U.S. patent application number 14/183015 was filed with the patent office on 2014-10-02 for diagnosis and monitoring of renal failure using peptide biomarkers.
This patent application is currently assigned to PXBiosciences LLC. The applicant listed for this patent is PXBiosciences LLC. Invention is credited to Leif HONDA, Michael JURGENS, Andrew PECK, Harald TAMMEN.
Application Number | 20140296481 14/183015 |
Document ID | / |
Family ID | 41707653 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140296481 |
Kind Code |
A1 |
TAMMEN; Harald ; et
al. |
October 2, 2014 |
Diagnosis and Monitoring of Renal Failure Using Peptide
Biomarkers
Abstract
Methods for the determination of renal failure, especially
chronic renal failure and acute kidney injury, by measurement of
peptide or protein biomarkers are described. The methods are useful
to determine stages of renal failure, especially the early stages
such as stage 1, 2, and 3 of chronic renal failure and stages R and
I of acute kidney injury. Furthermore there are described peptides
and test kits used in the invention. The described methods are
intended to replace or complement the measurement of creatinine
and/or cystatin C and/or NGAL for diagnosis of renal failure.
Inventors: |
TAMMEN; Harald; (Hanover,
DE) ; HONDA; Leif; (Rye, NH) ; JURGENS;
Michael; (Hanover, DE) ; PECK; Andrew;
(Waltham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PXBiosciences LLC |
Watham |
MA |
US |
|
|
Assignee: |
PXBiosciences LLC
Watham
MA
|
Family ID: |
41707653 |
Appl. No.: |
14/183015 |
Filed: |
February 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12544605 |
Aug 20, 2009 |
8673574 |
|
|
14183015 |
|
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61090853 |
Aug 21, 2008 |
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Current U.S.
Class: |
530/326 |
Current CPC
Class: |
G01N 2800/347 20130101;
G01N 2333/70546 20130101; G01N 33/6893 20130101; G01N 2333/78
20130101; G01N 2800/52 20130101; G01N 2800/50 20130101 |
Class at
Publication: |
530/326 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A peptide biomarker consisting of SEQ ID NO: 2.
2. A kit for diagnosing, monitoring the progression or monitoring
the treatment of a renal disorder, comprising: a. a peptide
biomarker consisting of SEQ ID NO: 2; and b. instructions to detect
said biomarker in a biological sample.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/544,605, filed Aug. 20, 2009, which claims the benefit of
priority to U.S. Provisional Application No. 61/090,853, filed Aug.
21, 2008, all of which are incorporated herein by reference in
their entireties.
FIELD OF THE INVENTION
[0002] The application relates to biomarkers and methods for the
diagnosis of renal failure. The invention targets to diagnose all
stages of chronic renal failure, including early stages which are
difficult to determine with commonly used diagnostic methods known
in the art. The biomarkers and methods of the invention are also
used for the diagnosis of acute renal failure or acute kidney
injury. The advantage of the invention is that the kidney function
can be described accurately and therefore renal failure can be
detected at early stages and tracked through all stages of
progression.
BACKGROUND OF THE INVENTION
[0003] Renal failure is distinguished between acute renal failure
or acute kidney injury and chronic renal failure. The basic
difference between acute and chronic renal failure is the
progression of the disease, which is fast for acute renal failure
(usually within days or weeks) and slow for chronic renal failure
(usually in the range of years). Both acute renal failure and
chronic renal failure can result in a complete loss of renal
function, causing the subject to depend on renal replacement
therapy, such as hemodialysis or kidney transplantation. The
incidence of renal failure has doubled since 1990, most likely
caused by the increase of the incidence of diabetes and
hypertension, the two major causes for kidney failure.
[0004] Chronic renal failure is an irreversible destruction of
renal function and usually is associated with the decreased size of
the kidneys. It does not cause pain, which is an important reason
that it is often not diagnosed until late stages, reducing the
success rate of therapy. Treatment usually is started when the
kidneys are already largely and irreversibly destroyed. In
contrast, acute renal failure can be reversible, depending on its
cause and on the success of the therapy. The success of the therapy
depends, to a large extent, on the early diagnosis.
[0005] Renal failure can be determined by calculating the
glomerular filtration rate (GFR). The GFR describes the total
volume of primary urine which is filtered by both kidneys within a
distinct time interval. The GFR is normally >90 ml/min/body
surface. It can be calculated by measuring either exogenic markers
or endogenic markers.
[0006] Specifically, the GFR can be determined by injecting known
amounts of exogenic diagnostic substances such as Inulin,
radioactive substances (such as Chromium (Cr51-EDTA), Technetium
(99mTC-DTPA), and Cobalt (Co57-vitamin B12)), or radiopaque
material (such as Iohexol and Iothalamate), and subsequently
measuring the excretion of these substances.
[0007] However, these tests are time-consuming, inconvenient, and
expensive as they require collecting and measuring the blood and/or
the urine excreted repeatedly over a specified time period. Other
disadvantages are the use of potentially harmful materials, such as
radioactive or radiopaque materials, and the use of expensive and
difficult to measure substances, such as Inulin. Thus, calculating
the GFR using exogenic substances is usually only used in
scientific studies and not clinical practice.
[0008] Renal failure can also be determined using endogenic
biomarkers, among which serum creatinine clearance is the most
commonly used in clinical practice. Creatinine originates from
muscle tissue and is increasingly secreted by renal tubules with
decreasing renal function. Creatinine clearance can be used to
diagnose chronic, as well as acute, renal failure.
[0009] Serum creatinine levels depend on age, sex, diet, muscle
mass, ethnic background, physical activity, and disease. It is also
secreted by the tubuli of the kidneys independent of kidney disease
(Nephrol Dial Transplant, 2002, 17 (Suppl. 7):7-15) or can be
secreted via the gut. All these factors impair the reliability of
creatinine clearance for diagnosis of renal failure.
[0010] Various correction formulas are known to improve the
accuracy of creatinine-based GFR determinations, by including
additional metadata such as race, weight, body height, and body
surface. However, these formulas may not work correctly under
certain circumstances. For example, if the subjects are
bodybuilders, vegetarians, obese, or suffer from diabetes,
hypertension, or chronic liver disease. In addition, these formulas
are not useful in determining the GFR of healthy subjects or of
subjects suffering from only mild or moderate renal failure.
Finally, the GFR may decline as much as 50% before serum creatinine
starts to increase above reference levels. These limitations
demonstrate that correction formulas often are not suitable for all
stages of renal failure (from mild GFR reduction to renal
failure).
[0011] Another endogenic biomarker for renal failure diagnosis is
Cystatin C, a 120 amino acid, 20 kDa cysteine-protease inhibitor
present in most body fluids. It is excreted by the kidneys. In
contrast to creatinine, the expression of Cystatin C is relatively
constant, independent of, e.g., muscle mass, body weight and age.
Consequently Cystatin C is used as a biomarker of chronic renal
failure. Some newer studies also indicate that it might also be
useful as a diagnostic marker for acute renal failure (Kidney
Int'l., 2004, 66:1115-1122). However, this has to be further
investigated.
[0012] Cystatin C can be determined directly from blood samples.
Therefore, there is no need for the time-consuming, inconvenient
and error prone urine collection. Nevertheless, there are still
limitations for timely and sensitive diagnosis of renal failure
using Cystatin C. In addition, the suitability of Cystatin C for
diagnosis of acute renal failure has not yet been validated.
[0013] NGAL is another promising endogeneous marker that appears to
detect early stages of acute renal injury. NGAL has been shown to
correlate with acute kidney injury in a variety of disease
modalities and is detectable at an early stage of disease. The
validity of NGAL for diagnosis of acute renal failure has yet to be
fully determined.
[0014] Early detection and intervention is critical to effective
treatment of effective treatment. However, the existing diagnosis
methods have many disadvantages and limitations as described.
Therefore, a need exists for novel methods for early diagnosis of
renal failure.
BRIEF SUMMARY OF THE INVENTION
[0015] The present invention provides biomarkers, methods, and test
kits for diagnosis of renal failure. In one aspect, the renal
failure is chronic renal failure. In another aspect, the renal
failure is acute renal failure.
[0016] The present invention provides a peptide biomarker having
the sequence of SEQ ID NO:1 or 2. The present invention also
provides a nucleic acid encoding a peptide biomarker having the
sequence shown in SEQ ID NO:1 or 2. The present invention further
provides an antibody that specifically binds to a peptide biomarker
having the sequence shown in SEQ ID NO:1 or 2. Also disclosed is a
nucleic acid molecule, or a recombinant nucleic acid molecule
consisting of a polynucleotide encoding the biomarker.
[0017] The present invention provides a method of identifying a
subject having or at risk of developing a renal disorder. The
method comprises obtaining a test biological sample from the
subject, and determining the quantity of a peptide biomarker in the
test biological sample. In one embodiment, the biomarker is
selected from the group consisting of SEQ ID NOs: 1 and 2.
[0018] In one aspect, the method further comprises the steps of
obtaining a control biological sample from a control subject not
having and not at risk of developing a renal disorder, determining
the quantity of the peptide biomarker in the control sample, and
comparing the quantity of the biomarker in the test sample and in
the control sample. A variance of the quantity of the biomarker in
the test sample relative to the control sample indicates that the
subject has or is at risk of developing the renal disorder.
[0019] In another aspect, the method comprises comparing the
quantity of the biomarker with a predetermined reference value. A
variance of the quantity of the biomarker in the test sample
relative to the reference value indicates that the subject has or
is at risk of developing a renal disorder.
[0020] The present invention also provides a method of monitoring
the progression or regression of a renal disorder in a subject in
need thereof, the method comprises obtaining a test biological
sample from a subject and determining the quantity of a peptide
biomarker in the test biological sample over a period of time. The
quantity of the biomarker correlates to a stage of the renal
disorder. A difference of the quantities of the biomarker over a
period of time is indicative of the progression or regression of
the renal disorder. In one embodiment, the biomarker is selected
from the group consisting of SEQ ID NOs: 1 and 2.
[0021] The present invention further provides a method of
monitoring the efficacy of a renal disorder therapy in a subject in
need thereof, the method comprises obtaining a test biological
sample from a subject, and determining the quantity of the peptide
biomarker in the test biological sample prior to and subsequent to
the therapy. The quantity of the peptide biomarker obtained prior
to therapy is compared to the quantity of the peptide biomarker
obtained subsequent to therapy. The difference is indicative of the
efficacy of the renal disorder therapy. In one embodiment, the
renal disorder therapy is the administration of medication. In
another embodiment, the renal disorder therapy is hemodialysis. In
yet another embodiment, the renal disorder therapy is kidney
transplantation. The biomarker is selected from the group
consisting of SEQ ID NOs: 1 and 2, and the quantity of the
biomarker correlates to a stage of the renal disorder.
[0022] The present invention further provides a method of
determining the renal-toxic side effects of substances (such as
medications, chemicals, and microbial or other toxins), living
organisms (such as plant, animal, and microbes), and environmental
influences (such as food ingestion, alcohol ingestion, consumption
of tobacco products, stress, and physical activity), on a human or
an animal. A first test biological sample is obtained from the
subject prior to the event, and a second test biological sample
from the subject is obtained after the event. The quantities of a
peptide biomarker in the first test sample and the second test
sample are determined. The quantity of the biomarker correlates to
a stage of the renal disorder. The difference between the
quantities is indicative of the effect of the event on renal
function.
[0023] In certain embodiments of all the aspects, the subject is
selected from the group consisting of a human, a primate, a mouse,
a dog, a pig, and a rat. The methods of the present invention can
be used to determine the stages of renal failure. In one aspect,
the methods of the present invention can be used to determine
stages 1, 2, 3, 4, and 5 of chronic renal failure. In another
aspect, the methods of the present invention can be used to
determine stages Normal, R, I, F, L, and E of acute renal
failure.
[0024] In certain embodiments of all the aspects, the quantity of
the biomarker is determined by calculating glomerular filtration
rate. The biological sample is selected from the group consisting
of blood, plasma, serum, hemofiltrate, urine, kidney tissue, in
vitro cultured kidney cell lines, in vitro cultured primary kidney
cells, in vitro cultured kidney tissue, in vitro cultured kidney
organ, and supernatants from in vitro cell culture, tissue culture,
and organ culture.
[0025] Certain embodiments of the aspects may further include a
method of injecting the subject with an exogenic substance and
calculating glomerular filtration rate by measuring the clearance
of the exogenic substance. The exogenic substance can be Inulin, a
radioactive substance (e.g., Cr51-EDTA, 99mTC-DTPA, and
Co57-vitamin B12), or a radiopaque material (e.g., Diatrizoates,
Iodipamide, Iohexol, Iopamidol, Ioversol, Iothalamate, Ioxaglate,
and Metrizamide). The glomerular filtration rate can be calculated
by measuring the clearance of an endogenic substance. The endogenic
substance can be selected from the group consisting of serum
creatinine, Cystatin C, NGAL, urea, interleukin 18, intestinal form
of alkaline phosphatease, N-acetyl-beta-gulcosaminidase,
alanine-aminopeptidase, kidney injury molecule 1, parathyroid
hormone, creatol, creatine kinase, methylguanidine, and
1,5-anhydroglucitol (1,5-AG).
[0026] In certain embodiments, the subject has a condition selected
from the group consisting of injury to a kidney, type 2 diabetes,
hypertension, cardiovascular disease, sepsis, hemorrhage, massive
blood loss, congestive heart failure, decompensated liver
cirrhosis, damaged kidney blood vessels, obstructions of urine
collection systems or extra-renal drainage, vasculitis, malignant
hypertension, acute glomerulonephritis, acute interstitial
nephritis, and acute tubular necrosis. Further, in certain
embodiments, the event is selected from the group consisting of
exposure to a substance, exposure to a living organism, food
ingestion, alcohol ingestion, consumption of tobacco products,
exposure to stress, and physical activity. The substance can be
selected from the group consisting of a medication, a chemical, and
a toxin. The living organism can be selected from the group
consisting of a plant, an animal, a microbe, and a virus.
[0027] In certain embodiments, the size of the kidneys is
determined, urine output is measured, urine sediments are analyzed,
and excretion of sodium or urea is analyzed. Kidney size can be
determined using an imaging technique, e.g., ultrasound.
[0028] In yet another aspect, the step of determining the quantity
of the biomarker is accomplished by an immunological method (e.g.,
an ELISA assay, an RIA assay, an ELI-Spot assay, a flow cytometry
assay, an immunohistochemistry assay, a Western blot analysis, and
a protein chip assay), a molecular biologic method (e.g., PCR
analysis, a RT-PCR analysis, a TaqMan PCR analysis, a nucleic acid
chip assay, in situ hybridization, a nucleic acid dot blot
analysis, a nucleic acid slot blot analysis, a Southern blot
analysis or a Northern blot analysis), or a physical method (e.g.,
mass spectrometric method, a FRET assay, a chromatographic assay,
or a dye-detection assay). The mass spectrometric method can be a
MALDI, ESI, ionspray, thermospray, MCI, FAB, SELDI, ICAT, iTRAQ, or
affinity mass spectrometric method. In certain aspects, the step of
determining the quantity of the biomarker can be accomplished by
nuclear magnetic resonance (NMR), fluorometry, colorimetry,
radiometry, luminometry, liquid chromatography, capillary
chromatography, thin-layer chromatography, plasmon-resonance (e.g.
BIACORE), or one- or two-dimensional gel electrophoresis.
[0029] The biological sample can be fractionated prior to the
detection step. Fractionating can be done through a method selected
from the group consisting of a chromatography method, a filtration
method, a capillary electrophoresis method, a gel electrophoresis
method, a liquid extraction method, a precipitation method, and an
immunoprecipitation method. Further, the chromatography method can
be reverse phase chromatography.
[0030] The methods of the present invention can be combined with
known diagnostic methods using markers or procedures such as
creatinine clearance, Cystatin C, or other markers for renal
failure, or with measuring urine output, measuring the size of the
kidneys, analysis of urine sediments and excretion of sodium or
urea to further improve the overall sensitivity and/or specificity
of the diagnosis of renal failure.
[0031] Another aspect of the invention is the pre-analysis of a
diagnostic sample intended for other non-renal failure diagnostic
procedures. The pre-analysis determines whether renal failure is
present, as renal failure might impair other markers for non-renal
failure diseases or conditions, which are intended to be measured
using the same sample.
[0032] The present invention further provides a test kit for
diagnosing, monitoring the progression or regression, or monitoring
the treatment of a renal disorder. In one aspect, the kit comprises
a peptide biomarker, a nucleic acid molecule consisting of a
polynucleotide encoding a peptide biomarker, or a recombinant
nucleic acid molecule consisting of a polynucleotide encoding a
peptide biomarker having the sequence of SEQ ID NO:1 or 2. In yet
another aspect, the kit comprises an antibody specifically binds to
a peptide biomarker having the sequence shown in SEQ ID NO:1 or 2.
In one embodiment, the kit of the present invention may optionally
comprise instructions for diagnosing, monitoring the progression or
regression, or monitoring the treatment of the renal disorder. In
certain aspects, instructions to detect the biomarker in the
biological sample are included in the kit.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1 depicts Peptide Displays of different stages of
chronic renal failure.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention provides biomarkers, methods, and test
kits for diagnosis of renal failure. In one aspect, the renal
failure is chronic renal failure. In another aspect, the renal
failure is acute renal failure. "Chronic renal failure" is used
interchangeably with chronic renal disease, and "acute renal
failure" is used interchangeably for acute renal disease, and/or
acute kidney injury. Renal failure is regarded as chronic if it
persists for at least 3 months.
[0035] The stages (alternatively termed states, classes or grades)
of chronic renal failure are defined by the GFR as stated in Table
1 (Kidney Int'l., 2005, 67:2089-2100).
TABLE-US-00001 TABLE 1 Stages of chronic renal failure Stage GFR
(mL/min) Description 1 .gtoreq.90 Normal or elevated GFR 2 60-89
Mild GFR reduction 3 30-59 Moderate GFR reduction 4 15-29 Severe
GFR reduction 5 <15 Renal failure
[0036] Acute renal failure is classified into 5 distinct stages
using the "RIFLE" (Risk, Injury, Failure, Loss, End-stage renal
disease) staging system as shown in Table 2 (The Lancet, 2005,
365:417-430). The first 3 stages are distinguished based on both
increased serum creatinine levels and reduced urine output.
TABLE-US-00002 TABLE 2 Stages of acute renal failure GFR (based on
Stage serum creatinine) criteria Urine output criteria Risk Serum
creatinine increased 1.5 times <0.5 mL/kg/h for 6 h Injury Serum
creatinine increased 2.0 times <0.5 mL/kg/h for 12 h Failure
Serum creatinine increased 3.0 times, <0.3 mL/kg/h for 24 h, or
creatinine >355 mM/L when there or anuria for 12 h was an acute
rise of >44 mM/L Loss Persistent acute renal failure -- >4
weeks End-stage End-stage renal disease >3 months --
[0037] Other staging methods for renal failure resulting in similar
or comparable classifications of different stages of renal failure
may be used according to the invention.
[0038] Determining the presence or absence of renal failure means
to determine if renal failure, including chronic renal failure or
acute renal failure, is present or is not present in a subject.
This includes the determination of any stage of the chronic renal
failure, e.g., stages 1-5 of the chronic renal failure shown in
Table 1. It also includes the determination of any stage of the
acute renal failure, e.g., stages RIFLE of the acute renal failure
shown in Table 2. It further includes distinguishing the stages of
renal failure, e.g., distinguishing stage 1 from stages 2 and/or 3,
or distinguishing stages 1, 2, or 3 from stages 4 and/or 5.
Samples and Subjects
[0039] Any sample containing peptides and/or proteins from a
subject to be tested for the presence or absence of renal failure
can be used according to the invention. The sample can be whole
blood, plasma, serum, hemofiltrate, urine, kidney or other tissue,
or a fraction thereof or a combination of the samples. The sample
may also originate from in vitro cultured cell lines, or primary
cells, or from in vitro cultured tissue or organs, especially
kidneys, or from cell culture, tissue culture, or organ culture
supernatants.
[0040] The sample may be pooled samples collected during a certain
period of time or of more than one subject. It can be used directly
or after storage for various time periods at various storage
conditions. The sample may or may not be pre-processed, e.g.,
fractionated by chromatography, filtration, capillary
electrophoresis, precipitation, liquid or other extraction methods,
and immunoprecipitation.
[0041] The subject from which the sample originates can be a human
or an animal used for experimental testing, preferably a mammal,
such as a primate, a dog, a pig, or a rodent, including, e.g., a
mouse or a rat.
[0042] Specifically, subjects having vascular diseases, including
bilateral renal artery stenosis, ischemic nephropathy,
hemolytic-uremic syndrome, and vasculitis, are more likely to
develop chronic renal failure. Other causes of chronic renal
failure include, but not limited to, focal segmental
glomerulosclerosis, IgA nephritis, diabetic nephropathy, lupus
nephritis, polycystic kidney disease, drug and toxin-induced
chronic tubulointerstitial nephritis, kidney stones, and prostate
diseases.
[0043] Causes of acute renal failure are divided into 3 main
groups: pre-renal, post-renal and intra-renal. Pre-renal causes
include lack of sufficient blood supply of the kidneys, which in
turn may be caused by hemorrhage, massive blood loss, congestive
heart failure, decompensated liver cirrhosis (liver cirrhosis with
complications such as bleedings, ascites), damaged kidney blood
vessels, sepsis, or systemic inflammation due to infection.
Post-renal causes include obstructions of urine collection systems
or extra-renal drainage, which in turn may be caused by medication
interfering with normal bladder emptying, prostate diseases, kidney
stones, abdominal malignancy (such as ovarian cancer or colorectal
cancer), or obstructed urinary catheter. Intra-renal causes are
renal tissue-destroying effects, such as vasculitis, malignant
hypertension, acute glomerulonephritis, acute interstitial
nephritis and acute tubular necrosis. They can be caused by, e.g.,
ischaemic events (such as hemoglobinuria, myoglobinuria, and
myoloma) or by nephrotoxic substances (such as antibiotics, radio
contrast agents, uric acid, and oxalate). Subjects suffering from
these diseases and conditions are more likely to develop acute
renal failure.
Biomarkers as Diagnostics for Renal Failure
[0044] A biomarker is an organic biomolecule present in a sample
used to determine the phenotypic status of the subject or predict a
physiological outcome (e.g., health or disease state). A biomarker
is differentially present between different phenotypic statuses if
the mean or median expression level of the biomarker in the
different groups is calculated to be statistically significant. A
single biomarker, or a combination of particular biomarkers,
provides measures of relative risk or probability that a subject
belongs to one phenotypic status or another or will develop
membership to a specific phenotypic status. Therefore, they are
useful as biomarkers for disease (diagnostics), therapeutic
effectiveness of a drug (theranostics), drug toxicity, and
predicting and identifying the immune response.
[0045] Biomarkers according to the invention include proteins,
protein fragments, peptides, and nucleic acids.
Peptides and Proteins
[0046] In one aspect, the present invention provides a peptide
biomarker having the sequence of
AGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGP (SEQ ID NO:1). This peptide is
a fragment of the full length protein Collagen alpha-1(III)
(P02461). The present invention also provides a peptide biomarker
having the sequence of ELEPPEQQEPGERQEPS (SEQ ID NO:2). This
peptide is a fragment of the full length protein Integrin alpha-7
(Q13683).
[0047] The term "peptide" refers to compounds consisting of two or
more amino acids joined covalently by peptide bonds having a
molecular mass of 0.5 to 20 kDa. The term "protein" refers to a
polypeptide bigger than a peptide.
[0048] A full length protein refers to the complete sequence of a
protein with the signal sequence. For example, full length human
cystatin C (NP.sub.--000090) has 146 amino acid residues. Fragments
of full length proteins are considered peptides, including signal
sequences and other sequences created during proteolytic or other
processing, fragments such as pro-peptide sequences, as long as the
fragments have at least 7 amino acid residues and can be identified
as originating form the parent protein sequence by use of standard
sequence alignment algorithms as known in the art, such as the
BLAST or FASTA algorithms. For example, amino acids 1-26 of the
full length human cystatin C (NP.sub.--000090) is a signal
peptide.
[0049] The terms "peptide" and "protein" according to the invention
include compounds containing only amino acids, as well as compounds
also containing non-amino acid constituents such as carbohydrates
and lipid structures and include compounds containing only peptide
bonds as well as compounds containing other bonds, e.g. ester,
thioether or disulfide.
[0050] The peptides of the present invention can be fragments of
proteins present in nature. Peptides and proteins may contain
postranslational modifications such as phosphate groups,
carbohydrate groups or lipid moieties. Also included are peptides
and proteins that comprise amino acids different from the standard
set of 20 amino acids coded by the genetic code.
[0051] Modifications of peptides or proteins according to the
invention may comprise modifications due to postranslational
modifications, chemical modifications, enzymatic modifications and
modifications due to other mechanisms. Examples of possible
modifications include but are not limited to: glycosylation,
phosphorylation, sulphatation, pyroglutamate modification,
cystein-disulfide bridges, methylation, acetylation, acylation,
farnesylation, formylation, geranylgeranylation, biotinylation,
stearoylation, palmitylation, lipolyation, C-mannosylation,
miristoyliation, amidation, deamidation, methylation,
demethylation, carboxylation, hydroxylation, iodination, oxidation,
pegylation, prenylation, ADP-ribosylation, addition of lipids, of
phosphatidylinositol, of glycosylphosphatidylinositol (GPI)-anchor,
of pyridoxal phosphate, modification of cystein residues resulting
in carboxyamidomethylcysteine, resulting in carboxymethylcysteine,
or resulting in pyridylethylcysteine, modification of lysine
residues resulting in liponic acid, modification of glutamic acid
resulting in pyroglutamic acid.
[0052] Modifications of peptides or proteins according to the
invention may comprise unusual amino acids, chemically or
enzymatically modified amino acids including, but not limited to:
alpha amino butyric acid, beta amino butyric acid, beta amino
iso-butyric acid, beta alanine, gamma butyric acid, alpha amino
adipic acid, 4-amino benzoic acid, amino ethyl cysteine, alpha
amino penicillanic acid, allysine, 4-carboxy glutamic acid,
cystathionine, carboxy glutamic acid, carboxy amido methyl
cysteine, carboxy methyl cysteine, cystein acid, citrulline,
dehydroalanine, di-amino butyric acid, dehydro amino-2-butyric
acid, ethionine, glycine-proline di-peptide, 4-hydroxyproline,
hydroxylysine, hydroxyproline, homoserine, homo cysteine,
histamine, iso-valeine, lysinoalanine, lanthionine, norvaline,
norleucine, ornithine, 2-pipiridine-carboxylic acid, pyroglutamic
acid, pyrrolysine, proline-hydroxy proline di-peptide, sarcosine,
4-selenocysteine, syndesine, and thioproline. Further examples can
be found in databases such as "Delta Mass" at
http://www.abrf.org/index.cfm/dm.home?AvgMass=all.
Nucleic Acids, Complementary and Degenerated Nucleic Acids and
Derivatives
[0053] In another aspect, the present invention provides a nucleic
acid encoding the peptide biomarker having sequence of SEQ ID NO:1
or 2. The present invention also provides a recombinant nucleic
acid containing the nucleic acid sequence encoding the peptide
biomarker of SEQ ID NO:1 or 2.
[0054] Nucleic acids are deoxyribonucleic acids (DNA), ribonucleic
acids (RNA), or combinations thereof, regardless if the nucleic
acid molecules are single or double-stranded, linear, circular or
branched. It will be understood that when a nucleotide sequence is
represented by a DNA sequence (i.e., A, T, G, C), this also
includes an RNA sequence (i.e., A, U, G, C).
[0055] The nucleic acid molecules are purified nucleic acid
molecules, i.e., the preparation contains at least 50%, and
preferably higher, of a certain nucleic acid molecule or of a
certain mixture of more than one certain nucleic acid molecule.
Examples of nucleic acid molecules are genomic DNA, cDNA
(complementary DNA), mRNA (messenger RNA), recombinantly produced
nucleic acid molecules corresponding to the natural sequences,
chemically synthesized nucleic acid molecules corresponding to the
natural sequences, or nucleic acid molecules generated by enzymatic
methods (such as by use of polymerase chain reaction (PCR)), or
nucleic acid molecules purified from natural sources (such as
prokaryotic or eukaryotic cells, blood cells, body fluids such as
plasma, serum, urine, biopsy material (especially kidney biopsy
material), using methods known in the art.
[0056] Furthermore, nucleic acid molecules according to the
invention can be partial or complete derivatives of nucleic acids,
including nucleotides or nucleotide-like molecules not found in
nature (such as phosphorothioates, peptide nucleic acids (PNAs),
N3', P5'-phosphoramidates, morpholino phosphoroamidates,
2'-O-methoxyethyl nucleic acids, 2'-fluoro-nucleic acids,
arabino-nucleic acids, and locked nucleic acids (LNA,
ribonucleotides containing a methylene bridge that connects the
2'-oxygen of ribose with the 4'-carbon)).
Antibodies
[0057] In another aspect, the present invention provides an
antibody that specifically binds to the peptide biomarker having
the sequence of SEQ ID NO:1 or 2. Such an antibody may be
polyclonal or monoclonal. The antibodies of the present invention
are useful in the methods and test kits of the invention. For
example, after an antibody is provided, a peptide marker can be
detected and/or quantified using any of a number of well recognized
immunological binding assays.
[0058] One skilled in the art would readily carry out known
techniques in order to raise antibodies against the biomarkers of
the invention. See, e.g., Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988);
Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such
techniques include, but are not limited to, antibody preparation by
selection of antibodies from libraries of recombinant antibodies in
phage or similar vectors, as well as preparation of polyclonal and
monoclonal antibodies by immunizing rabbits or mice (see, e.g.,
Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature
341:544-546 (1989)).
Determining a Biomarker
[0059] Determining a biomarker includes the qualitative and/or
quantitative determination and measurement of biomarkers that are
present in biological samples. Depending on the sensitivity of the
measurement method used, the determination may detect the presence
of a substance, i.e., the quantity of the substance is above the
detection limit of the method used, or it may be that the substance
is not detectable, i.e., its quantity is below the detection limit
of the method used.
[0060] If a quantitative determination is performed, the quantity
of the substance may be determined as a relative or as an absolute
value, depending on the method used. If the determination is
absolute, it may be expressed as an exact quantity of the biomarker
(e.g., in mg/mL, mmol or M). If the determination is relative, it
may be relative to a reference measurement, to a reference or
normal value, or to a reference sample. The relative quantity may
be expressed, e.g., as a percentage of the reference or in
arbitrary units.
[0061] Generally, all methods suitable to detect and analyze
biomarkers in a sample can be used in the methods of the invention.
These methods include physical, immunological, and molecular
biology methods.
[0062] Suitable physical methods include mass spectrometric
methods, fluorescence resonance energy transfer (FRET) assays,
chromatographic assays (such as liquid chromatography, capillary
chromatography, and thin-layer chromatography), and dye-detection
assays. Mass spectrometric methods include, but are not limited to,
matrix-assisted laser desorption ionisation (MALDI), continuous or
pulsed electrospray ionization (ESI), and related methods such as
ionspray or thermospray or massive cluster impact (MCI). The ion
sources can be matched with detection formats including linear or
non-linear reflection time-of-flight (TOF), single or multiple
quadrupole, single or multiple magnetic sector, fourier transform
ion cyclotron resonance (FTICR), ion trap, and combinations
thereof, e.g. ion-trap/time-of-flight. For ionization, numerous
matrix/wavelength combinations (MALDI) or solvent combinations
(ESI) can be used. Other suitable mass spectrometric methods are,
e.g., fast atom bombardment (FAB) mass spectrometry, Surface
Enhanced Laser Desorption/Ionization (SELDI) mass spectrometry,
isotope coded affinity tag (ICAT) mass spectrometry, iTRAQ mass
spectrometry, and affinity mass spectrometric methods.
[0063] Suitable immunologic methods include, but are not limited
to, enzyme linked immuno assays (ELISA), sandwich, direct,
indirect, or competitive ELISA assays, enzyme-linked immunospot
assays (ELISPOT), radio immuno assays (RIA), flow cytometry assays
(FACS=fluorescence activated cell sorting), immunohistochemistry,
Western blot, protein-chip assays using for example antibodies,
antibody fragments, receptors, ligands, or other agents binding the
substances, such as the peptides of the present invention. Binding
agents can be used to analyze and determine the presence or absence
of biomarkers in a sample. The biological sample is contacted with
one or more agents that bind to the biomarker to determine the
level of expression of the biomarker in the sample. One or more of
the agents may be operably linked to a detectable label.
[0064] Molecular biology methods that can be used according to the
invention include, but are not limited to, Northern or Southern
blot hybridization, nucleic acid dot- or slot-blot hybridization,
in situ hybridization, nucleic acid chip assays, PCR, reverse
transcriptase PCR (RT-PCR), or real time PCR (taq-man PCR).
[0065] Other methods to detect biomarkers include, e.g., nuclear
magnetic resonance (NMR), fluorometry, colorimetry, radiometry,
luminometry, or other spectrometric methods, plasmon-resonance
(e.g. BIACORE), and one- or two-dimensional gel
electrophoresis.
[0066] Furthermore, different methods may be combined to determine
a biomarker. For example, the combination of liquid chromatography
and mass spectrometry (LC-MS) or the combination of capillary zone
electrophoresis and mass spectrometry (CE-MS), or the combination
of extraction of certain classes of substances such as peptides,
from a sample prior to LC-MS or prior to CE-MS or prior to other
methods.
Combination of Methods
[0067] The methods of the present invention can be combined with
other diagnostic methods for renal failure to improve specificity
and selectivity. For example, other diagnostic methods include
measurements of exogenic markers, e.g., Inulin, radioactive
materials (such as Cr51-EDTA, 99mTC-DTPA, and Co57-vitamin B12),
and radiopaque materials (such as Diatrizoates, Iodipamide,
Iohexol, Iopamidol, Ioversol, Iothalamate, Ioxaglate, and
Metrizamide).
[0068] Other diagnostic methods also include measurements of
endogenic markers, such as serum creatinine and Cystatin C.
Additional biomarkers for renal failure have been described in the
literature. For example, biomarkers for acute renal failure include
NGAL, urinary interleukin 18, tubular enzymes (such as the
intestinal form of alkaline phosphatase,
N-acetyl-beta-gulcosaminidase and alanine-aminopeptidase), and
kidney injury molecule 1. Some biomarkers, such as parathyroid
hormone (parathormone), a peptide hormone, may be used to
distinguish acute from chronic renal failure (Ren. Fail., 2007,
29:509-512). In addition, known modified forms of biomarkers for
renal failure, such as creatinine modified by hydroxyl radical
addition resulting in Creatol (Ren. Fail., 2007, 29:279-283), or
enzymes modifying creatine, such as creatine kinase may also be
suitable for diagnosis of renal failure. Examples of non-peptide
markers for renal failure include methylguanidine (used in
combination with creatol), and 1,5-anhydroglucitol (1,5-AG) (used
in combination with serum creatinine) (Nephron, 1996,
73:707-709).
[0069] Further diagnostic methods include imaging techniques to
determine the size of the kidneys (such as ultrasound), measurement
of urine volume, analysis of urine sediments, and analysis of
urinary chemistry (such as excretion of sodium or urea).
[0070] It is possible to combine one or more than one of these
diagnostic methods with the method of the invention. In addition,
the diagnostic procedures can be done simultaneously or
successively to confirm the diagnosis or to increase the
sensitivity and/or specificity of the diagnosis.
Use of the Methods and Biomarkers of the Invention
[0071] The methods and biomarkers of the invention can be used,
among others, for diagnosis of renal failure, including chronic and
acute renal failure. The methods and biomarkers of the invention
can be used for determining the stages of renal failure, especially
early stages of renal failure which are difficult or not at all
possible to diagnose using methods known in the art.
[0072] The present invention provides a method of identifying a
subject having or at risk of developing a renal disorder, the
method comprises obtaining a test biological sample from the
subject and determining the quantity of a peptide biomarker in the
test sample. In one embodiment, the biomarker is selected from the
group consisting of SEQ ID NOs: 1 and 2.
[0073] In one aspect, the method further comprises the steps of
obtaining a control biological sample from a control subject not
having and not at risk of developing a renal disorder, and
comparing the quantity of the biomarker in the test sample and in
the control sample. A variance of the quantity of the biomarker in
the test sample relative to the control sample indicates that the
subject has or is at risk of developing the renal disorder.
[0074] In another aspect, the method further comprises comparing
the quantity of the biomarker with a predetermined reference value.
A variance of the quantity of the biomarker in the test sample
relative to the reference value indicates that the subject has or
is at risk of developing a renal disorder.
[0075] The methods and biomarkers of the invention can also be used
to determine the stages of renal failure. In one aspect, the
methods and biomarkers of the invention can be used to distinguish
chronic renal failure stages 1, 2, 3, 4 and 5 from each other. In
another aspect, the methods and biomarkers of the invention can be
used to distinguish acute renal failure stages R, I, F, L and E
from Normal and from each other.
[0076] Peptide Displays from different stages of chronic renal
failure are shown in FIG. 1. To generate Peptide Displays, samples
are prepared and separated by means of RP-HPLC (reversed phase high
pressure liquid chromatography) and peptides eluting from the HPLC
column are collected into 96 fractions. Each fraction is subjected
to MALDI-TOF-MS (matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry) and the mass spectra of all 96
fractions are combined, resulting in an in silico two-dimensional
display of peptide masses, where the x-axis displays the
mass-to-charge ratio, the y-axis is determined by the retention
time on the RP-HPLC or the fraction number and signal intensity is
depicted by color saturation. FIG. 1 demonstrates the accumulation
of peptides in blood plasma of subjects.
[0077] The present invention also provides a method of monitoring
the progression or regression of a renal disorder in a subject in
need thereof, the method comprises obtaining a test biological
sample from the subject; determining the quantity of a peptide
biomarker in the test sample; repeating the steps over a period of
time; and comparing the quantity of the biomarker over the period
of time. In one embodiment, the biomarker is selected from the
group consisting of SEQ ID NOs: 1 and 2.
[0078] The present invention further provides a method of
monitoring the efficacy of a renal disorder therapy in a subject in
need thereof, the method comprises determining the quantity of a
peptide biomarker in a test biological sample obtained from the
subject prior to and subsequent to the therapy. In one embodiment,
the renal disorder therapy is the administration of medication. In
another embodiment, the renal disorder therapy is hemodialysis. In
yet another embodiment, the renal disorder therapy is kidney
transplantation.
[0079] The present invention further provides a method of
determining the renal-toxic side effects of substances (such as
medications, chemicals, and microbial or other toxins), living
organisms (such as plant, animal, and microbes), and environmental
influences (such as food ingestion, alcohol ingestion, consumption
of tobacco products, stress, and physical activity), on a human or
an animal.
[0080] The methods and biomarkers of the invention can also be used
to pre-test a sample to determine if the subject from which the
sample originates suffers from kidney failure and for this reason
shows an altered excretion profile of other biomarkers intended to
be measured for diagnostic purposes. After the methods and
biomarkers of the invention have confirmed that the subject from
which the sample originates, does not suffer from kidney failure,
additional diagnostic biomarkers can be determined, without being
in doubt that a possibly underlying kidney failure impairs
measurement of the additional diagnostic biomarker. Alternatively
the pre-test for kidney failure and the test for the additional
biomarker can be done simultaneously, or the results of the
pre-test for kidney failure can be converted into a correction
factor used to correct the result of the renal-failure-impaired
measurement of the additional biomarker, based on the extent to
renal failure, or based on correction factors determined for
individual biomarkers by empirical testing.
Test Kits
[0081] The present invention further provides a test kit for
diagnosing, monitoring the progression or regression, or monitoring
the treatment of a renal disorder. In one embodiment, the kit
comprises a peptide biomarker having the sequence of SEQ ID NO:1 or
2. In another embodiment, the kit comprises a nucleic acid encoding
a peptide biomarker having the sequence shown in SEQ ID NO:1 or 2.
In yet another embodiment, the kit comprises an antibody that
specifically binds to a peptide biomarker having the sequence shown
in SEQ ID NO:1 or 2.
[0082] The test kit may optionally comprise substances for use as
standard and/or controls. The test kit may further optionally
comprise known markers for renal failure, such as creatinine or
Cystatin C, to further increase overall sensitivity and/or
specificity of the test.
[0083] In one aspect, the present invention provides test kits for
determining the absolute concentration or the relative
concentration, or determining the presence or absence of the
biomarkers of the present invention, thereby enabling the
measurement of renal function and/or diagnosis of renal failure.
The test kit can be used to diagnose the absence or presence of
renal failure/renal disease. The test kit can also be used to
predict the occurrence or to predict the stage of renal failure.
The test kit can further be used to predict and/or monitor the
success of a therapy for renal failure such as therapy by
medications, therapy by dialysis, or therapy by kidney
transplantation. Furthermore, the test kits of the present
invention can be used for the pre-analysis of a diagnostic sample
intended for other non-renal failure diagnostic procedures. The
test kits for pre-analysis determines whether renal failure is
present, as renal failure might impair other markers for non-renal
failure diseases or conditions, which are intended to be measured
using the same sample.
[0084] The test kit can be used to diagnose and/or predict chronic
renal failure at a stage earlier than other diagnostics tests
currently available (e.g. at Stage 2), such as creatinine
clearance, Cystatin C. The test kit can also be used to early
diagnose and/or predict acute renal failure, such as at R-stage or
I-stage.
[0085] The test kit can be used to analyze samples from a subject
such as those samples described previously. The test can be done
with pooled samples collected during a certain period of time,
e.g., after kidney transplantation, during hemodialysis, or during
diseases often associated with renal failure such as sepsis. The
test can be done with pooled samples of one or of more than one
subject. The sample can be used directly, or after storage of the
sample for various time periods at various storage conditions, or
the sample can be a pre-processed, for example, the sample can be
fractionated by chromatography, filtration, capillary
electrophoresis, precipitation, liquid or other extraction methods,
and immunoprecipitation. The whole sample or subject or two or more
combined fractions of a pre-processed sample, originating from one
or more than one samples can be analyzed.
[0086] The test can be done once or several times over a period of
time to analyze the time course of qualitative or quantitative
changes of a biomarker of the invention, e.g., the peptides of Seq
ID NOs:1 and 2, the encoding nucleic acids, and fragments or
modifications of the peptides and nucleic acids.
[0087] Furthermore, the test kit may comprise binding agents for
the antibodies binding specifically to the biomarkers of the
invention, or antibody fragments binding specifically to their
antigen. The binding agents can be present in the test kit in
immobilized form, for example immobilized to membranes, to
microtiter plates, to mass spectrometric targets, to SELDI chips,
to protein chips, to nucleic acid chips, to chromatographic resins,
to magnetic particles, to metal particles, to agarose particles, or
to other polymer particles. Alternatively, or in addition, the
binding agents can be present in the test kits in a form to
facilitate their immobilization to surfaces and materials as noted
above. The binding agents may be present in a labeled form, for
example, labeled with enzymes, fluorescent dyes, fluorescent
proteins or protein fragments or peptides, with different isotopes
which can be used in mass spectrometry and/or for radioactive
measurements, with dyes measurable by chemiluminescence, photometry
or by other measurement methods, with organic or inorganic groups
such as biotin, chitin, sugars, with lectins, with ligands for
receptors or with ligands for other structures.
[0088] In a further embodiment, the kit of the present invention
may optionally comprise instructions for diagnosing, monitoring the
progression or regression, or monitoring the treatment of the renal
disorder. The instructions may be on how to use the test kit, how
to prepare the samples, what kind of samples to use, how to analyze
and interpret the results. The test kit may further comprise
instructions on how to use the kit for determining the presence or
absence or prognosis of renal failure and of distinct stages of
renal failure, especially of chronic and/or acute renal
failure.
EXAMPLES
Example 1
Study Design and Probands
[0089] Healthy adult humans and humans suspected or known to suffer
from renal failure of different stages were enrolled in this study
after they provided written informed consent. Laboratory and other
parameters, such as body weight, height, age, sex, creatinine in
serum and/or urine, and protein in serum and/or urine, were
measured. If no 24 h urine sample was available, the creatinine
clearance was calculated by use of the Cockcroft formula.
Laboratory values determined were leukocytes, nitrite, pH, protein,
glucose, ketone, urobilinogen, bilirubin, blood and hemoglobin
presence in urine measured using urine sticks. Not all measurements
were performed on all subjects. In addition, the past medical
history was considered.
[0090] The samples were subjected to a standardized protocol
including addition of high concentrations of chaotropic salt and
ultra filtration, followed by reverse-phase chromatography,
followed by MALDI-mass spectrometry of individual fractions of the
chromatography, followed by quantitative and qualitative analysis
of the resulting data representing fraction, molecular mass and
MALDI signal intensity for each detected substance. This procedure
led to the identification of mass spectrometric signals suitable to
distinguish the different stages of renal failure.
Example 2
Sample Collection and Plasma Preparation
[0091] Blood samples were drawn from subjects by venipuncture from
a cubital vein, using a 20 gauche needle and a butterfly system
with a maximum tubing length of 8 cm. If a tourniquet was applied,
it did not remain in place for longer than 1 min. As soon as the
blood flowed into the container the tourniquet was released
partially. Prior to collecting a blood sample for subsequent plasma
preparation, the blood sampling device was flushed with a few ml of
the blood not used for plasma preparation. Subsequently, blood was
drawn into standard EDTA-containing syringes (e.g., 9 ml
EDTA-Monovette, Sarstedt, Numbrecht, Germany). Within 30 min. of
the blood drawing, the blood sample was centrifuged for 10 min. at
2000.times.g at room temperature. After centrifugation, the plasma
was transferred to a fresh tube and subjected to a second
centrifugation for 15 min. at 2500.times.g at room temperature. The
resulting, platelet-depleted plasma was transferred to a second
fresh tube. Alternatively, aliquots of 1.5 mL plasma from the first
centrifugation step were filtered using a 10 mL syringe equipped
with a cellulose acetate filter unit with a 0.2 M pore size and 5
cm.sup.2 filtration area (e.g., Sartorius Minisart.RTM., Sartorius,
Gottingen, Germany). All plasma samples in this study were prepared
by the 2-step centrifugation method.
[0092] Within 30 min. of the preparation, the plasma samples were
transferred to a -80.degree. C. freezer and stored until further
analyzed.
Example 3
Ultra Filtration of Plasma Samples in the Presence of Chaotropic
Salt
[0093] After thawing in a water bath, the plasma samples were kept
at room temperature until all proteins are re-dissolved. The sample
(0.95 mL) was mixed with 3.92 mL of 8 M Guanidin hydrochloride
solution, 0.08 mL peptide standard. The pH was adjusted to pH 2-3
by the addition of 0.018 mL of 30% [v/v] HCl. The diluted sample
was centrifuged for 60 min. at 4000.times.g at 23.degree. C. in an
ultra filtration device (Amicon Ulta-15, Art. No. UFC 905096,
Millipore, Bedford, Mass.). Prior to use, the ultra filtration
devices were rinsed with water by adding 15 mL water into the
device, centrifugation at 4000.times.g for 2 min. at 23.degree. C.,
and discarding any water present in the device after this rinsing
step. The final volume of 5.5 mL included 4.3 mL of the resulting
filtrate (flow through of the ultrafiltration) diluted with 1.2 mL
of 0.1% Tri-Fluor-Acetic Acid (TFA). This sample was stored at
-80.degree. C. until further analyzed.
Example 4
Reverse Phase (RP) Chromatography
[0094] The separation method carried out was a reverse phase (RP)
chromatography. The separation of peptides and proteins was done
using a source 5RPC, 4.6.times.150 mm reverse phase chromatography
column (Amersham Biosciences Europe GmbH, Freiburg, Germany).
Mobile phases of the following compositions were used: mobile phase
A: 0.06% (v/v) tri-fluor-acetic-acid, mobile phase B: 0.05% (v/v)
tri-fluor-acetic-acid, 80% (v/v) acetonitrile. The chromatography
took place at 33.degree. C. using an HP 1100 with a micro flow
cell, both supplied by Agilent Technologies (Boblingen,
Germany).
[0095] After thawing, samples prepared according to example 3 were
centrifuged at 18000g for 10 minutes. The resulting 0.75 mL plasma
equivalent was loaded onto the chromatography column. The
chromatography conditions were as follows: 5% mobile phase B at
time 0 min, from time 1 to 45 min. continuous increase in the
mobile phase B concentration to 50%, from time 45 to 49 min.
continuous increase in the mobile phase B concentration to 100%,
and subsequently up to time 53 min. constant 100% buffer B. Seven
minutes after the start of the chromatography, 96 fractions (0.5 ml
each) were collected.
Example 5
Mass Spectrometric Analysis
[0096] For mass spectrometric analysis, typical positive ion
spectra of peptides were produced in a MALDI-TOF mass spectrometer
(matrix-assisted laser desorption ionization). Suitable MALDI-TOF
mass spectrometers, such as Voyager-DE, Voyager-DE PRO or
Voyager-DE STR, are manufactured by Applied Biosystems (Foster
City, Calif.). BIFLEX manufactured by Bruker Daltonik (Bremen,
Germany) can also be used.
[0097] For the mass spectrometric analysis, the samples were
prepared by mixing them with a matrix substance that consists of an
organic acid. Suitable matrix substances according to the invention
are 3,5-dimethoxy-4-hydroxycinnamic acid,
alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. A
lyophilized equivalent obtained by reverse phase chromatography and
corresponding to 0.015 mL plasma was used to measure the peptides
and/or proteins and/or standards. The chromatographed sample was
dissolved in 0.015 mL of a matrix solution. The matrix solution
contains, for example, 10 g/L alpha-cyano-4-hydroxycinnamic acid
and 10 g/L L(-)fucose dissolved in a solvent mixture consisting of
acetonitrile, water, trifluoroacetic acid and acetone in the ratio
49:49:1:1 by volume. After 0.0003 mL of this solution was
transferred to a MALDI carrier plate, the dried sample was analyzed
in a Voyager-DE STR MALDI mass spectrometer.
[0098] The measurement took place in linear mode with delayed
extractionTM. The MALDI-TOF mass spectrometer can be employed to
quantify peptides if these peptides are present in a concentration
which is within the dynamic measurement range of the mass
spectrometer, thus avoiding detector saturation. There is a
specific ratio between measured signal and concentration for each
peptide, which means that the MALDI mass spectrometry can
preferably be used for the relative quantification of peptides. It
is possible to measure the signal intensities of the standards and
of peptides originating from the sample.
Example 6
Data Analysis
[0099] Subsequent to fractionation as described in Example 4, each
fraction was individually analyzed by MALDI mass spectrometry as
described in Example 5 resulting in 96 mass spectra for each
sample. These 96 mass spectra were electronically combined to a so
called peptide display. The x-axis of these peptide displays
depicts the molecular mass, the y-axis depicts the fraction number
and the color intensity represents the mass spectrometric signal
intensity.
[0100] Data pre-processing involved absolute scaling of MALDI
profiles, baseline correction, and m/z-recalibration of the mass
spectrometric data. Then spectra were binned down to 1 Da
resolution. Subsequent data analyses were performed using 4352 of
these signals which had an intensity above 50 units in at least 20%
of the samples. The signal intensities were stored using the
chromatographic fraction and mass spectrometric m/z ratio as
labels. Considering redundancy due to mass/fraction shifts (e.g.
the same peptide is present in 2, 3 or more neighboring fractions),
oxidation (the same peptide with and without oxidation is present
in the sample, without the oxidation being present in the sample at
the time the sample was collected) and, double or triple charged
peptide forms (the same peptide being detected at multiple
positions due to various charge states of it resulting in different
mass spectrometric signals of the same molecule), the number of
distinct peptides in the analysis was estimated to be about
1500.
Example 7
Mass Spectrometric Sequence Determination
[0101] The data of peptide displays were pre-processed by adjusting
for background noise. Differences between peptide displays were
calculated by subtracting peptide displays from each other
electronically. Detection of qualitative or quantitative
differences between individual samples or between groups of samples
regarding substances present in the samples, such as peptides or
proteins, was done by comparison of the mass spectrometric data,
e.g. the signal intensities of the corresponding substances. This
was done using mass spectrometric signal intensities of individual
samples and groups of samples, wherein the groups were samples from
healthy versus samples from subjects suffering from different
stages of kidney failure.
[0102] Peptides were identified using nanoSpray-MS/MS. This
entailed a standard peptide ion being selected in the mass
spectrometer on the basis of its specific m/z (mass/charge) value
in a manner known to the skilled worker. This selected ion was then
fragmented by supplying collision energy with an collision gas,
e.g. helium or nitrogen, and the resulting fragments of the
standard peptide were detected in the mass spectrometer in an
integrated analysis unit, and corresponding m/z values were
determined (principle of tandem mass spectrometry). The
fragmentation behavior of peptides made unambiguous identification
of the peptides possible. In this specific case, the mass
spectrometric analysis was performed using a Quadrupol-TOF
Instrument, QStar-Pulsar model from Applied Biosystems.
[0103] While the invention has been illustrated and described in
the figures and foregoing description, the same is to be considered
as illustrative and not restrictive in character, it being
understood that only the preferred embodiments have been shown and
described and that all changes and modifications that come within
the spirit of the invention are desired to be protected. In
addition, all references and patents cited herein are indicative of
the level of skill in the art and hereby incorporated by reference
in their entirety.
Sequence CWU 1
1
2136PRTHomo sapiens 1Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly
Thr Ser Gly His Pro 1 5 10 15 Gly Ser Pro Gly Ser Pro Gly Tyr Gln
Gly Pro Pro Gly Glu Pro Gly 20 25 30 Gln Ala Gly Pro 35 217PRTHomo
sapiens 2Glu Leu Glu Pro Pro Glu Gln Gln Glu Pro Gly Glu Arg Gln
Glu Pro 1 5 10 15 Ser
* * * * *
References