding to the analyte labelled with the mass
label, detecting and producing a mass spectrum of fragment ions,
and identifying the fragment ions corresponding to the mass marker
groups of the mass labels; ii) determining the quantity of the
analyte in each test sample on the basis of the quantity of their
mass marker groups in a mass spectrum relative to the quantities of
the mass marker groups from the aliquots of the calibration sample
in the same mass spectrum.
52. A method according to claim 47, wherein the quantity of an
analyte in each aliquot in the calibration sample is a known
absolute quantity, preferably wherein the absolute quantity of an
analyte in a test sample is determined in step (b).
53. A method according to claim 47, wherein the quantity of analyte
in each aliquot in the calibration sample is a known qualitative
quantity, preferably wherein the qualitative quantity is an
expected range of quantities of analyte in a subject having a
particular state, and/or wherein the calibrating step comprises
calibrating the quantity of the analyte in the test sample against
the known qualitative and determined quantities of the analytes in
the aliquots of the calibration sample, more preferably wherein the
percentage change in the amount of the analyte in the test sample
is determined.
54. A method according to claim 47, wherein the quantity of analyte
in each different aliquot is selected to reflect the known or
suspected variation in the quantity of the analyte in the test
sample, preferably wherein aliquots are provided which comprise the
analyte in quantities which correspond to the upper and lower
limits, and optionally intermediate points within a range of the
known or suspected quantities of the analyte found in test samples
of healthy or diseased subjects.
55. A method according to claim 47, wherein the different
quantities of analyte present in the different aliquots correspond
to the known or suspected quantity of analyte present in a test
sample which has been incubated for different periods of time.
56. A method according to claim 47, wherein the aliquots are taken
from a sample which is a standardized form of the test sample.
57. A method according to claim 47, wherein the test sample and/or
the aliquots of the calibration sample are from a plant or an
animal, preferably wherein the animal is a human.
58. A method according to claim 47, wherein the calibration sample
comprises an analyte in a quantity that indicates the efficacy
and/or toxicity of a therapy, and/or wherein the test sample and/or
the calibration sample comprises human or animal tissue, blood,
plasma, serum, cerebrospinal fluid, synovial fluid, ocular fluid,
urine, tears, tear duct exudates, lung aspirates, breast milk,
nipple aspirate, semen, lavage fluid, cell extract, tissue culture
extract, plant tissue, plant fluid, plant cell culture extract, a
bacterial sample, a virus sample, fungus, fermentation broth, a
foodstuff or a pharmaceutical composition, and/or wherein the
analyte comprises a protein, a polypeptide, a peptide, an amino
acid or a nucleic acid, a peptide-nucleic acid, a sugar, starch, a
complex carbohydrate, a lipid, a polymer, or fragments thereof.
59. A method according to claim 47, which further includes the step
of separating the isobarically labelled analytes
electrophoretically or chromatographically after step (a) but
before step (b).
60. A method according to claim 47, wherein the calibration sample
comprises a further aliquot which comprises the analyte in a
quantity which serves as a trigger during an MS scan or during
non-scanning MS/MS to initiate an MS/MS scan, preferably wherein
the analyte in the further aliquot is labelled with an isobaric
mass label, or alternatively wherein the analyte in the further
aliquot is labelled with a mass label which is chemically identical
to but isotopically distinct and differing in mass from the
isobaric mass labels of the other analytes in the calibration
sample.
61. A method according to claim 47, wherein the analyte in the
sample is a protein, and the analyte in the calibration sample is a
recombinant form of the protein in the sample.
62. A method according to claim 47, wherein the mass label
comprises the following structure: X-L-M wherein X is a mass marker
moiety comprising the following group: ##STR00017## wherein the
cyclic unit is aromatic or aliphatic and comprises from 0-3 double
bonds independently between any two adjacent atoms; each Z is
independently N, N(R.sup.1), C(R.sup.1), CO, CO(R.sup.1),
C(R.sup.1).sub.2, O or S; X is N, C or C(R.sup.1); each R.sup.1 is
independently H, a substituted or unsubstituted straight or
branched C.sub.1-C.sub.6 alkyl group, a substituted or
unsubstituted aliphatic cyclic group, a substituted or
unsubstituted aromatic group or a substituted or unsubstituted
heterocyclic group; and y is an integer from 0-10, L is a cleavable
linker and M is a mass normalization moiety, preferably wherein the
cleavable linker attaching the mass marker moiety to the mass
normalization moiety is a linker cleavable by collision, more
preferably wherein the linker is cleavable by CID, ETD, ECD or SID
using mass spectrometry.
63. A method according to claim 50, wherein the labelling step
comprises a step of reacting the analyte with a reactive mass
label, wherein the reactive mass label comprises a mass label and a
reactive functionality, preferably wherein the reactive
functionality is capable of reacting with any amino group on a
polypeptide and comprises a nucleophile or an electrophile, more
preferably wherein the reactive functionality comprises the
following group: ##STR00018## wherein each R.sup.2 is independently
H, a substituted or unsubstituted straight or branched
C.sub.1-C.sub.6 alkyl group, a substituted or unsubstituted
aliphatic cyclic group, a substituted or unsubstituted aromatic
group or a substituted or unsubstituted heterocyclic group.
64. A method according to claim 62, wherein the mass label is a
mass label from a set of two or more mass labels, wherein each mass
normalization moiety ensures that a mass label has a desired
aggregate mass, and wherein the set comprises: mass labels having a
mass marker moiety, each mass marker moiety having a mass different
from that of all other mass marker moieties in the set, and each
label in the set having a common aggregate mass, and wherein all
the mass labels in the set are distinguishable from each other by
mass spectroscopy, and preferably wherein each mass label in the
set has a mass adjuster moiety, selected from: (a) an isotopic
substituent situated within the mass marker moiety and/or within
the mass normalization moiety, and (b) substituent atoms or groups
attached to the mass marker moiety and/or attached to the mass
normalization moiety, and/or further preferably wherein the mass
adjuster moiety is selected from a halogen atom substituent, a
methyl group substituent, and .sup.2H, .sup.15N, .sup.13C or
.sup.18O isotopic substituents, and more preferably wherein the
mass adjuster moiety is .sup.15N or .sup.13C and the set comprises
two mass labels having the following structures: ##STR00019## or
alternatively more preferably wherein the mass adjuster moiety is
.sup.15N and .sup.13C and the set comprises five mass labels having
the following structures: ##STR00020## or still alternatively more
preferably wherein the mass adjuster moiety is .sup.15N and
.sup.13O and the set comprises six mass labels having the following
structures: ##STR00021##
65. A calibration sample for use in a mass spectrometric assay for
an analyte which comprises at least two different aliquots of the
analyte, each aliquot having a different known quantity of the
analyte wherein each aliquot is differentially labelled with an
isobaric mass label as defined in claim 62.
Description
[0001] This invention relates to mass spectrometry methods of
assaying for an analyte by labelling test samples and calibration
samples with isobaric mass labels. Relative and/or absolute
quantitation of the analytes of interest is particularly
facilitated by the invention.
[0002] Various methods of labelling molecules of interest are known
in the art, including radioactive atoms, fluorescent dyes,
luminescent reagents, electron capture reagents and light absorbing
dyes. Each of these labelling systems has features which make it
suitable for certain applications and not others. For reasons of
safety, interest in non-radioactive labelling systems lead to the
widespread commercial development of fluorescent labelling schemes
particularly for genetic analysis. Fluorescent labelling schemes
permit the labelling of a relatively small number of molecules
simultaneously, typically four labels can be used simultaneously
and possibly up to eight. However the costs of the detection
apparatus and the difficulties of analysing the resultant signals
limit the number of labels that can be used simultaneously in a
fluorescence detection scheme.
[0003] More recently there has been development in the area of mass
spectrometry as a method of detecting labels that are cleavably
attached to their associated molecule of interest. In many
molecular biology applications one needs to be able to perform
separations of the molecules of interest prior to analysis. These
are generally liquid phase separations. Mass spectrometry in recent
years has developed a number of interfaces for liquid phase
separations which make mass spectrometry particularly effective as
a detection system for these kinds of applications. Until recently
Liquid Chromatography Mass Spectrometry was used to detect analyte
ions or their fragment ions directly, however for many applications
such as nucleic acid analysis, the structure of the analyte can be
determined from indirect labelling. This is advantageous
particularly with respect to the use of mass spectrometry because
complex biomolecules such as DNA have complex mass spectra and are
detected with relatively poor sensitivity. Indirect detection means
that an associated label molecule can be used to identify the
original analyte, where the label is designed for sensitive
detection and a simple mass spectrum. Simple mass spectra mean that
multiple labels can be used to analyse multiple analytes
simultaneously.
[0004] PCT/GB98/00127 describes arrays of nucleic acid probes
covalently attached to cleavable labels that are detectable by mass
spectrometry which identify the sequence of the covalently linked
nucleic acid probe, The labelled probes of this application have
the structure Nu-L-M where Nu is a nucleic acid covalently linked
to L, a cleavable linker, covalently linked to M, a mass label.
Preferred cleavable linkers in this application cleave within the
ion source of the mass spectrometer. Preferred mass labels are
substituted poly-aryl ethers. This application discloses a variety
of ionisation methods and analysis by quadrupole mass analysers,
TOF analysers and magnetic sector instruments as specific methods
of analysing mass labels by mass spectrometry.
[0005] PCT/GB94/01675 discloses ligands, and specifically nucleic
acids, cleavably linked to mass tag molecules. Preferred cleavable
linkers are photo-cleavable. This application discloses Matrix
Assisted Laser Desorption Ionisation (MALDI) Time of Flight (TOF)
mass spectrometry as a specific method of analysing mass labels by
mass spectrometry.
[0006] PCT/US97/22639 discloses releasable non-volatile mass-label
molecules, In preferred embodiments these labels comprise polymers,
typically biopolymers which are cleavably attached to a reactive
group or ligand, i.e. a probe. Preferred cleavable linkers appear
to be chemically or enzymatically cleavable. This application
discloses MALDI TOF mass spectrometry as a specific method of
analysing mass labels by mass spectrometry.
[0007] PCT/US97/01070, PCT/US97/01046, and PCT/US97/01304 disclose
ligands, and specifically nucleic acids, cleavably linked to mass
tag molecules. Preferred cleavable linkers appear to be chemically
or photo-cleavable, These applications disclose a variety of
ionisation methods and analysis by quadrupole mass analysers, TOF
analysers and magnetic sector instruments as specific methods of
analysing mass labels by mass spectrometry.
[0008] None of these prior art applications mention the use of
tandem or serial mass analysis for use in analysing mass
labels.
[0009] Gygi et al. (Nature Biotechnology 17: 994-999, "Quantitative
analysis of complex protein mixtures using isotope-coded affinity
tags" 1999) disclose the use of `isotope encoded affinity tags` for
the capture of peptides from proteins, to allow protein expression
analysis. In this article, the authors describe the use of a biotin
linker, which is reactive to thiols, for the capture peptides with
cysteine in them. A sample of protein from one source is reacted
with the biotin linker and cleaved with an endopeptidase. The
biotinylated cysteine-containing peptides can then be isolated on
avidinated beads for subsequent analysis by mass spectrometry. Two
samples can be compared quantitatively by labelling one sample with
the biotin linker and labelling the second sample with a deuterated
form of the biotin linker. Each peptide in the samples is then
represented as a pair of peaks in the mass spectrum. Integration of
the peaks in the mass spectrum corresponding to each tag indicates
the relative expression levels of the peptide linked to the
tags.
[0010] PCT/GB01/01122 discloses a set of two or more mass labels,
each label in the set comprising a mass marker moiety attached via
a cleavable linker to a mass normalisation moiety, the mass marker
moiety being fragmentation resistant. The aggregate mass of each
label in the set may be the same or different and the mass of the
mass marker moiety of each label in the set may be the same or
different. In any group of labels within the set having a mass
marker moiety of a common mass each label has an aggregate mass
different from all other labels in that group, and in any group of
labels within the set having a common aggregate mass each label has
a mass marker moiety having a mass different from that of all other
mass marker moieties in that group, such that all of the mass
labels in the set are distinguishable from each other by mass
spectrometry. This application also discloses an array of mass
labels, comprising two or more sets of mass labels as defined
above. The aggregate mass of each of the mass labels in any one set
is different from the aggregate mass of each of the mass labels in
every other set in the array. This application further discloses
methods of analysis comprising detecting an analyte by identifying
by mass spectrometry a mass label or a combination of mass labels
unique to the analyte. This application discloses a vast number of
different specific mass labels. Preferred mass labels have the
structure M-L-X, where M is the mass normalization group, L is the
cleavable linker and X is the mass marker moiety. The nature of M
and X is not particularly limited.
[0011] PCT/GB02/04240 discloses a set of two or more mass labels,
each label in the set comprising a mass marker moiety attached via
at least one amide bond to a mass normalisation moiety.
[0012] The mass marker moiety comprises an amino acid and the mass
normalisation moiety comprises an amino acid. As for PCT/GB01/01122
the aggregate mass of each label in the set may be the same or
different and the mass of the mass marker moiety of each label in
the set may be the same or different such that all of the mass
labels in the set are distinguishable from each other by mass
spectrometry. As for PCT/GB01/01122 this application also discloses
an array of mass labels and a method of analysis. This application
is specifically directed to the analysis of peptides and mass
labels with mass normalisation moieties and mass marker moieties
comprising at least one amino acid.
[0013] Whilst the mass labels and methods of analysis disclosed in
PCT/GB01/01122 and PCT/GB02/04240 are by and large successful,
there is still a requirement to provide improved reagents and
methods of relatively or absolutely quantifying an analyte by
providing a mass labelled reference corresponding to the said
analyte, which labelled reference material can be added to the
sample containing the analyte and wherein the analyte and the
reference material can be simultaneously quantified and identified
by tandem mass spectrometry.
[0014] The development of isobaric mass tags in the late 1990's has
revolutionised biomarker discovery. The ability to analyse multiple
samples in theoretically unlimited numbers in a single LC-MS/MS
workflow increases throughput whilst at the same time reducing
analytical variability. Whilst application of these methodologies
provides enhanced biomarker discovery there remains a significant
bottleneck in biomarker validation and development of routine
assays capable of analysing large numbers of samples. This
bottleneck is created by the need to obtain high specificity
reagents, typically in the form of antibodies, against each
candidate biomarker. The production of antibodies is laborious,
costly and takes several months with no guarantee of success.
[0015] In addition to cost and time constraints, use of antibody
based methods for biomarker validation are also hampered by the
limit of such methods to detect analytes with widely different
normal and regulated concentration ranges. For example it is seldom
possible to measure more than 10 up to 20 different analytes in a
single multiplex assay using antibody arrays. Where the normal
concentration of such proteins is separated by more than one log
(i.e. micromolar to nanomolar) it is even less likely that such
multiplex antibody arrays can accurately quantify each analyte and
multiplexing rates are consequently significantly lower.
[0016] There remains therefore a need for improved methods of
quantitatively detecting and routinely measuring analytes by mass
spectrometry in a wide range of samples.
[0017] The majority of protein biomarker discovery is performed
using mass spectrometry linked to various methods of protein
separation. More recently a number of groups have proposed using
mass spectrometers to provide absolute quantitation of proteins
based on one or more isotopically labelled reference peptides. WO
03/016861 discloses one such embodiment termed `AQUA` which uses
synthetic peptides incorporating one or more stable isotope
labelled amino acids as a reference standard. Such peptides are
normally selected based on a number of criteria including their
ionisation behaviour, physicochemical properties, and ease and cost
of manufacture. In an Aqua experiment the reference peptides are
spiked into the sample of interest at a defined concentration.
Because they are labelled with stable isotopes the reference
peptide will produce a distinct peak from the naturally occurring
form of the peptide in the sample of interest. Typically the AQUA
peptide mass will be separated by an increased mass of about 5-50
daltons compared to the natural peptide. By comparing the relative
peak intensities of the natural peptide and its AQUA equivalent the
absolute concentration of the parent peptide in the sample can be
determined.
[0018] Whilst AQUA is able to measure absolute quantities of
multiple proteins in a single experiment, it is not suitable for
development of reference standard curves to cover a range of
naturally occurring concentrations. For biomarker validation
studies this may be problematic since regulation of protein
expression may result in a ten-fold or greater range of
concentrations for a given protein. Using a single reference
standard may lead to inaccurate quantitation of natural peptide
levels at the extremes of regulation and it would be desirable to
provide a means of including readily distinguishable reference
peptides at several different concentrations to cover the
physiological range and which provide an appropriate standard curve
against which the level of the target peptide in a sample can be
read. Producing such curves using AQUA would be difficult since
each added peptide increases the complexity of the MS profile. In
addition, to ensure that the standard curve is built only on the
reference peptides it would be advisable if not essential to
perform sequence confirmation by MS/MS of each reference peptide as
well as for the target peptide in the sample.
[0019] It is an aim of the present invention to solve one or more
of the problems with the prior art described above. Specifically,
it is an aim or the present invention to provide an improved mass
spectrometric method of assaying for an analyte.
[0020] To overcome the limitations of the art the inventors have
developed a method of quantifying molecules of interest using
isobarically tagged reference biomolecules or complex biological
materials, for example peptides, that allow construction of
multi-point standard curves for each analyte without increasing MS
complexity.
[0021] Accordingly, the present invention provides a method of
assaying for an analyte, which method comprises: [0022] a)
combining a test sample, which may comprise the analyte, with a
calibration sample comprising at least two different aliquots of
the analyte, each aliquot having a different known quantity of the
analyte, wherein the sample and each aliquot are differentially
labelled with one or more isobaric mass labels each with a mass
spectrometrically distinct mass marker group, such that the test
sample and each aliquot of the calibration sample can be
distinguished by mass spectrometry; [0023] b) determining by mass
spectrometry the quantity of the analyte in the test sample and the
quantity of analyte in each aliquot in the calibration sample, and
calibrating the quantity of the analyte in the test sample against
the known and determined quantities of the analytes in the aliquots
in the calibration sample.
[0024] The different aliquots each have a known quantity of the
analyte. The term "known quantity" means that the absolute
quantity, or a qualitative quantity of the analyte in each aliquot
of the calibration sample is known. A qualitative quantity in the
present context means a quantity which is not known absolutely, but
may be a range of quantities that are expected in a subject having
a particular state, for example a subject in a healthy or diseased
state, or some other expected range depending on the type of test
sample under investigation. Each aliquot is "different" since it
contains a different quantity of the analyte. Typically this is
achieved by taking different volumes from a standard sample,
especially for qualitative quantities where taking different
volumes will ensure that different quantities are present in each
aliquot in a desired ratio, without needing to know the absolute
quantities.
[0025] Preferably, step (b) comprises: [0026] i) in a mass
spectrometer selecting and fragmenting ions of a mass to charge
ratio corresponding to the analyte labelled with the mass label,
detecting and producing a mass spectrum of fragment ions, and
identifying the fragment ions corresponding to the mass marker
groups of the mass labels; [0027] ii) determining the quantity of
the analyte in each test sample on the basis of the quantity of
their mass marker groups in a mass spectrum relative to the
quantities of the mass marker groups from the aliquots of the
calibration sample in the same mass spectrum.
[0028] Typically, the fragmentation is caused by Collision Induced
Dissociation (CID), Surface Induced Dissociation (SID), Electron
Capture Dissociation (ECD), Electron Transfer Dissociation (ETD),
or Fast Atom Bombardment.
[0029] Electron capture dissociation (ECD) is a method of
fragmenting multiply charged (protonated) peptide or proteins ions
for tandem mass spectrometric analysis (structural elucidation). In
this method multiply protonated peptide or proteins are confined in
the Penning trap of a Fourier transform ion cyclotron resonance
(FTICR) mass spectrometer and exposed to electrons with
near-thermal energies. The capture of a thermal electron by a
protonated peptide is exothermic (.apprxeq.6 eV; 1
eV=1.602.times.10.sup.-19 J), and causes the peptide backbone to
fragment by a nonergodic process (i.e., a process that does not
involve intramolecular vibrational energy redistribution).
[M+nH].sup.n++e.sup.-.fwdarw.[[M+nH].sup.(n-1)+]*.fwdarw.fragments
[0030] In addition, one or more protein cations can be neutralized
with low energy electrons to cause specific cleavage of bonds to
form c, z products, in contrast to b, y products formed by other
techniques such as collisionally activated dissociation (CAD; also
known as collision-induced dissociation, CID). Since thermal
electrons introduced into the RF fields of RF 3D quadrupole ion
trap (QIT), quadrupole time-of-flight, or RF linear 2D quadrupole
ion trap (QLT) instruments maintain their thermal energy only for a
fraction of a microsecond and are not trapped in these devices, ECD
remains a technique exclusively used with FTICR, the most expensive
type of MS instrumentation.
[0031] Electron transfer dissociation (ETD) is a method of
fragmenting multiply protonated peptide or proteins ions for tandem
mass spectrometric analysis (structural elucidation). Similar to
electron capture dissociation (ECD), ETD induces fragmentation of
cations (e.g. multiple charged peptide or proteins) by transferring
electrons to them. In contrast to ECD, ETD does not use free
electrons but employs radical anions for this purpose (e.g.
anthracene or azobenzene anions which possess sufficiently low
electron affinities to act as electron donors).
[M+nH].sup.n++A.sup.-.fwdarw.[[M+nH].sup.(n-1)+]*+A.fwdarw.fragments
[0032] After the electron transfer, ETD results in a similar
fragmentation pattern as ECD, i.e. the formation of so called c and
z ions. Based on the different way of electron transfer, ETD can be
implemented on various "lower cost" mass spectrometers like
quadrupole ion trap (QIT) or RF linear 2D quadrupole ion trap (QLT)
instruments which are not appropriate for ECD. For an appropriate
reference see John E. P. Syka, Joshua J. Coon, Melanie J.
Schroeder, Jeffrey Shabanowitz, and Donald F. Hunt, PNAS, Vol. 101,
no. 26, pp. 9528-9533.
[0033] The most preferred embodiment is where the fragmentation is
caused by collision-induced dissociation. Collision-induced
dissociation occurs during an MS/MS experiment. The term `MS/MS` in
the context of mass spectrometry refers to an experiment which
involves selecting ions, subjecting selected ions to CID and
subjecting the fragment ions to further analysis.
[0034] This method enables multi-point calibration of the quantity
of each analyte without increasing MS complexity. Analyte
quantitation is obtained in the MS/MS profile, and the analyte in
the sample and in the calibration sample can be simultaneously
quantified and identified by tandem mass spectrometry. This method
provides means for the measurement of up to 10, up to 20, up to 50
or more analytes in a single LC-MS/MS experiment.
[0035] The method may comprise a further step prior to step (a) of
differentially labelling each test sample or each aliquot of the
calibration sample with one or more isobaric mass labels. In a
preferred embodiment the method also comprises a further step of
combining the differentially labelled aliquots to produce a
calibration sample prior to step (a).
[0036] The test sample may comprise a plurality of different
analytes, and in this case a calibration sample is provided for
each different analyte, and step (b) is repeated for each different
analyte. In one embodiment the plurality of analytes are peptide
fragments of a protein or polypeptide which are produced by
chemical or enzymatic processing of the protein or polypeptide
prior to step (a). In a particular embodiment, the plurality of
analytes are peptides from the same protein or polypeptide.
[0037] In one embodiment, a plurality of test samples is assayed
for an analyte. In a particular embodiment, each of the plurality
of test samples is assayed for the same analyte. In this case, each
of the test samples may be differentially labelled with one or more
isobaric mass labels and combined with a single calibration sample
in step (a), and the quantity of the analyte in each sample is
determined simultaneously in step (b). In another embodiment, each
test sample is labelled with the same mass label, and steps (a) and
(b) are repeated for each different sample. The same calibration
sample can be used for each test sample to be assayed. Typically,
the same known volume of the calibration sample comprising at least
two aliquots of the analyte is added to each different test sample.
This method is particularly useful in clinical studies involving
multiple samples from patients. If a large quantity of the
calibration sample is prepared and fractions taken, the same
calibration sample can be used by multiple laboratories,
facilitating cross-study and cross-laboratory comparisons.
[0038] In a method according to the invention, the quantity of
analyte in each aliquot in the calibration sample is a known
absolute quantity. This allows for the absolute quantity of an
analyte in a test sample to be determined in step (b).
[0039] In an alternative method, the absolute quantity of an
analyte in each aliquot in the calibration sample is not known. In
this embodiment, the quantity of analyte in each aliquot in the
calibration sample is a known qualitative quantity. The calibrating
step comprises calibrating the quantity of the analyte in the test
sample against the qualitative and determined quantities of the
analytes in the aliquots of the calibration sample. In a particular
embodiment, the qualitative quantity is an expected range of
quantities of analyte in a subject having a particular state, such
as a healthy or diseased state.
[0040] In a preferred embodiment, the quantity of analyte in each
different aliquot is selected to reflect the known or suspected
variation in the quantity of the analyte in the test sample. In a
yet further preferred embodiment, aliquots are provided which
correspond to the upper and lower limits, and optionally
intermediate points within a range of the known or suspected
quantities of the analyte found in test samples of healthy or
diseased subjects. The different quantities of analyte present in
the different aliquots may correspond to the known or suspected
quantity of analyte present in a test sample which has been
incubated for different periods of time.
[0041] The calibration sample may comprise an analyte in a quantity
that indicates the presence and/or stage of a particular disease.
The calibration sample may also comprise the analyte in a quantity
which indicates the efficacy and/or toxicity of a therapy.
[0042] The method according to the present invention may comprise a
further step of separating the components of the samples prior to
step (a). The method may also comprise a step of digesting each
sample with at least one enzyme to digest components of the samples
prior to step (a). In one embodiment the samples are labelled with
the isobaric mass labels prior to digestion. In another embodiment,
the labeling step occurs after the digestion step. The enzyme
digestion step may also occur after step (a) but before step
(b).
[0043] In another embodiment, the mass labels used in the method
further comprise an affinity capture ligand. The affinity capture
ligand of the mass label binds to a counter-ligand so as to
separate the isobarically labeled analytes from the unlabelled
analytes after step (a) but before step (b). The affinity capture
ligand provides a means of enrichment of the analytes of interest,
thereby increasing analytical sensitivity.
[0044] The method according to the invention may further include
the step of separating the isobarically labeled analytes
electrophoretically or chromatographically after step (a) but
before step (b).
[0045] Although the structure of the mass labels used in the
present invention is not especially limited, providing that they
are isobaric and have mass spectrometrically distinct mass marker
groups (moieties), in preferred embodiments the mass label
comprises the following structure:
X-L-M
wherein X is a mass marker moiety, L is a cleavable linker and M is
a mass normalisation moiety. L may be a single bond, or part of X,
or part of M. These mass labels may be attached at any point to the
analyte in the test or calibration samples, e.g. through M, L or X.
Preferably, they are attached through M, e.g. the label would
comprise the structure:
(X-L-M)-
[0046] This is typically effected by including a reactive
functionality in the mass label to allow it to bind to the analyte,
e.g:
X-L-M-reactive functionality
[0047] When the labels comprise a reactive functionality these are
termed reactive mass labels.
[0048] In preferred embodiments X is a mass marker moiety
comprising the following group:
##STR00001##
wherein the cyclic unit is aromatic or aliphatic and comprises from
0-3 double bonds independently between any two adjacent atoms; each
Z is independently N, N(R.sup.1), C(R.sup.1), CO, CO(R.sup.1) (i.e.
--O--C(R.sup.1)-- or --C(R.sup.1)--O--), C(R.sup.1).sub.2, O or S;
X is N, C or C(R.sup.1); each R.sup.1 is independently H, a
substituted or unsubstituted straight or branched C.sub.1-C.sub.6
alkyl group, a substituted or unsubstituted aliphatic cyclic group,
a substituted or unsubstituted aromatic group or a substituted or
unsubstituted heterocyclic group; and y is an integer from
0-10.
[0049] The reactive functionality for attaching the mass label to
the analyte is not especially limited and may comprise any
appropriate reactive group.
[0050] The term mass label used in the present context is intended
to refer to a moiety suitable to label an analyte for
determination. The term label is synonymous with the term tag.
[0051] The term mass marker moiety used in the present context is
intended to refer to a moiety that is to be detected by mass
spectrometry. The term mass marker moiety is synonymous with the
term mass marker group or the term reporter group.
[0052] The term mass normalisation moiety used in the present
context is intended to refer to a moiety that is not necessarily to
be detected by mass spectrometry, but is present to ensure that a
mass label has a desired aggregate mass. The mass normalisation
moiety is not particularly limited structurally, but merely serves
to vary the overall mass of the mass label.
[0053] In the above general formula, when Z is C(R.sup.1).sub.2,
each R.sup.1 on the carbon atom may be the same or different (i.e.
each R.sup.1 is independent). Thus the C(R.sup.1).sub.2 group
includes groups such as CH(R.sup.1), wherein one R.sup.1 is H and
the other R.sup.1 is another group selected from the above
definition of R.sup.1.
[0054] In the above general formula, the bond between X and the
non-cyclic Z may be single bond or a double bond depending upon the
selected X and Z groups in this position. For example, when X is N
or C(R.sup.1) the bond from X to the non-cyclic Z must be a single
bond. When X is C, the bond from X to the non-cyclic Z may be a
single bond or a double bond depending upon the selected non-cyclic
Z group and cyclic Z groups. When the non-cyclic Z group is N or
C(R.sup.1) the bond from non-cyclic Z to X is a single bond or if y
is 0 may be a double bond depending on the selected X group and the
group to which the non-cyclic Z is attached. When the non-cyclic Z
is N(R.sup.1), CO(R.sup.1), CO, C(R.sup.1).sub.2, O or S the bond
to X must be a single bond. The person skilled in the art may
easily select suitable X, Z and (CR.sup.1.sub.2).sub.y groups with
the correct valencies (single or double bond links) according to
the above formula.
[0055] The present inventors have discovered that the mass labels
defined above can be easily identified in a mass spectrometer and
also allow sensitive quantification.
[0056] In a preferred embodiment the aggregate molecular weight of
the mass label is 600 Daltons or less, more preferably 500 Daltons
or less, still more preferably 400 Daltons or less, most preferably
from 300 to 400 Daltons. Particularly preferred molecular weights
of the mass labels are 324, 338, 339 and 380 Daltons. These
preferred embodiments are particularly advantageous because the
small size of the mass labels means that the size of the peptide to
be detected is minimally increased when labelled with the mass
label. Therefore, the peptide labelled with the mass label may be
viewed in the same mass spectrum window as unlabelled peptide when
analysed by mass spectroscopy. This facilitates identification of
peaks from the mass label itself.
[0057] In a preferred embodiment, the molecular weight of the mass
marker moiety is 300 Daltons or less, preferably 250 Daltons or
less, more preferably 100 to 250 Daltons, most preferably 100-200
Daltons. These preferred embodiments are particularly advantageous
because the small size of the mass marker moiety means that it
produces a peak in the silent region of a mass spectrum, which
allows the mass marker to be easily identified from the mass
spectrum and also allows sensitive quantification.
[0058] The term silent region of a mass spectrum (such as an MS/MS
spectrum) used in the present context is intended to refer to the
region of a mass spectrum with low background "noise" caused by
peaks relating to the presence of fragments generated by
fragmentation of the labelled peptides. An MS/MS spectrum is
obtained by the fragmentation of one peak in MS-mode, such that no
contaminants, such as buffering reagents, denaturants and detergent
should appear in the MS/MS spectrum. In this way, quantification in
MS/MS mode is advantageous. Thus, the term silent region is
intended to refer to the region of the mass spectrum with low
"noise" caused by peaks relating to the peptide to be detected. For
a peptide or protein, the silent region of the mass spectrum is
less than 200 Daltons.
[0059] The present inventors have also discovered that the reactive
mass labels defined above are easily and quickly reacted with a
protein to form a labelled protein.
[0060] In the present invention a set of two or more mass labels is
employed. The labels in the sets are isobaric mass labels each
having a mass marker of a different mass. Thus, each label in the
set is as defined above and wherein each mass normalisation moiety
ensures that a mass label has a desired aggregate mass, and wherein
the set comprises:
mass labels having a mass marker moiety, each mass marker moiety
having a mass different from that of all other mass marker moieties
in the set, and each label in the set having a common aggregate
mass; and wherein all the mass labels in the set are
distinguishable from each other by mass spectroscopy.
[0061] The term "isobaric" means that the mass labels have
substantially the same aggregate mass as determined by mass
spectrometry. Typically, the average molecular masses of the
isobaric mass labels will fall within a range of .+-.0.5 Da of each
other. The term "labels" shall be synonymous with the term "tags".
In the context of the present invention, the skilled addressee will
understand that the term "mass marker moiety" and the term
"reporter group" can be used interchangeably.
[0062] The number of labels in the set is not especially limited,
provided that the set comprises a plurality of labels. However, it
is preferred if the set comprises two or more, three or more, four
or more, or five or more labels, more preferably six or more
labels, most preferably eight or more labels.
[0063] The term aggregate mass in the present context refers to the
total mass of the mass label, i.e. the sum of the masses of the
mass marker moiety, the cleavable linker, the mass normalisation
moiety and any other components of the mass label.
[0064] The mass of the mass normalisation moiety will be different
in each mass label in the set. The mass of the mass normalisation
moiety in each individual mass label will be equal to the common
aggregate mass minus the mass of the particular mass marker moiety
in that mass label and minus the mass of the cleavable linker.
[0065] All mass labels in the set are distinguishable from each
other by mass spectroscopy. Therefore, a mass spectrometer can
discriminate between the mass labels, i.e. the peaks derived from
individual mass labels can be clearly separated from one another.
The difference in mass between the mass marker moieties means that
a mass spectrometer can discriminate between ions derived from
different mass labels or mass marker moieties.
[0066] The present invention may also employ an array of mass
labels, comprising two or more sets of mass labels as defined
above, wherein the aggregate mass of each of the mass labels in any
one set is different from the aggregate mass of each of the mass
labels in every other set in the array.
[0067] In preferred embodiments of the invention, the array of mass
labels are preferably all chemically identical (substantially
chemically identical). The term "substantially chemically
identical" means that the mass labels have the same chemical
structure, into which particular isotopic substitutions may be
introduced or to which particular substituents may be attached.
[0068] In further preferred embodiments of this invention, the mass
labels may comprise a sensitivity enhancing group. The mass labels
are preferably of the form: [0069] sensitivity enhancing group
-X-L-M- reactive functionality
[0070] In this example the sensitivity enhancing group is usually
attached to the mass marker moiety, since it is intended to
increase the sensitivity of the detection of this moiety in the
mass spectrometer. The reactive functionality is shown as being
present and attached to a different moiety than the sensitivity
enhancing group. However, the mass labels need not be limited in
this way and in some cases the sensitivity enhancing group may be
attached to the same moiety as the reactive functionality.
[0071] In a further aspect, the present invention provides a method
of assaying a low abundance analyte in a sample, which method
comprises the method of mass spectrometric analysis as defined
above, wherein the calibration sample comprises a large quantity of
the analyte to be assayed, and the sample may comprise the analyte
in low abundance. In this method, the analyte is present in the
calibration sample in a quantity such that it can be readily
detected and separated together with the analyte in the sample by a
method such as one or two-dimensional gel electrophoresis,
free-flow electrophoresis, capillary electrophoresis, off-gel
isoelectric focusing or liquid chromatography mass spectrometry
prior to step (b). Preferably, the analyte in the sample is a
protein, and the analyte in the calibration sample is a recombinant
form of the protein in the sample.
[0072] The present invention will now be described further by way
of example only with reference to the accompanying drawings, in
which:
[0073] FIG. 1 shows a schematic of a method according to the
present invention.
[0074] FIG. 2 shows the MS/MS profile of one BSA tryptic peptide.
Upper panel shows the full MS/MS spectrum. Lower panel shows an
expansion of the isobaric mass marker moiety region, the different
intensities reflecting different abundances of the same peptide in
the study sample (126) and calibration sample (128, 129, 130,
131).
[0075] FIG. 3 shows the four point calibration curve for a set of
isobarically-labelled bovine serum albumin aliquots in a
calibration sample.
[0076] FIG. 4 shows a schematic of a method of preparing a plasma
sample for use in the present invention.
[0077] FIG. 5 shows a schematic of a method according to the
present invention wherein prior to mass spectrometry analysis a
mixture comprising a sample and a calibration sample is run on a 1D
PAGE gel, and an appropriate spot on the gel is picked and
digested.
[0078] FIG. 6 shows a schematic of a method according to the
present invention for assaying an analyte in a plurality of
samples.
[0079] FIG. 7 shows an accumulated MS from the retention profile of
a labelled peptide from clusterin as described in Example 3 below.
Inset-zoom view of m/z region 915-935 showing the peptide of
interest.
[0080] FIG. 8 shows an accumulated MS/MS spectrum of the labelled
peptide from clusterin. Insert: Zoom view at the mass marker
region.
[0081] FIG. 9 shows a calibration curve of the chosen clusterin
peptide.
[0082] FIG. 10 shows a MALDI MS/MS spectrum of peptide FQVDNNNR as
described in Example 4 below.
[0083] FIG. 11 shows a MALDI MS/MS spectrum of peptide
GAYPLSIEPIGVR as described in Example 4 below.
[0084] FIG. 12 shows a MALDI MS/MS spectrum of peptide GQYCYELDEK
as described in Example 4 below.
[0085] The present invention will now be described in detail.
[0086] This invention provides useful reagents for determining
relative and/or absolute quantities of analytes such as peptides,
proteins, nucleotides and nucleic acids and means of their
production. Specifically this invention relates to isobarically
labelled analytes and/or calibration samples for detection by
tandem mass spectrometry and associated methods of analysing test
samples into which such calibration samples have been added.
Relative and/or absolute quantitation of the analytes is
particularly facilitated by the invention.
[0087] This invention provides new methods for assaying analytes by
mass spectrometry in a variety of settings including measurement of
protein, lipid, carbohydrate and nucleic acid changes in cells,
tissues and fluids in human, veterinary, plant, microbial,
pharmaceutical, environmental and security sciences.
[0088] In the methods according to the present invention the
quantity of the analyte in the test sample and in each aliquot of
the calibration sample is determined by mass spectrometry. A
calibration function is used to relate the quantity of the analyte
in the test sample as measured by mass spectrometry to the actual
quantity of the analyte in the test sample. This calibration
function uses the quantities of the analyte in each aliquot of the
calibration sample (both the actual quantities in the aliquots
prior to analysis and the corresponding quantities as measured by
mass spectrometry) as variables.
[0089] In a preferred embodiment, the method comprises a step of
plotting a graph of the quantity of the analyte in each aliquot
versus the quantity of the analyte in each aliquot as determined by
mass spectrometry. This step may instead simply involve
calculation. The quantity of the analyte in the sample is then
calculated by measuring the quantity in the sample as determined by
mass spectrometry against the calibration graph. In the context of
this invention, a reference to "a quantity as measured by mass
spectrometry" is typically an ion abundance, ion intensity, or
other signal measured by mass spectrometry which relates to the
quantity of an analyte.
[0090] Typically, the method comprises: [0091] i) in a mass
spectrometer selecting and fragmenting ions of a mass to charge
ratio corresponding to the analyte labelled with the mass label,
detecting and producing a mass spectrum of fragment ions, and
identifying the fragment ions corresponding to the mass marker
groups of the mass labels; [0092] ii) determining the quantity of
the analyte in each test sample on the basis of the quantity of
their mass marker groups in a mass spectrum relative to the
quantities of the mass marker groups from the aliquots of the
calibration sample in the same mass spectrum.
[0093] In a particular embodiment, the method comprises the steps
of: [0094] 1. Optionally preparing the isobarically labelled
reference material containing a reference biomolecule or mixture of
reference biomolecules by reacting with a set of mass labels
according to this invention; [0095] 2. Labelling a sample in which
the quantity of the biomolecule or mixture of biomolecules is to be
quantified by reacting with one of the same set of mass labels as
used in step 1 above according to this invention; [0096] 3. Adding
a known amount of the isobarically labelled reference material into
the isobarically labelled test sample prepared in step 2; [0097] 4.
Optionally separating the isobarically labelled biomolecules
electrophoretically or chromatographically; [0098] 5. Ionising the
labelled biomolecules in a mass spectrometer; [0099] 6. Selecting
ions of a predetermined mass to charge ratio corresponding to the
mass to charge ratio of the preferred ions of the labelled
biomolecule in a mass analyser; [0100] 7. Inducing dissociation of
these selected ions by collision or electron transfer; [0101] 8.
Detecting the collision products to identify collision product ions
that are indicative of the mass labels; [0102] 9. Producing a
standard curve of ion intensity versus biomolecule amount based on
intensity of the collision product ions that are indicative of the
mass labels; [0103] 10. Calculating the absolute or relative
abundance of the biomolecule or mixture of biomolecules.
[0104] In relation to this invention the term "mass spectrometry"
shall include any type of mass spectrometry capable of
fragmentation analysis. The mass spectrometers suitable for use in
the present invention include instruments that comprise any form of
MS/MS analyser such as a triple quadropole mass spectrometer
equipped with a collision chamber, an ion trap mass spectrometer
capable of fragmenting selected precursor ions by fast atom
bombardment, collision induced dissociation, electron transfer
dissociation or any other form of parent ion fragmentation, and
matrix assisted laser desorption/ionisation (MALDI) mass
spectrometers fitted with a dual time of flight (TOF/TOF) analyser
and a means of parent ion fragmentation.
[0105] In certain embodiments the step of selecting the ions of a
predetermined mass to charge ratio is performed in the first mass
analyser of a serial instrument. The selected ions are then
channeled into a separate collision cell where they are collided
with a gas or a solid surface. The collision products are then
channeled into a further mass analyser of a serial instrument to
detect collision products. Typical serial instruments include
triple quadrupole mass spectrometers, tandem sector instruments and
quadrupole time of flight mass spectrometers.
[0106] In other embodiments, the step of selecting the ions of a
predetermined mass to charge ratio, the step of colliding the
selected ions with a gas and the step of detecting the collision
products are performed in the same zone of the mass spectrometer.
This may be effected in ion trap mass analysers and Fourier
Transform Ion Cyclotron Resonance mass spectrometers, for
example.
[0107] In the present invention, matrix assisted laser
desorption/ionisation (MALDI) techniques may be employed. MALDI
requires that the biomolecule solution be embedded in a large molar
excess of a photo-excitable `matrix`. The application of laser
light of the appropriate frequency results in the excitation of the
matrix which in turn leads to rapid evaporation of the matrix along
with its entrapped biomolecule. Proton transfer from the acidic
matrix to the biomolecule gives rise to protonated forms of the
biomolecule which can be detected by positive ion mass
spectrometry, particularly by Time-Of-Flight (TOF) mass
spectrometry. Negative ion mass spectrometry is also possible by
MALDI TOF. This technique imparts a significant quantity of
translational energy to ions, but tends not to induce excessive
fragmentation despite this. The laser energy and the timing of the
application of the potential difference used to accelerate the ions
from the source can be used to control fragmentation with this
technique. This technique is highly favoured due to its large mass
range, due to the prevalence of singly charged ions in its spectra
and due to the ability to analyse multiple peptides simultaneously.
The TOF/TOF technique may be employed in the present invention.
[0108] The photo-excitable matrix comprises a `dye`, i.e. a
compound that strongly absorbs light of a particular frequency, and
which preferably does not radiate that energy by fluorescence or
phosphorescence but rather dissipates the energy thermally, i.e.
through vibrational modes. It is the vibration of the matrix caused
by laser excitation that results in rapid sublimation of the dye,
which simultaneously takes the embedded analyte into the gas
phase.
[0109] Although MALDI techniques are useful in the context of the
present invention, the invention is not limited to this type of
technique, and other techniques common to the art can be employed
by the skilled person in the present invention, if desired. For
example electrospray or nanoelectrospray mass spectrometry may be
employed.
[0110] The term "analyte" is not particularly limiting, and the
methods according to the present invention may be employed to assay
any type of molecule provided that it can be analysed by mass
spectrometry, and is capable of being labelled by an isobaric mass
label with a mass spectrometrically distinct mass marker group.
Analytes include amino acids, peptides, polypeptides, proteins,
glycoproteins, lipoproteins, nucleic acids, polynucleotides,
oligonucleotides, DNA, RNA, peptide-nucleic acids, sugars, starches
and complex carbohydrates, fats and complex lipids, polymers and
small organic molecules such as drugs and drug-like molecules.
Preferably the analyte is a peptide, protein, nucleotide or nucleic
acid.
[0111] In relation to this invention the term protein shall
encompass any molecule comprising two or more amino acids including
di-peptides, tri-peptides, peptides, polypeptides and proteins.
[0112] In relation to this invention the term nucleic acid shall
encompass any molecule comprising two or more nucleotide bases
including di-nucleotides, tri-nucleotides, oligonucleotides,
deoxyribonucleic acids, ribonucleic acids and peptide nucleic
acids.
[0113] In relation to this invention the term analyte shall be
synonymous with the term biomolecule.
[0114] The mass labels employed to tag the analytes in the present
invention will now be described in more detail.
[0115] The skilled artisan will understand that the nature of the
isobaric mass label is not particularly limiting. Various suitable
isobaric mass labels are known in the art such as Tandem Mass Tags
(Thompson et al., 2003, Anal. Chem. 75(8): 1895-1904 (incorporated
herein by reference) disclosed in WO 01/68664 (incorporated herein
by reference) and WO 03/025576 (incorporated herein by reference),
iPROT tags disclosed in U.S. Pat. No. 6,824,981 (incorporated
herein by reference) and iTRAQ tags (Pappin et al., 2004, Methods
in Clinical Proteomics Manuscript M400129-MCP200 (incorporated
herein by reference)). Any of these isobaric mass labels are
suitable for preparation of the samples and calibration samples and
performing the methods of the current invention.
Mass Marker Moiety
[0116] In a preferred embodiment, the present invention uses a mass
label as defined above wherein the molecular weight of the mass
marker moiety is 300 Daltons or less, preferably 250 Daltons or
less, more preferably 100 to 250 Daltons, most preferably 100-200
Daltons. Particularly preferred molecular weights of the mass
marker moiety are 125, 126, 153 and 154 Daltons, or weights in
which one or more or all of the 12C atoms are replaced by 13C
atoms, e.g. for a non-substituted mass marker moiety having a
weight of 125, masses for its substituted counterparts would be
126, 127, 128, 129, 130 and 131 Daltons for substitution with 1, 2,
3, 4, 5 and 6 13C atoms respectively and/or one or more or all of
the 14N atoms are replaced by 15N atoms.
[0117] The components of the mass marker moiety of this invention
are preferably fragmentation resistant so that the site of
fragmentation of the markers can be controlled by the introduction
of a linkage that is easily broken by Collision Induced
Dissociation (CID), Surface Induced Dissociation, Electron Capture
Dissociation (ECD), Electron Transfer Dissociation (ETD), or Fast
Atom Bombardment. In the most preferred embodiment, the linkage is
easily broken by CID.
[0118] The mass marker moiety used in the present invention
typically comprises the following group:
##STR00002##
wherein the cyclic unit is aromatic or aliphatic and comprises from
0-3 double bonds independently between any two adjacent atoms; each
Z is independently N, N(R.sup.1), C(R.sup.1), CO, CO(R.sup.1) (i.e.
--O--C(R1)- or --C(R1)-O--), C(R.sup.1).sub.2, O or S; X is N, C or
C(R.sup.1); each R.sup.1 is independently H, a substituted or
unsubstituted straight or branched C.sub.1-C.sub.6 alkyl group, a
substituted or unsubstituted aliphatic cyclic group, a substituted
or unsubstituted aromatic group or a substituted or unsubstituted
heterocyclic group; and y is an integer from 0-10.
[0119] The substituents of the mass marker moiety are not
particularly limited and may comprise any organic group and/or one
or more atoms from any of groups IIIA, IVA, VA, VIA or VIIA of the
Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen
atom (e.g. F, Cl, Br or I).
[0120] When the substituent comprises an organic group, the organic
group preferably comprises a hydrocarbon group. The hydrocarbon
group may comprise a straight chain, a branched chain or a cyclic
group. Independently, the hydrocarbon group may comprise an
aliphatic or an aromatic group. Also independently, the hydrocarbon
group may comprise a saturated or unsaturated group.
[0121] When the hydrocarbon comprises an unsaturated group, it may
comprise one or more alkene functionalities and/or one or more
alkyne functionalities. When the hydrocarbon comprises a straight
or branched chain group, it may comprise one or more primary,
secondary and/or tertiary alkyl groups. When the hydrocarbon
comprises a cyclic group it may comprise an aromatic ring, an
aliphatic ring, a heterocyclic group, and/or fused ring derivatives
of these groups. The cyclic group may thus comprise a benzene,
naphthalene, anthracene, indene, fluorene, pyridine, quinoline,
thiophene, benzothiophene, furan, benzofuran, pyrrole, indole,
imidazole, thiazole, and/or an oxazole group, as well as
regioisomers of the above groups.
[0122] The number of carbon atoms in the hydrocarbon group is not
especially limited, but preferably the hydrocarbon group comprises
from 1-40 C atoms. The hydrocarbon group may thus be a lower
hydrocarbon (1-6 C atoms) or a higher hydrocarbon (7 C atoms or
more, e.g. 7-40 C atoms). The number of atoms in the ring of the
cyclic group is not especially limited, but preferably the ring of
the cyclic group comprises from 3-10 atoms, such as 3, 4, 5, 6 or 7
atoms.
[0123] The groups comprising heteroatoms described above, as well
as any of the other groups defined above, may comprise one or more
heteroatoms from any of groups IIIA, IVA, VA, VIA or VIIA of the
Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen
atom (e.g. F, Cl, Br or I). Thus the substituent may comprise one
or more of any of the common functional groups in organic
chemistry, such as hydroxy groups, carboxylic acid groups, ester
groups, ether groups, aldehyde groups, ketone groups, amine groups,
amide groups, imine groups, thiol groups, thioether groups,
sulphate groups, sulphonic acid groups, and phosphate groups etc.
The substituent may also comprise derivatives of these groups, such
as carboxylic acid anhydrydes and carboxylic acid halides.
[0124] In addition, any substituent may comprise a combination of
two or more of the substituents and/or functional groups defined
above.
[0125] The cleavable linker of the mass label used in the present
invention is not especially limited. Preferably, the cleavable
linker is a linker cleavable by Collision Induced Dissociation,
Surface Induced Dissociation, Electron Capture Dissociation (ECD),
Electron Transfer Dissociation (ETD), or Fast Atom Bombardment. In
the most preferred embodiment, the linkage is cleavable by CID. The
linker may comprise an amide bond.
Linker
[0126] In the discussion above and below reference is made to
linker groups which may be used to connect molecules of interest to
the mass label compounds used in this invention. A variety of
linkers is known in the art which may be introduced between the
mass labels of this invention and their covalently attached
biological molecule. Some of these linkers may be cleavable. Oligo-
or poly-ethylene glycols or their derivatives may be used as
linkers, such as those disclosed in Maskos, U. & Southern, E.
M. Nucleic Acids Research 20: 1679-1684, 1992. Succinic acid based
linkers are also widely used, although these are less preferred for
applications involving the labelling of oligonucleotides as they
are generally base labile and are thus incompatible with the base
mediated de-protection steps used in a number of oligonucleotide
synthesisers.
[0127] Propargylic alcohol is a bifunctional linker that provides a
linkage that is stable under the conditions of oligonucleotide
synthesis and is a preferred linker for use with this invention in
relation to oligonucleotide applications. Similarly 6-aminohexanol
is a useful bifunctional reagent to link appropriately
functionalised molecules and is also a preferred linker.
[0128] A variety of known cleavable linker groups may be used in
conjunction with the compounds employed in this invention, such as
photocleavable linkers. Ortho-nitrobenzyl groups are known as
photocleavable linkers, particularly 2-nitrobenzyl esters and
2-nitrobenzylamines, which cleave at the benzylamine bond. For a
review on cleavable linkers see Lloyd-Williams et al., Tetrahedron
49, 11065-11133, 1993, which covers a variety of photocleavable and
chemically cleavable linkers.
[0129] WO 00/02895 discloses the vinyl sulphone compounds as
cleavable linkers, which are also applicable for use with this
invention, particularly in applications involving the labelling of
polypeptides, peptides and amino acids. The content of this
application is incorporated by reference.
[0130] WO 00/02895 discloses the use of silicon compounds as
linkers that are cleavable by base in the gas phase. These linkers
are also applicable for use with this invention, particularly in
applications involving the labelling of oligonucleotides. The
content of this application is incorporated by reference.
Mass Normalisation Moiety
[0131] The structure of the mass normalization moiety of the mass
label used in the present invention is not particularly limited
provided that it is suitable for ensuring that the mass label has a
desired aggregate mass. However, the mass normalization moiety
preferably comprises a straight or branched C.sub.1-C.sub.20
substituted or unsubstituted aliphatic group and/or one or more
substituted or unsubstituted amino acids.
[0132] Preferably, the mass normalization moiety comprises a
C.sub.1-C.sub.6 substituted or unsubstituted aliphatic group, more
preferably a C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5
substituted or unsubstituted aliphatic group, still more preferably
a C.sub.1, C.sub.2, or C.sub.5 substituted or unsubstituted
aliphatic group or a C.sub.1 methyl substituted group.
[0133] The one or more substituted or unsubstituted amino acids may
be any essential or non-essential naturally occurring amino acids
or non-naturally occurring amino acids. Preferred amino acids are
alanine, .beta.-alanine and glycine.
[0134] The substituents of the mass normalisation moiety are not
particularly limited and may comprise any organic group and/or one
or more atoms from any of groups IIIA, IVA, VA, VIA or VIIA of the
Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen
atom (e.g. F, Cl, Br or I).
[0135] When the substituent comprises an organic group, the organic
group preferably comprises a hydrocarbon group. The hydrocarbon
group may comprise a straight chain, a branched chain or a cyclic
group. Independently, the hydrocarbon group may comprise an
aliphatic or an aromatic group. Also independently, the hydrocarbon
group may comprise a saturated or unsaturated group.
[0136] When the hydrocarbon comprises an unsaturated group, it may
comprise one or more alkene functionalities and/or one or more
alkyne functionalities. When the hydrocarbon comprises a straight
or branched chain group, it may comprise one or more primary,
secondary and/or tertiary alkyl groups. When the hydrocarbon
comprises a cyclic group it may comprise an aromatic ring, an
aliphatic ring, a heterocyclic group, and/or fused ring derivatives
of these groups. The cyclic group may thus comprise a benzene,
naphthalene, anthracene, indene, fluorene, pyridine, quinoline,
thiophene, benzothiophene, furan, benzofuran, pyrrole, indole,
imidazole, thiazole, and/or an oxazole group, as well as
regioisomers of the above groups.
[0137] The number of carbon atoms in the hydrocarbon group is not
especially limited, but preferably the hydrocarbon group comprises
from 1-40 C atoms. The hydrocarbon group may thus be a lower
hydrocarbon (1-6 C atoms) or a higher hydrocarbon (7 C atoms or
more, e.g. 7-40 C atoms). The number of atoms in the ring of the
cyclic group is not especially limited, but preferably the ring of
the cyclic group comprises from 3-10 atoms, such as 3, 4, 5, 6 or 7
atoms.
[0138] The groups comprising heteroatoms described above, as well
as any of the other groups defined above, may comprise one or more
heteroatoms from any of groups IIIA, IVA, VA, VIA or VITA of the
Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen
atom (e.g. F, Cl, Br or I). Thus the substituent may comprise one
or more of any of the common functional groups in organic
chemistry, such as hydroxy groups, carboxylic acid groups, ester
groups, ether groups, aldehyde groups, ketone groups, amine groups,
amide groups, imine groups, thiol groups, thioether groups,
sulphate groups, sulphonic acid groups, and phosphate groups etc.
The substituent may also comprise derivatives of these groups, such
as carboxylic acid anhydrydes and carboxylic acid halides.
[0139] In addition, any substituent may comprise a combination of
two or more of the substituents and/or functional groups defined
above.
Reactive Mass Label
[0140] The reactive mass labels typically used in the present
invention for labelling and detecting a biological molecule by mass
spectroscopy comprise a reactive functionality for facilitating
attachment of or for attaching the mass label to a biological
molecule and a mass label as defined above. In preferred
embodiments of the present invention, the reactive functionality
allows the mass label to be reacted covalently to an analyte,
preferably an amino acid, peptide or polypeptide. The reactive
functionality may be attached to the mass labels via a linker which
may or may not be cleavable. The reactive functionality may be
attached to the mass marker moiety of the mass label or the mass
normalization moiety of the mass label.
[0141] A variety of reactive functionalities may be introduced into
the mass labels used in this invention. The structure of the
reactive functionality is not particularly limited provided that it
is capable of reacting with one or more reactive sites on the
biological molecule to be labelled. The reactive functionality is
preferably a nucleophile or an electrophile.
[0142] In the simplest embodiments this may be an
N-hydroxysuccinimide ester. An N-hydroxysuccinimide activated mass
label could also be reacted with hydrazine to give a hydrazide
reactive functionality, which can be used to label periodate
oxidised sugar moieties, for example. Amino-groups or thiols can be
used as reactive functionalities in some applications. Lysine can
be used to couple mass labels to free carboxyl functionalities
using a carbodiimide as a coupling reagent. Lysine can also be used
as the starting point for the introduction of other reactive
functionalities into the mass labels of this invention. The
thiol-reactive maleimide functionality can be introduced by
reaction of the lysine epsilon amino group with maleic anhydride.
The cysteine thiol group can be used as the starting point for the
synthesis of a variety of alkenyl sulphone compounds, which are
useful protein labelling reagents that react with thiols and
amines. Compounds such as aminohexanoic acid can be used to provide
a spacer between the mass modified mass marker moiety or mass
normalization moiety and the reactive functionality.
[0143] Table 1 below lists some reactive functionalities that may
be reacted with nucleophilic functionalities which are found in
biomolecules to generate a covalent linkage between the two
entities. Any of the functionalities listed below could be
introduced into the compounds of this invention to permit the mass
markers to be attached to a biological molecule of interest. A
reactive functionality can be used to introduce a further linker
groups with a further reactive functionality if that is desired.
Table 1 is not intended to be exhaustive and the present invention
is not limited to the use of only the listed functionalities.
TABLE-US-00001 TABLE 1 Nucleophilic Reactive Resultant Linking
Functionality Functionality Group --SH --SO.sub.2--CH.dbd.CR.sub.2
--S--CR.sub.2--CII.sub.2--SO.sub.2-- --NH.sub.2
--SO.sub.2--CH.dbd.CR.sub.2
--N(CR.sub.2--CH.sub.2--SO.sub.2--).sub.2 or
--NH--CR.sub.2--CH.sub.2--SO.sub.2-- --NH.sub.2 ##STR00003##
--CO--NH-- --NH.sub.2 ##STR00004## --CO--NH-- --NH.sub.2 --NCO
--NH--CO--NH-- --NH.sub.2 --NCS --NH--CS--NH-- --NH.sub.2 --CHO
--CH.sub.2--NH-- --NH.sub.2 --SO.sub.2Cl --SO.sub.2--NH--
--NH.sub.2 --CH.dbd.CH-- --NH--CH.sub.2--CH.sub.2-- --OH
--OP(NCH(CH.sub.3).sub.2).sub.2 --OP(.dbd.O)(O)O--
[0144] In a preferred embodiment of the present invention the
reactive functionality comprises the following group:
##STR00005##
wherein each R.sup.2 is independently H, a substituted or
unsubstituted straight or branched C.sub.1-C.sub.6 alkyl group, a
substituted or unsubstituted aliphatic cyclic group, a substituted
or unsubstituted aromatic group or a substituted or unsubstituted
heterocyclic group.
[0145] The substituents of the reactive functionality are not
particularly limited and may comprise any organic group and/or one
or more atoms from any of groups IIIA, IVA, VA, VIA or VIIA of the
Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen
atom (e.g. F, Cl, Br or I).
[0146] When the substituent comprises an organic group, the organic
group preferably comprises a hydrocarbon group. The hydrocarbon
group may comprise a straight chain, a branched chain or a cyclic
group. Independently, the hydrocarbon group may comprise an
aliphatic or an aromatic group. Also independently, the hydrocarbon
group may comprise a saturated or unsaturated group.
[0147] When the hydrocarbon comprises an unsaturated group, it may
comprise one or more alkene functionalities and/or one or more
alkyne functionalities. When the hydrocarbon comprises a straight
or branched chain group, it may comprise one or more primary,
secondary and/or tertiary alkyl groups. When the hydrocarbon
comprises a cyclic group it may comprise an aromatic ring, an
aliphatic ring, a heterocyclic group, and/or fused ring derivatives
of these groups. The cyclic group may thus comprise a benzene,
naphthalene, anthracene, indene, fluorene, pyridine, quinoline,
thiophene, benzothiophene, furan, benzofuran, pyrrole, indole,
imidazole, thiazole, and/or an oxazole group, as well as
regioisomers of the above groups.
[0148] The number of carbon atoms in the hydrocarbon group is not
especially limited, but preferably the hydrocarbon group comprises
from 1-40 C atoms. The hydrocarbon group may thus be a lower
hydrocarbon (1-6 C atoms) or a higher hydrocarbon (7 C atoms or
more, e.g. 7-40 C atoms). The number of atoms in the ring of the
cyclic group is not especially limited, but preferably the ring of
the cyclic group comprises from 3-10 atoms, such as 3, 4, 5, 6 or 7
atoms.
[0149] The groups comprising heteroatoms described above, as well
as any of the other groups defined above, may comprise one or more
heteroatoms from any of groups IIIA, IVA, VA, VIA or VIIA of the
Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen
atom (e.g. F, Cl, Br or I). Thus the substituent may comprise one
or more of any of the common functional groups in organic
chemistry, such as hydroxy groups, carboxylic acid groups, ester
groups, ether groups, aldehyde groups, ketone groups, amine groups,
amide groups, imine groups, thiol groups, thioether groups,
sulphate groups, sulphonic acid groups, and phosphate groups etc.
The substituent may also comprise derivatives of these groups, such
as carboxylic acid anhydrydes and carboxylic acid halides.
[0150] In addition, any substituent may comprise a combination of
two or more of the substituents and/or functional groups defined
above.
[0151] In a more preferred embodiment the reactive functionality
comprises the following group:
##STR00006##
[0152] In a preferred embodiment of the present invention the
reactive mass label has one of the following structures:
##STR00007## [0153]
3-[2-(2,6-Dimethyl-piperidin-1-yl)-acetylamino]-propanoic
acid-(2,5-dioxo-pyrrolidine-1-yl)-ester (DMPip-.beta.Ala-OSu)
[0153] ##STR00008## [0154]
3-[2-(Pyrimidin-2-ylsulfanyl)-acetylamino]-propanoic
acid-(2,5-dioxo-pyrrolidine-1-yl)-ester (Pyrm-.beta.Ala-OSu)
[0154] ##STR00009## [0155]
6-[(Pyrimidin-2-ylsulfanyl)-acetylamino]-hexanoic
acid-(2,5-dioxo-pyrrolidine-1-yl)-ester (Pyrm-C6-OSu)
[0155] ##STR00010## [0156]
2-[2-(2,6-Dimethyl-piperidin-1-yl)-acetylamino]-propanoic
acid-(2,5-dioxo-pyrrolidine-1-yl)-ester (DMPip-Ala-OSu)
[0156] ##STR00011## [0157]
[2-(2,6-Dimethyl-piperidin-1-yl)-acetylamino]-acetic
acid-(2,5-dioxo-pyrrolidine-1-yl)-ester (Pyrm-Gly-OSu).
[0158] In the method according to the present invention, each label
in the set has a common aggregate mass and each label in the set
has a mass marker moiety of a unique mass.
[0159] It is preferred that, each mass marker moiety in the set has
a common basic structure and each mass normalisation moiety in the
set has a common basic structure, and each mass label in the set
comprises one or more mass adjuster moieties, the mass adjuster
moieties being attached to or situated within the basic structure
of the mass marker moiety and/or the basic structure of the mass
normalisation moiety. In this embodiment, every mass marker moiety
in the set comprises a different number of mass adjuster moieties
and every mass label in the set has the same number of mass
adjuster moieties.
[0160] Throughout this description, by common basic structure, it
is meant that two or more moieties share a structure which has
substantially the same structural skeleton, backbone or core. The
skeleton comprises the mass marker moiety of the formula given
above or the mass normalisation moiety as defined above. The
skeleton may additionally comprise a number of amino acids linked
by amide bonds. Other units such as aryl ether units may also be
present. The skeleton or backbone may comprise substituents pendent
from it, or atomic or isotopic replacements within it, without
changing the common basic structure.
[0161] In a preferred embodiment the set of mass labels or reactive
mass labels according to the invention comprise mass labels having
the following structure:
M(A)y-L-X(A)z
wherein M is a mass normalisation moiety, X is a mass marker
moiety, A is a mass adjuster moiety, L is a cleavable linker, y and
z are integers of 0 or greater, and y+z is an integer of 1 or
greater. Preferably M is a fragmentation resistant group, L is a
linker that is susceptible to fragmentation on collision with
another molecule or atom and X is preferably a pre-ionised,
fragmentation resistant group.
[0162] The sum of the masses of M and X is the same for all members
of the set. Preferably M and X have the same basic structure or
core structure, this structure being modified by the mass adjuster
moieties. The mass adjuster moiety ensures that the sum of the
masses of M and X is the same for all mass labels in a set, but
ensures that each X has a distinct (unique) mass.
[0163] The mass adjuster moiety (A) is preferably selected from:
[0164] (a) an isotopic substituent situated within the mass marker
moiety and/or within the mass normalisation moiety, and [0165] (b)
substituent atoms or groups attached to the mass marker moiety
and/or attached to the mass normalisation moiety.
[0166] Preferably the mass adjuster moiety is selected from a
halogen atom substituent, a methyl group substituent, and .sup.2H,
.sup.15N, .sup.18O, or .sup.13C isotopic substituents.
[0167] In one preferred embodiment the present invention, each mass
label in the set of mass labels as defined above has the following
structure:
X.sup.(*.sup.)n-L-M.sup.(*.sup.)m
wherein X is the mass marker moiety, L is the cleavable linker, M
is the mass normalisation moiety, * is an isotopic mass adjuster
moiety, and n and m are integers of 0 or greater such that each
label in the set comprises a mass marker moiety having a unique
mass and each label in the set has a common aggregate mass.
[0168] It is preferred that X comprises the following group:
##STR00012##
wherein R.sup.1, Z, X and y are as defined above and each label in
the set comprises 0, 1 or more * such that each label in the set
comprises a mass marker moiety having a unique mass and each label
in the set has a common aggregate mass.
[0169] In a further preferred embodiment, the reactive mass labels
of the present invention comprise the following reactive
functionality group:
##STR00013##
wherein R.sup.2 is as defined above and the set comprises 0, 1 or
more * such that each label in the set comprises a mass marker
moiety having a unique mass and each label in the set has a common
aggregate mass.
[0170] In all of the above preferred formulae, it is particularly
preferred that the isotopic species * is situated within the mass
marker moiety and/or the linker and/or the mass adjuster moiety,
rather than on any reactive moiety that is present to facilitate
attaching the label to an analyte. The number of isotopic
substituents is not especially limited and can be determined
depending on the number of labels in the set. Typically, the number
of * species is from 0-20, more preferably from 0-15 and most
preferably from 1-10, e.g. 1, 2, 3, 4, 5, 6, 7 or 8. In a set of
two labels, it is preferred that the number of species * is 1,
whilst in a set of 5 labels, it is preferred that the number is 4,
whilst in a set of 6 labels it is preferred that the number is 5.
However, the number may be varied depending upon the chemical
structure of the label.
[0171] If desired, isotopic variants of S may also be employed as
mass adjuster moieties, if the labels contain one or more sulphur
atoms.
[0172] In a particularly preferred embodiment wherein the mass
adjuster moiety is .sup.15N or .sup.13C the set of reactive mass
labels comprises two mass labels having the following
structures:
##STR00014##
[0173] In an alternative particularly preferred embodiment wherein
the mass adjuster moiety is .sup.15N or .sup.13C the set of
reactive mass labels comprises the set comprises five mass labels
having the following structures:
##STR00015##
[0174] In an alternative particularly preferred embodiment wherein
the mass adjuster moiety is .sup.15N or .sup.13C the set of
reactive mass labels comprises six mass labels I-VI having the
following structures, or stereoisomers of these structures:
##STR00016##
[0175] The method according to the present invention may comprise a
further step of separating the components of the samples prior to
step (a). The method may also comprise a step of digesting each
sample with at least one enzyme to digest components of the samples
prior to step (a). The enzyme digestion step may also occur after
step (a) but before step (b).
[0176] In a further embodiment, the mass labels used in the method
further comprise an affinity capture ligand. The affinity capture
ligand of the mass label binds to a counter-ligand so as to
separate the isobarically labeled analytes from the unlabelled
analytes after step (a) but before step (b).
[0177] Affinity capture ligands are ligands which have highly
specific binding partners. These binding partners allow molecules
tagged with the ligand to be selectively captured by the binding
partner. Preferably a solid support is derivitised with the binding
partner so that affinity ligand tagged molecules can be selectively
captured onto the solid phase support. A preferred affinity capture
ligand is biotin, which can be introduced into the mass labels of
this invention by standard methods known in the art. In particular
a lysine residue may be incorporated after the mass marker moiety
or mass normalization moiety through which an amine-reactive biotin
can be linked to the mass labels (see for example Geahlen R. L. et
al., Anal Biochem 202(1): 68-67, "A general method for preparation
of peptides biotinylated at the carboxy terminus." 1992; Sawutz D.
G. et al., Peptides 12(5): 1019-1012, "Synthesis and molecular
characterization of a biotinylated analogue of [Lys]bradykinin."
1991; Natarajan S. et al., Int J Pept Protein Res 40(6): 567-567,
"Site-specific biotinylation. A novel approach and its application
to endothelin-1 analogues and PTH-analogue.", 1992). Iminobiotin is
also applicable. A variety of avidin counter-ligands for biotin are
available, which include monomeric and tetrameric avidin and
streptavidin, all of which are available on a number of solid
supports.
[0178] Other affinity capture ligands include digoxigenin,
fluorescein, nitrophenyl moieties and a number of peptide epitopes,
such as the c-myc epitope, for which selective monoclonal
antibodies exist as counter-ligands. Metal ion binding ligands such
as hexahistidine, which readily binds Ni.sup.2+ ions, are also
applicable. Chromatographic resins, which present iminodiacetic
acid chelated Ni.sup.2+ ions are commercially available, for
example. These immobilised nickel columns may be used to capture
mass labels. As a further alternative, an affinity capture
functionality may be selectively reactive with an appropriately
derivatised solid phase support. Boronic acid, for example, is
known to selectively react with vicinal cis-diols and chemically
similar ligands, such as salicylhydroxamic acid.
[0179] The method according to the invention may further include
the step of separating the isobarically labeled analytes
electrophoretically or chromatographically after step (a) but
before step (b). In a preferred embodiment, strong cation exchange
chromatography is used.
[0180] The term "test sample" refers to any specimen in which an
analyte may be present. The test sample may comprise only one
analyte. Alternatively, the test sample may comprise a plurality of
different analytes. In this embodiment, a calibration sample is
provided for each different analyte. The test sample may be from a
natural source or may be produced synthetically. An example of a
synthetic sample is a mixture of recombinant proteins. In one
embodiment, the test sample is a complex mixture, for example a
sample from a plant or an animal. In a preferred embodiment the
sample is from a human.
[0181] Examples of test samples assayed in the present invention
include: mammalian tissue, fluids such as blood, plasma, serum
cerebrospinal fluid, synovial fluid, ocular fluid, urine, tears and
tear duct exudate, lung aspirates including bronchioalveolar lavage
fluid, breast milk, nipple aspirate, semen, lavage fluids, cell
extracts, cell lines and sub-cellular organelles, tissues such as
solid organ tissues, cell culture supernatants or preparations
derived from mammals, fish, birds, insects, annelids, protozoa and
bacteria, tissue culture extracts, plant tissues, plant fluids,
plant cell culture extracts, bacteria, viruses, fungi, fermentation
broths, foodstuffs, pharmaceuticals and any intermediary
products.
[0182] In a preferred embodiment the test sample is blood plasma.
In a particularly preferred embodiment the test sample is depleted
blood plasma. This is blood plasma which has been purified to
remove the most abundant plasma proteins, such as albumin, so as to
reduce the protein load in the sample, hence reducing the number of
analytes in the sample.
[0183] The term "calibration sample" refers to a sample which
comprises at least two different aliquots of the analyte. The
different aliquots each have a known quantity of the analyte. The
term "known quantity" means that the absolute quantity, or a
qualitative quantity of the analyte in each aliquot of the
calibration sample is known. A qualitative quantity in the present
context means a quantity which is not known absolutely, but may be
a range of quantities that are expected in a subject having a
particular state, for example a subject in a healthy or diseased
state, or some other expected range depending on the type of test
sample under investigation.
[0184] Each aliquot is "different" since it contains a different
quantity of the analyte. Typically this is achieved by taking
different volumes from a standard sample, especially for
qualitative quantities where taking different volumes will ensure
that different quantities are present in each aliquot in a desired
ratio, without needing to know the absolute quantities. As an
alternative, each aliquot is prepared separately and is not taken
from the same sample. In one embodiment, each different aliquot has
the same volume, but comprises a different quantity of the
analyte.
[0185] The calibration sample may be a natural sample such as a
body fluid or a tissue extract or may be synthetic, as for the
sample to be assayed. The calibration sample may comprise a
recombinantly expressed protein, synthetically manufactured peptide
or oligonucleotide. In addition it is possible to produce a number
of different peptides by recombinant protein expression in a
concatenated sequence. European patent application EP 1736480
discloses methods of producing multiple reference peptides as a
concatenated recombinant protein for use in multiple reaction
monitoring experiments in a manner analogous to the AQUA
methodology. Such methods of production may be combined with
isobaric mass labels to provide the calibration samples according
to any of the various aspects of this invention.
[0186] The calibration sample may be a standardised form of the
sample to be assayed. The calibration sample may comprise all of
the components of the sample to be assayed but in particular
quantities. For example, the calibration sample may comprise a
standardised preparation of mammalian tissue, fluids such as blood,
plasma, serum cerebrospinal fluid, synovial fluid, ocular fluid,
urine, tears and tear duct exudate, lung aspirates including
bronchioalveolar lavage fluid, breast milk, nipple aspirate, semen,
lavage fluids, cell extracts, cell lines and sub-cellular
organelles, tissues such as solid organ tissues, cell culture
supernatants or preparations derived from mammals, fish, birds,
insects, annelids, protozoa and bacteria, tissue culture extracts,
plant tissues, plant fluids, plant cell culture extracts, bacteria,
viruses, fungi, fermentation broths, foodstuffs, pharmaceuticals
and any intermediary products. If the analytes of interest are
proteins, since all proteins in the calibration sample are
labelled, the entire proteome of such a sample may be used as a
reference for all proteins of the study sample.
[0187] Alternatively, the calibration sample may comprise only
analytes to be assayed in the sample, and not any other components
of the sample. The calibration sample comprising one or more
analytes may be produced and isobarically labelled exogenously and
added to the complex mixture containing the analyte. For example,
if the sample is a plasma sample, but only a particular protein is
to be assayed in the plasma sample, a calibration sample can be
prepared which comprises different aliquots of the recombinant form
of the protein.
[0188] In a method according to the invention, the quantity of
analyte in each aliquot in the calibration sample is a known
absolute quantity. This allows for the absolute quantity of an
analyte in a test sample to be determined in step (b).
[0189] In an alternative method, the absolute quantity of an
analyte in each aliquot in the calibration sample is not known. In
this embodiment, the quantity of analyte in each aliquot in the
calibration sample is a known qualitative quantity. The calibrating
step comprises calibrating the quantity of the analyte in the test
sample against the qualitative and determined quantities of the
analytes in the aliquots of the calibration sample. In a particular
embodiment, the qualitative quantity is an expected range of
quantities of analyte in a subject having a particular state, such
as a healthy or diseased state. Assays which provide such
calibration samples for relative quantitation have wide range of
applications including biomarker discovery, industrial
microbiology, pharmaceutical and food manufacture and the diagnosis
and management of human and veterinary disease
[0190] Relative quantitation experiments are often useful when
analysing complex biological samples such as blood plasma. In a
specific embodiment, a large amount of entire human blood plasma is
split into several (i.e. four) aliquots and individually labelled
with different isobaric mass labels. For instance, one could
utilise the 6-plex Tandem Mass Tag reagents (see above) to produce
four labelled aliquots of blood plasma. 6TMT-128, 6TMT-129,
6TMT-130, 6TMT-131 would be used for labelling. All individual
samples of a blood plasma study are labelled with one further
different version of this isobaric mass tag, i.e. 6TMT-126. The
aliquots of blood plasma can now be used to generate a calibration
curve, for instance by mixing the 4 aliquots in a 0.5 to 1 to 2 to
5 .mu.L ratio to produce a calibration sample, and then adding 1
.mu.l of the study sample. By combining the sample with the
calibration sample comprising four differentially labelled
aliquots, virtually all MS/MS experiments performed with this
material will result in groups of five reporter-ions--four from the
calibration sample and one from the sample. Thus, the entire
proteome can be used in a 4-point calibration curve. If all plasma
samples of the study are spiked with the identical amount of the
calibration sample, relative quantification across all study
samples becomes possible. Since the calibration sample can be used
by multiple laboratories, cross-study and cross-laboratory
comparisons are possible.
[0191] Whereas the absolute quantity of an analyte might not be
known, the % change in quantity can be calculated from the
calibration curve. Depending on the application, the ratio and
width of the calibration curve can be adjusted.
[0192] In a preferred embodiment, the quantity of analyte in each
different aliquot of the calibration sample is selected to reflect
the known or suspected variation of the analyte in the test sample.
In a still further preferred embodiment, aliquots are provided
which comprise the analyte in quantities which correspond to the
upper and lower limits, and optionally intermediate points within a
range of the known or suspected quantities of the analyte found in
test samples of healthy or diseased subjects.
[0193] Because each analyte is quantified independently of all
other analytes in the sample it is conceivable to prepare multiple
sets of calibration samples each at widely different concentrations
to all other calibration samples, so enhancing the dynamic range of
the experiment. It is also possible to prepare a number of
reference biomolecules for each analyte wherein each biomolecule is
provided in a range of overlapping quantities thereby extending the
total range of the standard curve for a given analyte. As an
example a number of different tryptic peptides from a target
protein may be selected for use as reference standards based on
their performance in a tandem mass spectrometer. The reference
peptides may be selected on the basis of the ion intensity of the
ion corresponding to the peptide in a mass spectrum or on the basis
of the signal-to-noise ratio in the area of the spectrum in which
the ion corresponding to the peptide appears. Alternatively the
reference peptides may be selected so as to avoid peptides which
have isobaric species. The selection of proteotypic peptides, i.e.
peptides which are only present in a particular protein is
particularly favoured.
[0194] If each standard peptide is independently labelled with up
to five different members of a sixplex set of isobaric mass tags
these may be mixed in different ratios to provide a five-point
standard curve. The same isobaric mass labels may be used to label
second, third, fourth or more standard peptides each of which may
be mixed in different ratios covering a range of concentrations
different to that covered by each of the other reference peptides
for the same analyte.
[0195] A different calibration curve is produced for each peptide
derived from the target protein, each calibration curve covering a
different range of concentrations. The concentration of each
peptide is then determined from their respective calibration curve,
and this can be related back to the concentration of the target
protein. For some of the calibration curves, the quantity of the
peptide in the test sample may fall in the middle of the
calibration curve, providing an accurate determination of its
actual quantity in the sample. For other calibration curves
covering a different range in concentrations, the quantity of the
peptide in the test sample may fall outside the range of the
calibration curve. By using multiple peptides which are each
derived from a single analyte of interest, we can produce multiple
calibration curves which can be related to the same analyte and
then choose the most accurate calibration to determine the
concentration of the analyte in the test sample from the
concentration of one or more of the peptides. In this way a broad
dynamic range may be covered without compromising assay
sensitivity.
[0196] The calibration sample may comprise a normal quantity of an
analyte. The quantity of the analyte in the calibration sample may
indicate that a plant, animal, or preferably a human is healthy.
Alternatively, the calibration sample may comprise an analyte in a
quantity that indicates the presence and/or stage of a particular
disease, In a further embodiment, the calibration sample comprises
an analyte in a quantity that indicates the efficacy and/or
toxicity of a therapy. Standard panels of known markers of a
particular trait such as presence and/or stage of disease, response
to therapy, and/or toxicity are prepared. Calibration samples
comprising body fluids or tissue extracts labelled with an isobaric
mass tag could be prepared from patients with well characterised
disease including but not limited to tumours, neurodegeneration,
cardiovascular, renal, hepatic, respiratory, metabolic,
inflammatory, and infectious diseases. Known amounts of such
samples are added to multiple test samples in such a manner that
for a series of analyses ion intensities in the MS/MS scan can be
normalised based on the ion intensity of the common calibration
sample, thereby providing more accurate comparisons between the
separate analyses, reducing the analytical variability of the
study.
[0197] In the case of coronary medicine a series of peptides
derived from the tryptic digestion of known heart disease markers
such as myoglobin, troponin-I, CK-MB, BNP, pro-BNP and NT-pro-BNP
are produced synthetically and split into three aliquots. Each
aliquot of each reference peptide is labelled with one of three
isobaric mass tags from a set of such isobaric mass tags wherein
all tags in the set have substantially the same aggregate mass as
determined by mass spectrometry and wherein each tag in the set
releases a mass reporter ion of unique mass on collision induced
dissociation in a mass spectrometer. Each unique reference
peptide-mass tag molecule is then added to a carrier solution such
as a MS-compatible buffer at a known concentration such that the
concentration of the three differentially labelled aliquots of the
same reference peptide are different, and that the differences span
the normal biological concentrations of the parent protein in
patients with cardiac disease. The resultant reference peptide
panel is added at a defined volumetric ratio with a test sample
that has been labelled with a fourth isobaric mass tag from the
same set of isobaric mass tags used to label the reference
peptides, The spiked sample is then subjected to tandem mass
spectrometry wherein the survey scan is performed in a directed
manner to only identify those precursor ions of characteristic
retention time and mass correlating to each of the isobarically
labelled reference peptides. For each selected ion the MS/MS scan
will contain reporter ions derived from the high, medium and low
concentration reference peptides and the test sample.
[0198] A simple standard curve is easily constructed from the
reference peptide reporter ion intensities and the fourth reporter
ion from the same peptide in the test sample can be read against
the calibration curve. By this means the absolute concentration of
multiple biologically relevant proteins can be determined in a
single MS/MS experiment. The skilled artisan will be aware that the
number of different proteins for which reference peptides are
prepared need not be particularly limited and will be in the range
of 1-100 and most preferably 1-50. Similarly the number of
representative peptides may be in the range of 1-20, preferably
1-10, more preferably 1-5 and most preferably 1-3. It would be
understood by the skilled artisan that the example described above
is a general examplar and the principles described therein may be
applied to known markers of any disease and applied for disease
diagnosis, monitoring of disease progression or monitoring the
response of a patient to treatment.
[0199] A further application is in the use of these calibration
samples in time course experiments. The "Status" of a sample with
respect to time course can be established if the (4) different
aliquots are from 4 different time points, such as time zero, 1
hour, 8 hours, and 24 hours into an experiment (drug challenge in
mice and man, induction of fermentation in E. coli and yeast), also
on a longer time scale of weeks and months for development or
treatment response of chronic diseases.
[0200] In a further aspect of the invention, one of the aliquots of
the calibration sample comprises an analyte in a quantity which
serves as a trigger during an MS scan or during non-scanning MS/MS
to initiate an MS/MS scan.
[0201] Non-scanning MS/MS is when you do not select for any
particular ion with a given m/z ratio in a mass analyser in a mass
spectrometer, but instead essentially all of the analytes are
fragmented to produce an unspecific fragment spectrum. Typically,
this involves allowing all ions to pass from a first mass analyser
into a collision cell, where CID occurs on all of the analytes in
the sample instead of a particular selected ion as in conventional
MS/MS. Although the MS/MS spectrum will not be specific to a
particular analyte, the reporter ion from the trigger can be used
as an indicator that an analyte of interest is right now entering
the mass spectrometer. In a preferred embodiment, the presence of a
reporter ion from the trigger indicates that an analyte of interest
is eluting from an LC column during LC-MS. This would "trigger" the
execution of a pre-defined MS/MS experiment.
[0202] This trigger may not necessarily be an analyte labelled with
an isobaric mass label. The trigger may be any other labelled
analyte which co-elutes, or substantially co-elutes with the
labelled analyte of interest during LC-MS. The label of the trigger
analyte may have a different mass to that of the isobaric mass
labels of the calibration sample. For example, in one embodiment,
the calibration sample comprises aliquots of an analyte
differentially labelled with isobaric mass labels, and further
comprises an aliquot of the analyte which is labelled with a
chemically identical but isotopically distinct mass label,
preferably with a mass difference of 5 Da from that of the isobaric
mass labels. The isotopically distinct mass label could then serve
as the "trigger". During the MS phase of the analysis each analyte
present in the calibration sample bearing the isotopically distinct
and isobaric labels will appear as a pair of peaks separated by the
mass difference between the isobaric and isotopically distinct
labels and wherein the analyte bearing the isotopically distinct
label is present in a readily detectable amount. The mass
spectrometer is programmed to perform a dedicated MS/MS experiment
on the isobarically labelled analyte in such pairs, thereby
triggering the quantitative analysis of the analytes of
interest.
[0203] In a preferred embodiment, the isotopically distinct mass
label trigger comprises no isotopic substituents, and the isobaric
mass labels comprise a plurality of isotopic substituents,
preferably .sup.2H, .sup.15N, .sup.18O, or .sup.13C isotopic
substituents. This provides a mass difference between the analytes
of the calibration sample labelled with isobaric mass labels and
the analyte labelled with the trigger label. Since the trigger
label comprises no isotopic substituents, this label can be used in
large quantities if required without the need for costly isotope
labelling.
[0204] The present invention also provides a method for increasing
the detectability of low abundance analytes in a sample. For a low
abundance protein of interest a recombinant reference protein may
be expressed and then labelled with an isobaric mass tag. A test
sample is then labelled with a second member of the same set of
isobaric mass labels and a large amount of the isobarically
labelled recombinant reference protein is added to the test sample
at such a concentration as to be readily detectable by any chosen
method which might include one- or two-dimensional gel
electrophoresis, free-flow electrophoresis, capillary
electrophoresis, off-gel isoelectric focussing and LC-MS/MS,
LC-MS.sup.n and/or LC-TOF/TOF. Subsequent to detection of the
isobarically labelled reference material a MS/MS scan or TOF/TOF
analysis is performed and the reporter masses of the reference
material and test sample are quantified. By using several members
of a set of isobaric mass tags it is possible to provide a
multipoint calibration curve with more physiologically relevant
concentrations and so improving the overall accuracy of the
analysis. A non-isobaric label can also be used to label the
trigger analyte in this embodiment if it can be detected together
with the labelled analytes of interest, for example if the trigger
analyte and isobarically labelled analytes appear at the same spot
on a gel or co-elute during LC-MS.
[0205] The Invention is described by the following non-limiting
examples.
EXAMPLE 1
Preparation of a Four Point Absolute Quantitative Standard for
Bovine Serum Albumin
[0206] To demonstrate the principle of the invention a set of
reference reagents for bovine serum albumin (BSA) were prepared.
One milligram of BSA was dissolved in buffer, reduced, alkylated
and then digested by trypsin. The skilled artisan would appreciate
that any method suitable of preparing tryptic peptides compatible
for analysis by tandem mass spectrometry may be used.
[0207] The tryptic digest was split into four aliquots and each
aliquot was labelled with a different member of the sixplex TMT
labels of WO 2007/012849 whereby the first aliquot was labelled
with the TMT label whose mass marker moiety has a mass of 128 Da,
the second aliquot with the TMT label whose mass marker moiety has
a mass of 129 Da, the third aliquot with the TMT label whose mass
marker moiety has a mass of 130 Da and the final aliquot with the
TMT label whose mass marker moiety has a mass of 131 Da. Labelling
was performed by adding each respective TMT label reagent stock
solutions (60 mM in acetonitrile) to the respective sample to give
a final concentration of 15 mM TMT reagent.
[0208] Samples were then incubated at room temperature for 1 hour.
Finally, each sample was treated to reverse partial side reactions
and pooling of labelled samples by adding hydroxylamine stock (50%
w/v in water) to each protein sample (to reach a final
concentration of 0.25% [w/v] hydroxylamine) and incubated at room
temperature for 15 min.
[0209] To provide the BSA reference standard the different aliquots
of TMT-labelled digests were mixed to give the following final
concentrations:
TABLE-US-00002 128-TMT 15.6 .mu.g ml.sup.-1 129-TMT 46.9 .mu.g
ml.sup.-1 130-TMT 140.6 .mu.g ml.sup.-1 131-TMT 421.9 .mu.g
ml.sup.-1
[0210] For each analysis 10 .mu.l of reference material is spiked
into the analytical sample thereby providing reference amounts of
0.156, 0.469, 1.406 and 4.219 .mu.g.
EXAMPLE 2
Analysis of Bovine Serum Albumin Solutions by Tandem Mass
Spectrometry
[0211] The accuracy of quantitation of the BSA standard prepared in
Example 1 was determined by analyzing a series of solutions of
known BSA concentration into which the BSA standard solution was
spiked.
[0212] Individual solutions of BSA were prepared in [Buffer] and
treated as described above to prepare tryptic digests. Each tryptic
digest was labelled essentially as described above using the TMT
label whose mass marker moiety has a mass of 126 Da.
[0213] Prior to analysis by tandem mass spectrometry 10 .mu.l of
BSA standard stock solution was added to 10 .mu.l of each 126-TMT
labelled BSA solution and the total volume injected into the
ionisation source of the QTOF II electrospray mass
spectrometer.
LC-ESI-MS Protocol
[0214] MS/MS data were generated via our pipeline consisting of a
Waters Cap-LC with 75 .mu.m, 150 mm RP-C18 column with 3 .mu.m
particle size, flow rate 300 .mu.l/min coupled to Micromass QToF
II. MS/MS experiments were performed by data dependent acquisition
(DDA). During the run, MS/MS experiments were done using
acquisition time of 1.0 sec. for an MS-scan followed by 4
consecutive MS/MS scans of the four most abundant ion species of
1.4 sec. each. FIG. 2 shows an MS/MS profile for BSA tryptic
peptide AEFVEVTK. Upper panel shows the full MS/MS spectrum. Lower
panel shows zoom of isobaric mass marker moiety region and the
different intensities reflecting different abundance of the same
peptide in the study sample (126) and reference material (128, 129,
130 & 131).
[0215] MS/MS spectra are then analysed by Sequest.TM. and matched
to an actual release of IPI database. Protein ID (accession number
plus partial sequence as extracted from MS/MS scan), retention
times, as well as reporter ion intensities of all reporters (126,
128, 129, 130 & 131 Da) are exported into an excel
spreadsheet.
[0216] Depending on experimental conditions and the individual
behaviour of a peptide during analysis, the selection of 1, 4, 10
or more peptides to participate in quantification is done.
Preferably, peptides with low intensity reporters are excluded if
their analytical precision and quality are questionable, as well as
peptides where reporters are outside of defined intensity
thresholds.
[0217] The results of the analysis of 10 .mu.l of a BSA solution
containing 100 .mu.g ml.sup.-1 126-TMT labelled BSA spiked with 10
.mu.L of the four point BSA reference standard described above are
shown in Table 2.
[0218] The standard curve for this experiment was calculated by
adding all the TMT mass marker moiety intensities of the
BSA-derived tryptic peptides (128, 129, 130 & 131 Da
respectively) for each reference amount of BSA and plotting the
summed ion intensity against absolute BSA amount injected. The
standard curve is shown in FIG. 3. To calculate the amount of BSA
in the analytical sample the ion intensities of all of the 126 Da
TMT mass marker moiety intensities were added and this value read
against the standard curve. This gave a calculated BSA amount
injected of 0.892 .mu.g (one outlier peptide discarded for
analysis). If data for individual peptides were used the calculated
range was 0.751 to 1.016 .mu.g BSA.
TABLE-US-00003 TABLE 2 Relative ion intensity, slope
characteristics and calculation of absolute peptide amount in test
sample- four-point reference of BSA Absolute peptide concen- Peak
Sample tration retention ion Reference ion Slope (Actual time
intensity intensity characteristics input BSA peptide (min) 126 128
129 130 131 (y = mx + b) 1 (.mu.g) Sequence start end Da Da Da Da
Da m b r2 (.mu.g) K.A]EFVEVTK.L 36,16 36,16 489,39 84,34 225,73
690,79 1971,28 464,4 17,3 1,000 1,016 K.A]FDEK.L 28,66 28,66 222,49
46,29 101,51 396,07 1079,19 256,3 5,3 0,998 0,847 K.A]WSVAR.L 30,11
30,11 807,32 142,32 354,13 1285,76 3567,41 847,2 13,7 0,999 0,937
K.K]VPQVSTPTLVEVSR.N 35,92 35,92 29,43 14,26 45,00 148,96 421,26
100,1 1.0 0,999 0,284 K.L]GEYGFQNALIVR.Y 49,69 49,69 14,22 0,00
8,16 23,76 64,49 -- -- -- -- K.L]GEYGFQNALIVR.Y 53,12 53,12 2,10
0,00 0,00 0,00 19,12 -- -- -- -- K.L]VNELTEFAK.T 52,92 52,92 19,45
0,00 5,11 34,80 103,88 -- -- -- -- K.L]VTDLTK.V 39,48 39,48 410,41
77,59 241,53 715,05 2075,43 490,3 11,3 1,000 0,814 K.Q]NCDQFEK.L
26,67 26,67 214,62 31,42 112,63 285,43 1013,20 241,8 -17,1 0,997
0,958 K.Q]TALVELLK.H 50,03 50,03 144,95 29,74 77,77 288,61 810,59
193,2 -0,2 0,999 0,751 K.Q]TALVELLK.H 51,63 51,63 12,19 0,00 0,00
29,65 87,27 -- -- -- -- K.V]LTSSAR.Q 23,30 23,30 259,29 57,48
154,79 412,19 1249,77 293,1 10,7 1,000 0,848 K.Y]LYEIAR.R 40,62
40,62 221,01 31,09 101,50 332,90 955,92 227,5 -0,0 1,000 0,972
EXAMPLE 3
Analysis of a Plasma Sample to Detect a Specific Protein Biomarker
Candidate
[0219] 10 crude human plasma samples were used.
[0220] The analyte of interest to be quantified by using the
invention was the protein clusterin. One clusterin peptide with the
amino acid sequence depicted below was used as a reference:
VTTVASHTSDSDVPSGVTEVVVK
[0221] This peptide has a molecular weight of 2313.17 Da
(monoisotopic) or 2314.53 Da (average). This peptide corresponds to
residues 386-408 within the SwissProt entry (CLUS_HUMAN), and is a
part of the a chain of matured clusterin that has a molecular
weight of 25878 Da. The peptide is estimated to contain 3.times.TFA
counter ions, causing an increase of the molecular weight to 2655
g/mol when generating the peptide stock.
Generation of the Calibration Sample from the Peptide
[0222] A 1 .mu.g/.mu.L peptide stock was obtained from 1.66 mg
peptide. 4 portions of 200 .mu.L each were labelled with TMT6-128,
TMT6-129, TMT6-130 and TMT6-131 respectively.
[0223] The peptides were treated with NH.sub.2OH to reverse
possible Tyrosine, Serine and Threonine labelling. The
differentially labelled peptide samples were then mixed in a
1:2:4:8 ratio. FIG. 6 shows a schematic of the methodology used.
Initial analysis by LC-MS/MS showed that partial overlabelling was
not completely reversed, and therefore a second treatment was
necessary. The calibration sample was diluted by a factor of 528
prior to mixing with the TMT6-128 labelled plasma samples.
Processing of the Plasma Samples
[0224] 10 plasma samples were from chosen a cohort based on their
clusterin content as determined by ELISA. 1.66 .mu.L of each plasma
sample was diluted with 198.33 .mu.L buffer (100 mM TEAB, 0.1% SDS,
pH 8.5), providing 142-200 .mu.g protein in total (0.71-1.00
.mu.g/.mu.L protein concentration). Each plasma sample was then
labeled with TMT6-126, and an NH.sub.2OH treatment was carried out
according to the optimised conditions. 4 .mu.L of diluted
calibration sample was then added to 10% of each sample. The sample
was then purified by reverse phase as well as strong cation
exchange chromatography.
LC-MS/MS
[0225] LC-MS/MS was carried out using a CapLC coupled to a Qtof-2
.RTM. mass spectrometer (Waters, Manchester, UK)
[0226] 5 .mu.l of purified sample was injected per run
(corresponding to 40 nL crude plasma). FIG. 7 shows the mass
spectrum from the retention profile of the labelled peptide from
clusterin. Targeted MS/MS acquisition was then carried out using
include lists that contained the m/z of the TMT-labelled peptide
from clusterin. Optimisation of collision energy parameters was
performed to obtain increased mass marker group ion intensities.
FIG. 8 shows the MS/MS spectrum of the labelled peptide from
clusterin. As inset is shown the region of the MS/MS spectrum which
shows the mass marker ions.
Data Analysis
[0227] Manual accumulation of all corresponding MS/MS scans was
performed to obtain 1 MS/MS file per run. Peak processing and ID
was then performed using standard methodologies. A calibration
curve was generated for each MS/MS file based on the mass marker
ion intensities 128-131 (linear regression) (FIG. 9). The amount of
peptide present in each sample was then determined using the
calibration curve (Table 3). Finally, the clusterin concentration
per sample in .mu.g/mL was calculated based on the molecular weight
of clusterin .alpha. chain.
TABLE-US-00004 TABLE 3 R square of calculated amount of the peptide
from plasma concentration of concentration of Experimental
calibration sample on column peptide in plasma Clusterin in plasma
Sample ID code curve (fmol) samples (nmol/mL) samples (.mu.g/mL)*
PRG042 ZP191-1 0.996 291 7.0 182 TLS712 ZP191-2 1.000 236 5.7 148
LNDO137 ZP191-3 0.995 247 6.0 155 THSA046 ZP191-4 0.997 180 4.4 113
THSA034 ZP191-5 0.999 199 4.8 125 THSM044 ZP191-6 0.986 208 5.0 130
THSC021 ZP191-7 0.994 195 4.7 122 LDZC004 ZP191-8 0.995 197 4.8 124
THSA023 ZP191-9 0.987 327 7.9 205 K708A ZP191-10 0.995 178 4.3
111
EXAMPLE 4
Preparation of a Whole Proteome Qualitative Reference Standard
[0228] In many circumstances, for example in early biomarker
discovery workflows, it is not essential to have absolute
quantitative reference standards but rather a representative and
uniform standard covering the whole proteome to be analysed in
which the absolute quantity of any given analyte is unknown but is
deemed to be in the normal range of the reference sample. One
example of such a whole proteome standard is human plasma. Using
the present invention it is possible to prepare a universal and
uniform reference standard plasma in which all proteins and/or
peptides are present as isobarically labelled multiple qualitative
standards. When such a standard is added to an analytical sample
wherein all proteins and/or peptides have been labelled with a
different member of the same set of isobaric labels it is possible
to perform quantitative MS/MS assays on all precursor ions detected
in MS and to determine the relative abundance of the analyte in the
analytical sample compared to the reference standard.
[0229] The skilled addressee would well understand that this
concept can be applied to any qualitative standard including but
not limited to whole or depleted plasma, serum, cerebro spinal
fluid, synovial fluid, urine, semen, nipple aspirate, tissue
homogenate, cell culture supernatant, cell extracts, sub-cellular
fractions, membrane preparations etc. and that individual reference
materials representing a specific sample type may be prepared for
example to normalise biomarker studies across multi-centre clinical
trials.
[0230] As an example of such a reference material preparation of a
human reference plasma was performed, Using 4 different isobaric
mass tags, plasma was labeled and mixed to create a whole plasma
proteome calibration mixture. After chromatographic separation by
1. Strong cation exchanger (SCX) into 24 fractions and 2.
Reversed-phase HPLC into 480 spots on a stainless steel
MALDI-target. Spots were subsequently analysed by MS and MS/MS in a
4800 MALDI Tof/Tof mass spectrometer (Applied Biosystems, USA).
Materials and Methods
[0231] Human Plasma was purchased from Dade Behring (Standard
Plasma). The plasma was spiked with two proteins:
1) Ribonuclease A Type I-AS: From Bovine Pancreas (Sigma, R-5503),
MW 13.7 kDa; pI 9.6; 85% purity. 1.8 mg were dissolved in 170 .mu.L
water; 10 .mu.L of this solution was added to 1 ml Plasma prior to
depletion of high abundant proteins. 2) Trypsin Inhibitor, Type
I-S: From Soybean (Sigma, T-9003); MW 20.1 kDa; pI 4.5; 90% Protein
content; .alpha.-chain MW 20090 Da; .beta.-Chain MW 20036 Da;
.gamma.-Chain MW 20163 Da. 8.8 mg were dissolved in 196 .mu.L
water; 10 .mu.L of this solution was added to 1 ml Plasma prior to
depletion of high abundant proteins.
[0232] Prior to isobaric mass labeling six high abundant proteins
(human albumin, IgG, Antitrypsin, IgA, transferrin and haptoglobin)
were depleted using an Agilent high capacity MARS 4.6.times.100 mm
Column (Part-Nr. 518-5333) on a BioCAD Vision HPLC from Applied
Biosystems
Reduction, Alkylation and Digest with Trypsin
[0233] The Protein treatment was performed following standard
protocols. Protein was diluted to a 1 g/L protein solution pH 7.5
in 100 mM Borate buffer and 0.1% Sodium dodecylsulfate. Reduction
of the cysteins with 1 mM TCEP was performed for 30 min at room
temperature. The cysteins were alkylated with Iodoacetamide for one
hour at room temperature. 440 .mu.s Trypsin were added and
incubated for 18-24 hours at 37.degree. C.
Assembling of a 1:2:4:8 Proportion and Labelling with TMT.sup.6
[0234] After tryptic digestion depleted plasma was split into four
aliquots that were independently labelled with the different
isobaric label TMTsixplex reagents TMT.sup.6-128, TMT.sup.6-129,
TMT.sup.6-130 and TMT.sup.6-131 After labeling the aliquots were
mixed volumetrically in the ratio 1:2:4:8 respectively to generate
a plasma 4-point calibration mixture (FIG. 1). To determine the
proteome coverage of the reference material it was subjected to
multi-dimensional chromatography and tandem mass spectrometry
analysis
Collection of 24 Strong Cation Exchange Fractions
[0235] Prior to mass spectrometry a first separation was performed
on a BioCAD Vision HPLC from Applied Biosystems on a SCX column
(Poly LC 4.6 i.d..times.100 mm; Polysulfoethyl A). The sample was
trapped on a Waters Sunfire RP PreColumn (4.0 mm i.d..times.10 mm),
and eluted with a 50% Acetonitrile pulse to the SCX column. The
PreColumn was switched offline when the elution gradient for the
SCX started. The SCX gradient was formed with solvents C (Water 75%
Acetonitrile 25+5 mM KH.sub.2PO.sub.4, pH 3) and D (Water 75%
Acetonitrile 25%+5 mM KH.sub.2PO.sub.4, pH 3+500 mM KCl, pH 3) from
0 to 50% in 30 minutes. 24 SCX fractions were collected from this
separation step. Each fraction was subsequently subjected to
reverse-phase separation.
Reversed Phase HPLC
[0236] The second separation system was a reversed phase
chromatography on a Waters nanoAQUITY UPLC System. Due to the
decoupling of the chromatography and the MS measurements in the
MALDI workflow the HPLC conditions could be optimized to get a high
peak capacity. Column: 75 .mu.m I.D..times.250 mm filled with 1.7
.mu.m BEH 130 C18 packing material (Waters part Nr.: 186003545).
Column oven temperature 60.degree. C. 5 .mu.L of each SCX fraction
were injected directly without any precolumn on the UPLC
column.
Gradient:
TABLE-US-00005 [0237] TABLE 4 Time(min) % Acetonitrile 1. Initial 5
2. 31.00 5 3. 150.00 25 4. 210.00 50 5. 220.00 95 6. 225.00 95 7.
226.00 5
MALDI Target Spotting
[0238] The separation column of the nanoACQUITY UPLC System is
joined to the inlet of a Dionex Probot spotter to fractionate the
peptides in MALDI preparations on a Microliter format MALDI sample
target for the 4800 Tof/Tof MALDI instrument, On a MALDI target
1920 individual fractions can be collected. Four RP chromatograms
each with 480 spots were prepared per MALDI target. The spotting
starts at retention time 50 minutes and ends at retention time 210
min with a spotting duration of 20 seconds per spot. On the spotter
the eluent flow of 0.35 .mu.L per minute is mixed with a MALDI
matrix solution (5 g/L solution of .alpha.-cyan-4-Hydroxycinnamic
acid in 80% Acetonitrile, 19.8% water, 0.2% Trifluoracetic acid)
flow of 0.6 .mu.L per minute. Each MALDI preparation has a volume
of about 330 nL.
MALDI MS and MS/MS Analysis on the 4800 Tof/Tof Instrument
[0239] The spots were run in two modes on the 4800 Tof/Tof
instrument. In a first run in Reflector mode conventional MS
spectra were recorded. 1,000 MALDI Shots were summed up for each
individual MS Spectrum. The spectra were calibrated internally with
the matrix trimer signal at m/z 568.138 Th. The interpretation tool
of the "4000 Series Explorer" instrument software generated a
precursor list of MS/MS experiments using a LC MALDI strategy that
includes the calculation of elution profiles for the peptide peaks
(fraction to fraction mass tolerance 100 ppm, exclusion of
precursors within 200 resolution). Up to five precursors were
allowed per spot to be selected for subsequent MS/MS analysis. The
MS/MS acquisition was on the strongest precursors first. 1,000
laser shots were acquired per spectrum. The MS/MS spectra were
calibrated internally with the theoretical mass value of the
TMT.sup.6-131 fragment ion 131.1387 Th.
MASCOT Data Base Search for Identification
[0240] Data base search with 15818 queries was performed with
MASCOT Version 2.1.04.
[0241] Human proteins were identified in a search with the IP1
Human Data base (IPI_Human.sub.--20071024; 68348 sequences;
28969400 residues). Both spiked proteins were not present in the
IPI Human data base; they were searched in the Swissprot database
using the taxonomy key "Other mammalia". The peak areas of the
masses 126, 127, 128, 129, 130, 131 were extracted from the TOFTOF
Matcher from the Sequest Toolbox.
Results
Quantitative Analysis
[0242] The Quantitation of reporter peaks at the masses 128, 129,
130 and 131 Da was performed via the Extraction of the Peak areas
out of the GPS Oracle data base. After peak area calculation,
regression analysis was performed in order to check for quality of
the calibration curve (1:2:4:8 ratio). Each peptide MS/MS
experiment was checked by analysing the reporter peaks for the fit
to a straight line. A linear regression was performed for every
MS/MS spectrum.
[0243] FIGS. 10, 11 and 12 show representative examples of an MS/MS
spectrum. In the expanded view, the sequencing b- and y-ions are
seen. In the insert a zoom is displayed demonstrating the reporter
region with the reporters on 128, 129, 130 and 131 m/z with their
1:2:4:8 ratios.
[0244] The regression analysis showed that 12,000 MS/MS spectra
fulfil certain R.sup.2 values (see Table 5):
TABLE-US-00006 TABLE 5 R.sup.2 value distribution of 12,000 MS/MS
spectra from TMT-4-point calibration plasma. R.sup.2 Nr of MS/MS in
% 0.999 2368 17.0 0.995 6439 46.1 0.99 8589 61.6 0.98 10439 74.8
0.97 11361 81.4 0.96 11926 85.5 0.95 12308 88.2 0.94 12571 90.1
0.93 12805 91.8 0.92 12970 93.0 0.91 13124 94.1 0.9 13247 94.9 0.7
13835 99.2 0.6 13875 99.4 0.5 13904 99.6
Summary
[0245] In total, about 12,000 MS/MS spectra were generated which
showed reporter ion intensities fulfilling the intensity criteria
for quantification. The MALDI TofTof MS/MS spectra show the TMT tag
fragment ion in good intensity to allow for quantification
purposes. The y- and b-ion series in the MS/MS spectra of the
peptides were used for peptide ID. The data base search with
conservative thresholds gave 141 human protein identifications in
the IPI Human data base. Both spiked proteins were found using the
Swissprot database and species related filtering. Among the
identified human proteins there was for example Clusterin found
with 18 MS/MS spectra and 25% Sequence coverage. Regression
analysis shows that more than half of the MS/MS spectra have a
R.sup.2 value better than 0.99, 90% have a R.sup.2 value better
than 0.94.
[0246] In addition to the spiked proteins, 141 human proteins were
identified when a minimal peptide score threshold of 20 was applied
and a protein threshold greater than 45 (Table 6).
TABLE-US-00007 TABLE 6 Database: IPI_human 20071024 (68348
sequences; 28969400 residues) Significant hits: 1. IPI00164623
Gene_Symbol = C3 187 kDa protein 2. IPI00478003 Gene_Symbol = A2M
Alpha-2-macroglobulin precursor 3. IPI00022229 Gene_Symbol = APOB
Apolipoprotein B-100 precursor 4. IPI00021885 Gene_Symbol = FGA
Isoform 1 of Fibrinogen alpha chain precursor 5. IPI00298497
Gene_Symbol = FGB Fibrinogen beta chain precursor 6. IPI00414283
Gene_Symbol = FN1 fibronectin 1 isoform 4 preproprotein 7.
IPI00418163 Gene_Symbol = C4B; C4A C4B1 8. IPI00215894 Gene_Symbol
= KNG1 Isoform LMW of Kininogen-1 precursor 9. IPI00017601
Gene_Symbol = CP Ceruloplasmin precursor 10. IPI00032328
Gene_Symbol = KNG1 Isoform HMW of Kininogen-1 precursor 11.
IPI00305461 Gene_Symbol = IT1H2 Inter-alpha-trypsin inhibitor heavy
chain H2 precursor 12. IPI00021891 Gene_Symbol = FGG Isoform
Gamma-B of Fibrinogen gamma chain precursor 13. IPI00021841
Gene_Symbol = APOA1 Apolipoprotein A-I precursor 14. IPI00292530
Gene_Symbol = IT1H1 Inter-alpha-trypsin inhibitor heavy chain II1
precursor 15. IPI00029739 Gene_Symbol = CFH Isoform 1 of Complement
factor H precursor 16. IPI00218192 Gene_Symbol = ITIH4 Isoform 2 of
Inter-alpha-trypsin inhibitor heavy chain H4 precursor 17.
IPI00550991 Gene Symbol = SERPINA3 Alpha-1-antichymotrypsin
precursor 18. IPI00019591 Gene_Symbol = CFB Isoform 1 of Complement
factor B precursor (Fragment) 19. IPI00294193 Gene_Symbol = ITIH4;
TMEM110 Isoform 1 of Inter-alpha- trypsin inhibitor heavy chain H4
precursor 20. IPI00026314 Gene_Symbol = GSN Isoform 1 of Gelsolin
precursor 21. IPI00022895 Gene_Symbol = A1BG Alpha-1B-glycoprotein
precursor 22. IPI00793618 Gene_Symbol = C3 13 kDa protein 23.
IPI00022488 Gene_Symbol = HPX Hemopexin precursor 24. IPI00641737
Gene_Symbol = HP Haptoglobin precursor 25. IPI00022391 Gene_Symbol
= APCS Serum amyloid P-component precursor 26. IPI00019580
Gene_Symbol = PLG Plasminogen precursor 27. IPI00298828 Gene_Symbol
= APOH Beta-2-glycoprotein 1 precursor 28. IPI00019568 Gene_Symbol
= F2 Prothrombin precursor (Fragment) 29. IPI00218732 Gene_Symbol =
PON1 Serum paraoxonase/arylesterase 1 30. IPI00022431 Gene_Symbol =
AHSG Alpha-2-HS-glycoprotein precursor 31. IPI00032291 Gene_Symbol
= C5 Complement C5 precursor 32. IPI00829768 Gene_Symbol = IGHM
IGHM protein 33. IPI00477090 Gene_Symbol = IGHM IGHM protein 34.
IPI00844156 Gene_Symbol = SERPINC1 SERPINC1 protein 35. IPI00032179
Gene_Symbol = SERPINC1 Antithrombin III variant 36. IPI00304273
Gene_Symbol = APOA4 Apolipoprotein A-IV precursor 37. IPI00022426
Gene_Symbol = AMBP AMBP protein precursor 38. IPI00298971
Gene_Symbol = VTN Vitronectin precursor 39. IPI00025426 Gene_Symbol
= PZP Pregnancy zone protein precursor 40. IPI00022371 Gene_Symbol
= HRG Histidine-rich glycoprotein precursor 41. IPI00022394
Gene_Symbol = C1QC Complement C1q subcomponent subunit C precursor
42. IPI00477597 Gene_Symbol = HPR Isoform 1 of Haptoglobin-related
protein precursor 43. IPI00021857 Gene_Symbol = APOC3
Apolipoprotein C-III precursor 44. IPI00555812 Gene_Symbol = GC
Vitamin D-binding protein precursor 45. IPI00291262 Gene_Symbol =
CLU Clusterin precursor 46. IPI00029863 Gene_Symbol = SERPINF2
Alpha-2-antiplasmin precursor 47. IPI00021842 Gene_Symbol = APOE
Apolipoprotein E precursor 48. IPI00017696 Gene_Symbol = C1S
Complement C1s subcomponent precursor 49. IPI00291866 Gene_Symbol =
SERPING1 Plasma protease C1 inhibitor precursor 50. IPI00021727
Gene_Symbol = C4BPA C4b-binding protein alpha chain precursor 51.
IPI00006114 Gene_Symbol = SERPINF1 Pigment epithelium-derived
factor precursor 52. IPI00011261 Gene_Symbol = C8G Complement
component C8 gamma chain precursor 53. IPI00186903 Gene_Symbol =
APOL1 Isoform 2 of Apolipoprotein-L1 precursor 54. IPI00021854
Gene_Symbol = APOA2 Apolipoprotein A-II precursor 55. IPI00293925
Gene_Symbol = FCN3 Isoform 1 of Ficolin-3 precursor 56. IPI00292950
Gene_Symbol = SERPIND1 Heparin cofactor 2 precursor 57. IPI00386879
Gene_Symbol = IGHA1 CDNA FLJ14473 fis, clone MAMMA1001080, highly
similar to Homo sapiens SNC73 prot 58. IPI00007221 Gene_Symbol =
SERPINA5 Plasma serine protease inhibitor precursor 59. IPI00385264
Gene_Symbol =- Ig mu heavy chain disease protein 60. IPI00006662
Gene_Symbol = APOD Apolipoprotein D precursor 61. IPI00303963
Gene_Symbol = C2 Complement C2 precursor (Fragment) 62. IPI00291867
Gene_Symbol = CFI Complement factor I precursor 63. IPI00022395
Gene_Symbol = C9 Complement component C9 precursor 64. IPI00430820
Gene_Symbol = IGKV1-5 IGKV1-5 protein 65. IPI00009920 Gene_Symbol =
C6 Complement component C6 precursor 66. IPI00020996 Gene_Symbol =
IGFALS Insulin-like growth factor-binding protein complex acid
labile chain precursor 67. IPI00032220 Gene_Symbol = AGT
Angiotensinogen precursor 68. IPI00168728 Gene_Symbol = IGHM
FLJ00385 protein (Fragment) 69. IPI00019581 Gene_Symbol = F12
Coagulation factor XII precursor 70. IPI00020986 Gene_Symbol = LUM
Lumican precursor 71. IPI00294395 Gene_Symbol = C8B Complement
component C8 beta chain precursor 72. IPI00654888 Gene_Symbol =
KLKB1 Uncharacterized protein KLKB1 73. IPI00299503 Gene_Symbol =
GPLD1 Isoform 1 of Phosphatidylinositol- glycan-specific
phospholipase D precursor 74. IPI00022429 Gene_Symbol = ORM1
Alpha-1-acid glycoprotein 1 precursor 75. IPI00296608 Gene_Symbol =
C7 Complement component C7 precursor 76. IPI00009793 Gene_Symbol =
C1RL Complement C1r-like protein 77. IPI00019943 Gene_Symbol = AFM
Afamin precursor 78. IP100022417 Gene_Symbol = LRG1 Leucine-rich
alpha-2-glycoprotein precursor 79. IPI00296165 Gene_Symbol = C1R;
C17orf13; LOC442122; ACYP1; RP11- 114H20.1 Complement C1r
subcomponent precursor 80. IPI00020091 Gene_Symbol = ORM2
Alpha-1-acid glycoprotein 2 precursor 81. IPI00328609 Gene_Symbol =
SERPINA4 Kallistatin precursor 82. IPI00011264 Gene_Symbol = CFHR1
Complement factor H-related protein 1 precursor 83. IPI00179330
Gene_Symbol = UBB; UBC; RPS27A ubiquitin and ribosomal protein S27a
precursor 84. IPI00011252 Gene_Symbol = C8A Complement component C8
alpha chain precursor 85. IPI00019399 Gene_Symbol = SAA4 Serum
amyloid A-4 protein precursor 86. IPI00163207 Gene_Symbol = PGLYRP2
Isoform 1 of N-acetylmuramoyl-L- alanine amidase precursor 87.
IPI00022420 Gene_Symbol = RBP4 Plasma retinol-binding protein
precursor 88. IPI00791350 Gene_Symbol = CLEC3B 11 kDa protein 89.
IPI00218413 Gene_Symbol = BTD biotinidase precursor 90. IPI00294004
Gene_Symbol = PROS1 Vitamin K-dependent protein S precursor 91.
IPI00009028 Gene_Symbol = CLEC3B Tetranectin precursor 92.
IPI00382480 Gene_Symbol =- Ig heavy chain V-III region BRO 93.
IPI00296176 Gene_Symbol = F9 Coagulation factor IX precursor 94.
IPI00477992 Gene_Symbol = C1QB complement component 1, q
subcomponent, B chain precursor 95. IPI00154742 Gene_Symbol = IGL@
IGL@ protein 96. IPI00292946 Gene_Symbol = SERPINA7
Thyroxine-binding globulin precursor 97. IPI00382938 Gene_Symbol =
IGLV4-3 IGLV4-3 protein 98. IPI00299778 Gene_Symbol = PON3 Serum
paraoxonase/lactonase 3 99. IPI00410714 Gene_Symbol = HBA2; HBA1
Hemoglobin subunit alpha 100. IPI00296534 Gene_Symbol = FBLN1
Isoform D of Fibulin-1 precursor 101. IPI00027235 Gene_Symbol =
ATRN Isoform 1 of Attractin precursor 102. IPI00029193 Gene_Symbol
= HGFAC Hepatocyte growth factor activator precursor 103.
IPI00807428 Gene_Symbol =- Putative uncharacterized protein 104.
IPI00022463 Gene_Symbol = TF Serotransferrin precursor 105.
IPI00019576 Gene_Symbol = F10 Coagulation factor X precursor 106.
IPI00794397 Gene_Symbol = CHMP4A chromatin modifying protein 4A
107. IPI00022733 Gene_Symbol = PLTP 45 kDa protein 108. IPI00024825
Gene_Symbol = PRG4 Isoform A of Proteoglycan-4 precursor 109.
IPI00216882 Gene_Symbol = MASP1 mannan-binding lectin serine
protease 1 isoform 3 110. IPI00387115 Gene_Symbol =- Ig kappa chain
V-III region SIE 111. 1PI00003590 Gene_Symbol = QSOX1 Isoform 1 of
Sulfhydryl oxidase 1 precursor 112. IPI00022432 Gene_Symbol = TTR
Transthyretin precursor 113. IPI00029061 Gene_Symbol = SEPP1
Selenoprotein P precursor 114. IPI00028413 Gene_Symbol = ITIII3
Inter-alpha-trypsin inhibitor heavy chain H3 precursor 115.
IPI00479116 Gene_Symbol = CPN2 Carboxypeptidase N subunit 2
precursor 116. IPI00178926 Gene_Symbol = IGJ immunoglobulin J chain
117. IPI00166729 Gene_Symbol = AZGP1 alpha-2-glycoprotein 1, zinc
118. IPI00445707 Gene_Symbol = MAEA CDNA FLJ43512 fis, clone
PERIC2004028, moderately similar to Mus musculus erythroblast
macrophage protein EMP mRNA 119. IPI00329775 Gene_Symbol = CPB2
Isoform 1 of Carboxypeptidase B2 precursor 120. IPI00008603
Gene_Symbol = ACTA2 Actin, aortic smooth muscle 121. IPI00384938
Gene_Symbol = IGHG1 Putative uncharacterized protein DKFZp686N02209
122. IPI00784822 Gene_Symbol = IGHV4-31 IGHV4-31 protein 123.
IPI00027482 Gene_Symbol = SERPINA6 Corticosteroid-binding globulin
precursor 124. 1PI00795068 Gene_Symbol = RRBP1 Ribosome binding
protein 1 homolog 180 kDa 125. IPI00025204 Gene_Symbol = CD5L CD5
antigen-like precursor 126. IPI00003351 Gene_Symbol = ECM1
Extracellular matrix protein 1 precursor 127. IPI00163446
Gene_Symbol = IGHD IGHD protein 128. IPI000I0252 Gene_Symbol =
TRIM33 Isoform Alpha of E3 ubiquitin- protein ligase TRIM33 129.
IPI00041065 Gene_Symbol = HABP2 Hyaluronan-binding protein 2
precursor 130. IPI00297550 Gene_Symbol = F13A1 Coagulation factor
XIII A chain precursor 131. IPI00005439 Gene_Symbol = FETUB
Fetuin-B precursor 132. IPI00064667 Gene_Symbol = CNDP1
Beta-Ala-His dipeptidase precursor 133. IPI00018305 Gene_Symbol =
IGFBP3 Insulin-like growth factor-binding protein 3 precursor 134.
IPI00023019 Gene_Symbol = SHBG Isoform 1 of Sex hormone-binding
globulin precursor 135. 1PI00382748 Gene_Symbol = HYI Isoform 3 of
Putative hydroxypyruvate isomerase 136. IPI00004798 Gene_Symbol =
CRISP3 Cysteine-rich secretory protein 3 precursor 137. IPI00032956
Gene_Symbol = KIAA1166 Isoform 1 of Hepatocellular
carcinoma-associated antigen 127 138. IPI00022434 Gene_Symbol = ALB
Uncharacterized protein ALB 139. IPI00009276 Gene_Symbol = PROCR
Endothelial protein C receptor precursor 140. IPI00030739
Gene_Symbol = APOM Apolipoprotein M 141. IPI00032311 Gene_Symbol =
LBP Lipopolysaccharide-binding protein prec
* * * * *