U.S. patent application number 12/997210 was filed with the patent office on 2011-06-23 for methods and materials for monitoring myeloma using quantitative mass spectrometry.
This patent application is currently assigned to H. Lee Moffit Cancer & Research Institute. Invention is credited to Kaaron Benson, Mohamad Hussein, John Matthew Koomen, Elizabeth Renee Remily.
Application Number | 20110151494 12/997210 |
Document ID | / |
Family ID | 41466575 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110151494 |
Kind Code |
A1 |
Koomen; John Matthew ; et
al. |
June 23, 2011 |
METHODS AND MATERIALS FOR MONITORING MYELOMA USING QUANTITATIVE
MASS SPECTROMETRY
Abstract
The subject invention concerns methods and materials for
diagnosing, monitoring the progress, and/or providing a prognosis
for multiple myeloma and other conditions associated with antibody
production in a person or animal. The methods o f the invention
utilize mass spectrometry for quantitative monitoring and detection
of antibody produced by the plasma cells. The methods of the
invention can be utilized for diagnosis, monitoring, and/or
prognosis of multiple myeloma, monoclonal gammopathy, and other
immunological or hematological conditions and disorders. In
addition to detecting and quantifying antibody in a sample, other
biological markers, such as serum albumin and/or
beta-2-microglobulin, can also be detected and quantified using the
present invention, and in combination with detection and
quantification of antibody. Thus, in one embodiment, both antibody
and serum albumin and/or beta-2-microglobulin are detected and
quantified using mass spectrometry and a diagnosis or prognosis
made based on the results and levels detected.
Inventors: |
Koomen; John Matthew;
(Tampa, FL) ; Remily; Elizabeth Renee; (Lutz,
FL) ; Benson; Kaaron; (Apollo Beach, FL) ;
Hussein; Mohamad; (Odessa, FL) |
Assignee: |
H. Lee Moffit Cancer & Research
Institute
Tampa
FL
|
Family ID: |
41466575 |
Appl. No.: |
12/997210 |
Filed: |
June 30, 2009 |
PCT Filed: |
June 30, 2009 |
PCT NO: |
PCT/US09/49286 |
371 Date: |
March 3, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61076907 |
Jun 30, 2008 |
|
|
|
Current U.S.
Class: |
435/23 ; 436/86;
530/326; 530/327; 530/328 |
Current CPC
Class: |
G01N 2800/7028 20130101;
G01N 33/6857 20130101; A61P 35/00 20180101; G01N 2800/52 20130101;
G01N 2800/56 20130101; C07K 16/065 20130101 |
Class at
Publication: |
435/23 ; 530/328;
530/327; 530/326; 436/86 |
International
Class: |
C12Q 1/37 20060101
C12Q001/37; C07K 7/06 20060101 C07K007/06; C07K 7/08 20060101
C07K007/08; G01N 33/48 20060101 G01N033/48 |
Claims
1. An isolated peptide comprising an amino acid sequence shown in
any of SEQ ID NO:1 to SEQ ID NO:30, or a fragment thereof.
2. The isolated peptide according to claim 1, wherein the peptide
consists of an amino acid sequence shown in any of SEQ ID NO:1 to
SEQ ID NO:30, or a fragment thereof.
3. The isolated peptide according to claim 1, wherein the peptide
comprises a stable heavy isotope.
4. A method for diagnosing, monitoring the progress of, and/or
providing a prognosis of a disorder or condition associated with
antibody production in a person or animal, said method comprising
treating a biological sample from the person or animal to isolate a
target protein; fragmenting the target protein to create peptide
fragments thereof; subjecting the peptide fragments of the target
protein to quantitative mass spectrometry to identify and
quantitate the amount of target protein in the biological sample;
and correlating the identity and quantity of the target protein in
the biological sample to a disorder or condition in the person or
animal, whereby the disorder or condition can be diagnosed,
monitored for progress, and/or a prognosis provided.
5. The method according to claim 4, wherein the target protein is
an antibody.
6. The method according to claim 4, wherein the target protein is a
serum albumin and/or a beta-2-microglobulin.
7. The method according to claim 5, wherein the antibody comprises
human heavy chain of IgG, IgA, IgM, IgD, or IgE.
8. The method according to claim 5, wherein the antibody comprises
human kappa light chain or human lambda light chain.
9. The method according to claim 4, wherein the disorder or
condition is multiple myeloma or monoclonal gammopathy of
undetermined significance (MGUS).
10. The method according to claim 4, wherein the target protein is
identified and quantitated by spiking in a known amount of a
specific labeled peptide corresponding to a fragment of a target
protein during mass spectrometry, wherein the specific labeled
peptide comprises a heavy isotope label or has an amino acid
substitution to create a mass difference between the fragment of
the target protein and the specific labeled peptide.
11. The method according to claim 10, wherein the heavy isotope
label is .sup.2H, .sup.13C, or .sup.15N.
12. The method according to claim 10, wherein the specific labeled
peptide comprises an amino acid sequence of any of SEQ ID NO:1 to
SEQ ID NO:30.
13. The method according to claim 10, wherein the specific labeled
peptide corresponds to a fragment of a human antibody heavy chain
of IgG, IgA, IgM, IgD, or IgE.
14. The method according to claim 10, wherein the specific labeled
peptide corresponds to a fragment of a human antibody heavy chain
of IgG and comprises the amino acid sequence ALPAPIEK (SEQ ID
NO:4).
15. The method according to claim 10, wherein the specific labeled
peptide corresponds to a fragment of a human antibody kappa light
chain or lambda light chain.
16. The method according to claim 10, wherein the specific labeled
peptide corresponds to a fragment of a human antibody kappa light
chain and comprises the amino acid sequence DSTYSLSSTLTLSK (SEQ ID
NO:28).
17. The method according to claim 10, wherein the specific labeled
peptide corresponds to a fragment of a human serum albumin or human
beta-2-microglobulin.
18. The method according to claim 4, wherein the target protein is
denatured prior to fragmentation.
19. The method according to claim 18, wherein the target protein is
denatured by treatment with urea, disulfide reduction, and/or
cysteine alkylation.
20. The method according to claim 4, wherein the target protein is
fragmented by proteolytic enzyme digestion.
21. The method according to claim 20, wherein the proteolytic
enzyme is trypsin.
22. The method according to claim 4, wherein said treating step
comprises one or more of size exclusion chromatography, gel
electrophoresis, and/or affinity chromatography to isolate the
target protein.
23. The method according to claim 4, wherein the mass spectrometry
comprises liquid chromatography coupled to multiple reaction
monitoring (LC-MRM).
24. The method according to claim 23, wherein the mass spectrometry
is conducted on a triple quadrupole mass spectrometer.
25. The method according to claim 10, wherein the amino acid
substitution comprises substitution of alanine for glycine.
26. The method according to claim 4, wherein the method is used to
monitor for progression of monoclonal gammopathy of undetermined
significance in the person or animal to multiple myeloma; or
wherein the method is used to monitor the efficacy of a treatment
regimen on a person or animal with multiple myeloma or MGUS.
27. (canceled)
28. The isolated peptide according to claim 2, wherein the peptide
comprises a stable heavy isotope.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
Provisional Application Ser. No. 61/076,907, filed Jun. 30, 2008,
which is hereby incorporated by reference herein in its entirety,
including any figures, tables, nucleic acid sequences, amino acid
sequences, and drawings.
BACKGROUND OF THE INVENTION
[0002] Multiple myeloma (MM) is a cancer of the plasma cell, which
primarily develops in the elderly population. The progression of
the tumor is well understood, and it can be diagnosed by the
presence of multiple myeloma cells in the bone marrow and monitored
by the amount of antibody secretion from the clonal population of
plasma cells. A premalignant condition known as monoclonal
gammopathy of undetermined significance (MGUS) develops at certain
rates in the U.S. population: 3% at age 50, 5% at age 70, and 7% by
age 85; approximately 1% of MGUS patients progress to multiple
myeloma on an annual basis (Kyle et al., 2006). The molecular
causes for progression from MGUS to MM are unknown. After the onset
of the cancer, multiple myeloma patients suffer from several
symptoms, including calcium dysregulation, renal failure, anemia,
and bone lesions. A diagnosis of multiple myeloma is established
using blood and urine tests. For advanced stage patients, complete
skeletal surveys are also used to examine the damage caused by
multiple myeloma in the bone marrow. Staging with serum calcium,
creatinine, hemoglobin, and most importantly, the concentration of
the "monoclonal serum protein" was established in 1975 by Durie and
Salmon (Durie and Salmon, 1975). The International Staging System
determined in 2005 uses those markers as well as serum albumin and
.beta.-2-microglobulin (Greipp et al., 2005). The survival
statistics indicate the importance of early detection and proper
staging, and show the devastating impact of multiple myeloma. Stage
I patients have median survival times of 62 months, stage II 45
months, and stage III patient median survival is reduced to 29
months.
[0003] Despite the highly specific and easily detectable
biomarkers, many challenges still exist for MM treatment. Several
different treatment regimens are under investigation; these
strategies have been the subject of numerous recent reviews
(Fonseca and Stewart, 2007; Chanan-Khan and Lee, 2007; Thomas and
Alexanian, 2007; Falco et al., 2007). Novel therapeutic strategies
include proteasome inhibition with agents like bortezomib (Voorhees
and Orlowski, 2007; Manochakian et al., 2007) and a combination of
cancer cell targeting and immune modulation with thalidomide
derivatives like Lenalidomide (Singhal and Mehta, 2007). While each
of these agents can have some success against multiple myeloma
cells, proteasome inhibitors are the only molecularly guided
therapy to date: treatment is more effective for patients with
myelomas that secrete high levels of monoclonal antibodies (Meister
et al., 2007). The use of the other agents is directed by the
expected tolerance for side effects rather than molecular
targeting. Regardless, these agents improve the patient outcome
when compared to the current standard of care (Ma et al., 2003),
and drug combination strategies are currently in clinical trials
(Srikanth et al., 2008; Richardson et al., 2007; Merchionne et al.,
2007). Proteomic research may contribute to guidance of existing
and emerging therapies. Identification of novel targets including
c-Jun and the Fanconi anemia pathway (Chen et al., 2005) also
offers opportunities to examine protein expression, binding
partners, and post-translational modification. Furthermore, the
bone marrow microenvironment is critical for progression of
multiple myeloma and likely contributes to drug resistance; (Li and
Dalton, 2006; Hazlehurst et al., 2003; Dalton, 2003) this knowledge
has led to preclinical models examining multiple myeloma in the
context of the bone marrow microenvironment. Plausible targets in
the bone marrow microenvironment include cytokine signaling, e.g.
IL-6, (Chauhan et al., 1997; Urashima et al., 1997) and integrin
mediated drug resistance (Damiano et al., 1999). Proteome analysis
may make a significant contribution here as well.
[0004] Patient monitoring strategies present significant
challenges, particularly in the detection of MGUS patients most
likely to develop multiple myeloma and ongoing assessment of
relapse or recurrence in previously treated multiple myeloma
patients. Many MM patients who have undergone treatment are
repetitively checked at two week or four week intervals, leading to
high numbers of clinic visits and collection of large volumes of
blood. Methods for patient sampling and detection of the monoclonal
serum protein are presented from a process chemistry standpoint.
Process chemists use extensive background knowledge of synthesis,
analysis, and engineering to redesign industrial assembly lines or
improve individual steps in manufacturing.
BRIEF SUMMARY OF THE INVENTION
[0005] The subject invention concerns methods and materials for
diagnosing, monitoring the progress, and/or providing a prognosis
for multiple myeloma and other conditions associated with antibody
production in a person or animal. In one embodiment, quantitative
mass spectrometry is used to monitor the amount of multiple myeloma
cells in patients using serum samples. Each MM tumor secretes a
specific (monoclonal) antibody; the amount of the tumor in the
blood or bone marrow of a patient can be measured by the detection
of this protein. Current methods use gel or capillary
electrophoresis to monitor the relative amount and identify the
type of the antibody that is secreted by the MM cells. The
quantitative mass spectrometry techniques of the present invention
combines these two measurements and can provide for absolute
quantification for each of the antibody chains (A, D, E, G, and M,
as well as kappa and lambda) in MM patients. Proteolytic peptides
are used as surrogate biomarkers to measure the amount of the
monoclonal antibody expressed in patients' sera. The methods of the
present invention can be applied to MM patients, patients with the
premalignant condition, monoclonal gammopathy of undetermined
significance (MGUS), and other immune or blood disorders, such as
Waldenstrom's macroglobinemia or HIV/AIDS. Additional diagnostic
markers, including but not limited to serum albumin and
beta-2-microglobulin, can also be quantified using the present
invention.
[0006] In one embodiment of a method of the invention, monoclonal
antibody proteins were excised from serum protein electrophoresis
gels and digested with trypsin. Following trypsin digestion, the
resulting isolated proteolytic peptides were sequenced with liquid
chromatography coupled to tandem mass spectrometry. Using the
results from several patient samples, specific peptides were
selected to monitor each type of antibody (A, D, E, G, and M), as
well as kappa and lambda light chain and other diagnostic molecules
like serum albumin and beta-2-microglobulin (see Table 1). After
selecting peptides that were consistently detected in all patient
samples, a quantitative assay was developed using liquid
chromatography coupled to multiple reaction monitoring (LC-MRM) on
a triple quadrupole mass spectrometer. After overnight digestion of
patient serum, the peptides are analyzed in a 45-minute experiment
separating them by reverse phase and filtering them by molecular
weight and sequence specific fragment ions. Using these transitions
(pairs of intact molecules and fragments), individual peptide
molecules can be selectively quantified, even from a complex matrix
like human blood serum. The methods of the present invention have
been proven effective with control serum and patient samples.
Absolute quantification is obtained by spiking in known amounts of
synthetic peptide containing heavy isotope labels, e.g. .sup.13C
and .sup.15N or by creating a mass shift by substituting an amino
acid with one of a similar composition (such as Alanine for
Glycine).
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIGS. 1A and 1B depict the antibody production for detected
with conventional serum protein electrophoresis for use in multiple
myeloma diagnosis and prognosis.
[0008] FIG. 2 depicts the antibodies identified using
immunofixation electrophoresis for use in multiple myeloma
diagnosis and prognosis.
[0009] FIG. 3 depicts the multiple myeloma diagnosis and prognosis
method where monoclonal spike is an IgG with .kappa. light chain.
After LC-MS/MS, peptides, such as ALPAPIEK (SEQ ID NO:4) from IgG,
can be selected for quantitative mass spectrometry assays.
[0010] FIG. 4 depicts a schematic diagram of selected reaction
monitoring used in multiple myeloma diagnosis and prognosis.
[0011] FIG. 5 depicts the abilities of multiple myeloma diagnosis
and prognosis, where single molecule can be detected by filtering
the m/z values for peptide and specific fragments. Using the same
serum sample shown in FIG. 1B, the quantity and type of antibody
are determined in a mass spectrometry assay; high levels of
ALPAPIEK (SEQ ID NO:4) from IgG.
[0012] FIG. 6 depicts an assay to confirm the multiple myeloma
diagnosis and prognosis method DSTYSLSSTLTLSK (SEQ ID NO:28) from
.kappa. light chain were confirmed using Multiple Reaction
Monitoring.
BRIEF DESCRIPTION OF THE SEQUENCES
[0013] SEQ ID NO:1 is a peptide sequence of the invention (IGHA1,
2).
[0014] SEQ ID NO:2 is a peptide sequence of the invention (IGHA1,
2).
[0015] SEQ ID NO:3 is a peptide sequence of the invention
(IGHA1).
[0016] SEQ ID NO:4 is a peptide sequence of the invention (IGHG1,
3).
[0017] SEQ ID NO:5 is a peptide sequence of the invention (IGHG1,
2).
[0018] SEQ ID NO:6 is a peptide sequence of the invention (IGHG1,
2).
[0019] SEQ ID NO:7 is a peptide sequence of the invention
(IGHG1).
[0020] SEQ ID NO:8 is a peptide sequence of the invention
(IGHG2).
[0021] SEQ ID NO:9 is a peptide sequence of the invention
(IGHG3).
[0022] SEQ ID NO:10 is a peptide sequence of the invention (IGHG3,
4).
[0023] SEQ ID NO:11 is a peptide sequence of the invention
(IGHG4).
[0024] SEQ ID NO:12 is a peptide sequence of the invention
(IGHM).
[0025] SEQ ID NO:13 is a peptide sequence of the invention
(IGHM).
[0026] SEQ ID NO:14 is a peptide sequence of the invention
(IGHM).
[0027] SEQ ID NO:15 is a peptide sequence of the invention
(IGKC).
[0028] SEQ ID NO:16 is a peptide sequence of the invention
(IGKC).
[0029] SEQ ID NO:17 is a peptide sequence of the invention
(IGKC).
[0030] SEQ ID NO:18 is a peptide sequence of the invention
(LAC).
[0031] SEQ ID NO:19 is a peptide sequence of the invention
(LAC).
[0032] SEQ ID NO:20 is a peptide sequence of the invention
(IGHE).
[0033] SEQ ID NO:21 is a peptide sequence of the invention
(IGHE).
[0034] SEQ ID NO:22 is a peptide sequence of the invention
(IGHD).
[0035] SEQ ID NO:23 is a peptide sequence of the invention
(IGHD).
[0036] SEQ ID NO:24 is a peptide sequence of the invention
(IGHD).
[0037] SEQ ID NO:25 is a peptide sequence of the invention
(ALBU).
[0038] SEQ ID NO:26 is a peptide sequence of the invention
(ALBU).
[0039] SEQ ID NO:27 is a peptide sequence of the invention
(ALBU).
[0040] SEQ ID NO:28 is a peptide sequence of the invention.
[0041] SEQ ID NO:29 is a peptide sequence of the invention.
[0042] SEQ ID NO:30 is a peptide sequence of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The subject invention concerns methods and materials for
diagnosing, monitoring the progress, and/or providing a prognosis
for multiple myeloma and other diseases or conditions associated
with antibody production in a person or animal. In one embodiment,
the disease or condition is one characterized by excessive antibody
production, and in particular, excessive monoclonal antibody
production. The methods of the invention utilize mass spectrometry
for quantitative monitoring and detection of antibody produced by
the plasma cells. The methods of the invention can be utilized for
diagnosis, monitoring, and/or prognosis of multiple myeloma,
monoclonal gammopathy, and other immunological or hematological
conditions and disorders. In addition to detecting and quantifying
antibody in a sample, other biological markers, such as serum
albumin and/or beta-2-microglobulin, can also be detected and
quantified using the present invention, and in combination with
detection and quantification of antibody. Thus, in one embodiment,
both antibody and serum albumin and/or beta-2-microglobulin are
detected and quantified using mass spectrometry and a diagnosis or
prognosis made based on the results and levels detected.
[0044] In one embodiment of a method of the present invention, a
biological sample, such as a blood or serum sample, is treated to
isolate the target protein therein. In one embodiment, the
biological sample is subjected to size exclusion chromatography,
gel electrophoresis, and/or affinity chromatography to isolate the
target protein. In a specific embodiment, the target protein is an
antibody. The target protein is then subjected to proteolytic
fragmentation to create fragments of the target protein. In a
specific embodiment, the target protein fragments are prepared by
exposing the protein to trypsin for a sufficient period of time.
Other means for fragmentation of a target protein are known in the
art and can be used in the present methods. Optionally, the target
protein can be denatured prior to fragmentation. In one embodiment,
treatment of the target protein with urea and disulfide reduction
and cysteine alkylation can be performed. Following fragmentation
of the target protein into peptides, the peptides are subjected to
mass spectrometry to identify and quantify the levels of the target
protein. In one embodiment, following proteolytic fragmentation and
prior to quantitative mass spectrometry, the peptide fragments are
separated by reverse phase chromatography and/or filtering by
molecular weight. Using the results from several patient samples,
specific peptides were selected to monitor each type of antibody
(A, D, E, G, and M), as well as kappa and lambda light chain and
other diagnostic molecules like serum albumin and
beta-2-microglobulin (see Table 1). In a specific embodiment, the
fragmentation peptides of the target protein are ALPAPIEK (SEQ ID
NO:4) and/or DSTYSLSSTLTLSK (SEQ ID NO:28). Synthetic peptides
having an amino acid substitution or synthetic stable
isotope-labeled peptides (e.g., comprising .sup.2H, .sup.13C, or
.sup.15N atoms in the peptide molecule) having the same sequence as
the fragmentation peptides can be used as internal standards during
the mass spectrometry to provide for quantitation of the specific
peptide fragments. The ratio of the peptide fragment to the
isotope-labeled peptide standard can be used to calculate the
quantity of the target protein. In one embodiment, the peptides are
specific to heavy chains of human IgG, IgA, IgM, IgD, or IgE. In
another embodiment, the peptides are specific to human kappa
(.kappa.) or lambda (.lamda.) immunoglobulin light chains. In one
embodiment, the internal standard peptides can have an amino acid
sequence shown in any of SEQ ID NOs:1 to 30, or a fragment or
variant thereof. In a specific embodiment, the synthetic internal
standard peptide comprises the amino acid of SEQ ID NO:4 or SEQ ID
NO:28, or a fragment or variant thereof. In one embodiment, the
mass spectrometry methods comprise liquid chromatography coupled to
multiple reaction monitoring (LC-MRM) using a triple quadrupole
mass spectrometer.
[0045] The subject invention also concerns peptides of target
proteins, such as immunoglobulin heavy chain, kappa light chain,
lambda light chain, serum albumin, and beta-2-microglobulin, that
can be used in the methods of the present invention. In one
embodiment, a peptide corresponds to a proteolytic digestion
fragment of a human IgG, IgA, IgM, IgD, or IgE heavy chain, or a
human kappa or lambda immunoglobulin light chain. In one
embodiment, a peptide of the invention comprises one or more stable
heavy isotopes, such as .sup.2H, .sup.13C, or .sup.15N. In another
embodiment, a peptide of the invention comprises one or more amino
acid substitutions of similar composition (such as an alanine
substituted for a glycine) from that of the sequence of target
protein such that the subject peptide has a "mass shift" when
compared to the corresponding peptide fragment of the target
protein.
[0046] In a specific embodiment, a peptide of the invention
comprises an amino acid sequence shown in any of SEQ ID NOs:1 to
30, or a fragment or variant thereof. In an exemplified embodiment,
a peptide of the invention has the amino acid sequence of SEQ ID
NO:4 (for IgG heavy chain) or SEQ ID NO:28 (for kappa light
chain).
[0047] Biological samples refer to a fluid or tissue composition
obtained from a human or animal. Biological samples within the
scope of the invention include, but are not limited to, whole
blood, peripheral blood, blood plasma, bone marrow, spleen, serum,
urine, tears, saliva, sputum, exhaled breath, nasal secretions,
pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations,
interstitial fluid, lymph fluid, meningal fluid, amniotic fluid,
glandular fluid, feces, perspiration, mucous, vaginal or urethral
secretion, cerebrospinal fluid, and transdermal exudate. A
biological sample also includes experimentally separated fractions
of all of the preceding solutions or mixtures containing
homogenized solid material, such as feces, tissues, and biopsy
samples.
[0048] The methods of the present invention can be used with humans
and other animals. The other animals contemplated within the scope
of the invention include domesticated, agricultural, or zoo- or
circus-maintained animals. Domesticated animals include, for
example, dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs,
monkeys or other primates, and gerbils. Agricultural animals
include, for example, horses, mules, donkeys, burros, cattle, cows,
pigs, sheep, and alligators. Zoo- or circus-maintained animals
include, for example, lions, tigers, bears, camels, giraffes,
hippopotamuses, and rhinoceroses.
[0049] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0050] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
[0051] In multiple myeloma, because each plasma cell secretes a
unique antibody, the replication of the tumor cell and the
progression of disease can be monitored by measuring the serum
concentration of the monoclonal antibody it produces. Initial
qualitative measurements are made using serum protein
electrophoresis (SPEP) and dye visualization (see FIGS. 1A and 1B).
Separation of the serum proteins is achieved, isolating albumin
from four regions of globulins, termed alpha 1 (.alpha.1), alpha 2
(.alpha.2), beta (.beta.), and gamma (.gamma.), described by the
differences in their migration relative to albumin. Normally,
antibodies migrate into the .gamma. region, but are low in
intensity compared to albumin and are present only as diffuse bands
(FIG. 1A). The monoclonal antibodies produced in high concentration
by multiple myeloma plasma cells can be visualized as a single
narrow, discrete, dark band usually in the .gamma. region of the
gel (FIG. 1B). Patients with abnormally high levels of protein in
the gamma region can be diagnosed with multiple myeloma by
identifying the type of monoclonal antibody using immunofixation
electrophoresis (IFE), which is a separation similar to SPEP, but
with specific detection for each antibody chain (FIG. 2). Typical
screens test for immunoglobulin G, A, and M heavy chains, as well
as kappa (.kappa.) and lambda (.lamda.) light chains.
Immunoglobulin D or E myelomas are very rare; when suspected, lanes
of the standard IFE are replaced, enabling specific detection of
IgD or IgE heavy chain proteins. In the example, the patient has a
tumor that produces an IgG .kappa. monoclonal antibody protein
(FIG. 2). The combination of these two tests establishes the
relative amount and type of the antibody that is secreted by the
multiple myeloma tumor cells. Gel-based techniques have recently
been complemented by capillary array instruments that can analyze
eight samples in parallel, greatly increasing the throughput and
lowering the amount of sample preparation necessary (tubes of serum
are simply loaded into the instrument, which automatically dilutes
each sample in the buffer used for capillary electrophoresis). SPEP
and IFE are performed separately.
[0052] The quantitative mass spectrometry methods of the invention
can replace standard art methods with a single analysis. Protein
bands from SPEP have been processed for protein identification
using LC-MS/MS. Constant regions on an antibody are utilized in
detection for quantitative monitoring; as shown in FIG. 3, a
peptide sequence ALPAPIEK (SEQ ID NO:4) is used to detect
immunoglobulin G heavy chains. After generating peptides for
monitoring each of the types of antibodies, a comprehensive method
for antibody measurement was made. Briefly, minute volumes of serum
(1 to 10 .mu.l) were processed for detection of each of the
antibody chains: G, A, M, D, E, .kappa., and .lamda.. After protein
denaturation with urea, disulfide reduction, and cysteine
alkylation, trypsin digestion was performed. The sample was then
diluted and analyzed with liquid chromatography coupled to multiple
reaction monitoring (LC-MRM) on a triple quadrupole mass
spectrometer (FIG. 4). Methods for quantitative mass spectrometry
of other proteins, such as vitellogenin, thyroglobulin, C-reactive
protein, and others, have been described in U.S. Pat. Nos.
7,544,518 and 7,163,803; Published U.S. Patent Application Nos.
2009/0011447 and 2009/0148951; and publications by Anderson and
Hunter (2006) and Kuhn et al. (2004).
[0053] In one embodiment, the mass spectrometer instrument
selectively quantifies peptides by filtering the m/z of the intact
species in the first quadrupole (Q1), fragments the molecules in
the second quadrupole (Q2), and filters the m/z of a particular
fragment in the third quadrupole (Q3). Each of these peptide and
fragment pairs is known as a transition; the instrument measures
each transition as part of a cycle, continuously moving from one to
the next. For each peptide, multiple transitions are monitored; the
coincidence detection of multiple fragments from the peptide
increases the confidence in the assignment. Each target protein can
be quantified using more than one peptide. While several rules for
peptide selection have been put forward, selection of peptides in
biological or clinical context frequently deviates from those
guidelines. Examples of quantification with LC-MRM are shown in
FIGS. 5 and 6 using the ALPAPIEK (SEQ ID NO:4) peptide from
immunoglobulin G (IgG) heavy chain and DSTYSLSSTLTLSK (SEQ ID
NO:28) from the .kappa. light chain. The ion signals corresponding
to the y.sub.5 and y.sub.6 ions of ALPAPIEK (SEQ ID NO:4) were
detected at 22 minutes in FIG. 5; y.sub.4 were also monitored. The
y.sub.8, y.sub.11, and y.sub.12 ion signals for DSTYSLSSTLTLSK (SEQ
ID NO:28) were detected at 28 minutes, as shown in FIG. 6. These
ion signals were confirmed from the sample used for the SPEP and
IFE, illustrated with the diagrams in FIGS. 1 and 2.
[0054] The quantitative mass spectrometry assay of the present
invention is advantageous in animal models where limited amounts of
blood serum can be obtained. The implementation of a single
quantitative test provides advantages over the qualitative tests
currently used to follow multiple myeloma patients. The speed and
parallel processing that can be achieved with automated sample
handling and MS detection will also significantly improve the
throughput of patient samples. The adoption of the methods of the
invention at a tertiary cancer center will enable surrounding
primary care physicians and hospitals to send samples to a
centralized facility for processing and analysis. Point of care
patient sampling can be performed with rapid turnaround of results
to the treating physician (.about.1 day) even at a centralized
facility.
TABLE-US-00001 TABLE 1 Protein Peptide Sequence M/Z Transitions
IGHA1, 2 SAVQGPPER (SEQ ID NO: 1) 470.747 4 498.268 5 555.289 6
683.348 IGHA1, 2 WLQGSQELPR (SEQ ID NO: 2) 607.320 6 729.390 7
786.411 8 914.470 WLQGSQELPR (SEQ ID NO: 2) 610.820 6 736.390 7
793.411 8 921.470 IGHA1 TPLTATLSK (SEQ ID NO: 3) 466.277 5 519.314
6 620.362 7 733.446 TPLTATLSK (SEQ ID NO:3) 469.777 5 526.314 6
627.362 7 740.446 IGHG1, 3 ALPAPIEK (SEQ ID NO: 4) 419.756 4
486.293 5 557.330 6 654.383 IGHG1, 2 EPQVYTLPPSR (SEQ ID NO: 5)
643.841 4 456.257 6 670.389 7 883.452 DPQVYTLPPSR (SEQ ID NO: 6)
636.833 4 456.257 6 670.389 7 833.452 IGHG1 GPSVFPLAPSSK (SEQ ID
NO: 7) 593.828 8 846.473 9 945.541 10 1032.573 GPSVFPLAPSSK (SEQ ID
NO: 7) 596.828 8 852.472 9 951.540 10 1038.573 IGHG2 GLPAPIEK (SEQ
ID NO: 8) 412.748 4 486.293 5 557.240 6 654.383 GLPAPIEK (SEQ ID
NO: 8) 415.748 4 492.293 5 563.330 6 660.383 IGHG3 WYVDGVEVHNAK
(SEQ ID NO: 9) 708.850 6 697.363 9 968.480 11 1230.612 WYVDGVEVHNAK
(SEQ ID NO: 9) 711.850 6 703.363 9 974.480 11 1236.611 IGHG3, 4
VVSVLTVLHQDWLNGK (SEQ ID NO: 10) 904.507 5 617.341 10 1209.638 11
1310.686 IGHG4 SLSLSLGK (SEQ ID NO: 11) 402.746 5 517.335 6 604.367
7 717.451 IGHM DGFFGNPR (SEQ ID NO: 12) 455.215 4 443.237 5 590.305
7 794.394 DAFFGNPR (SEQ ID NO: 13) 462.223 4 443.237 5 590.305 7
808.410 IGHM QVGSGVTTDQVQAEAK (SEQ ID NO: 14) 809.408 8 888.443 9
989.490 10 1090.538 IGKC VDNALQSGNSQESVTEQDSK (SEQ ID NO: 15)
1068.489 6 707.321 14 1495.651 8 893.421 IGKC TVAAPSVFIFPPSDEQLK
(SEQ ID NO: 16) 973.518 8 913.463 9 1060.532 11 1320.684
TVGAPSVFIFPPSDEQLK (SEQ ID NO: 17) 966.510 8 913.463 9 1060.532 11
1320.684 LAC AAPSVTLFPPSSEELQANK (SEQ ID NO: 18) 993.513 10
1102.538 11 1199.591 12 1346.659 LAC AGVETTTPSK (SEQ ID NO: 19)
495.759 5 533.294 6 634.341 7 763.384 AGVETTTPSK (SEQ ID NO: 19)
498.759 5 539.294 6 640.341 7 769.384 IGHE GSGFFVFSR (SEQ ID NO:
20) 502.254 5 655.357 6 802.425 7 859.447 GSAFFVFSR (SEQ ID NO: 21)
509.262 5 655.357 6 802.425 7 873.462 IGHD EPAAQAPVK (SEQ ID NO:
22) 455.754 5 542.330 6 613.367 7 684.404 EPAGQAPVK (SEQ ID NO: 23)
448.746 5 542.330 6 599.351 7 670.388 IGHD VRPGGVEEGLLER (SEQ ID
NO: 24) 678.362 7 845.437 10 1058.548 11 1159.596 ALBU LVNEVTEFAK
(SEQ ID NO: 25) 575.312 6 694.378 7 823.420 8 937.463 LVNDVTEFAK
(SEQ ID NO: 26) 568.303 6 694.378 7 809.404 8 923.447 ALBU AEFAEVSK
(SEQ ID NO: 27) 440.725 5 533.294 6 680.362 7 809.404
Table 1 displays a list of monitored peptides of interest and their
corresponding internal standards (where applicable) along with the
Y-ion transitions used in MRM. The underlining designates an amino
acid that can be labeled with a stable heavy isotope.
[0055] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
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