U.S. patent application number 14/217716 was filed with the patent office on 2014-07-10 for biomarkers for diagnosing alzheimer's disease.
This patent application is currently assigned to BAYER INTELLECTUAL PROPERTY GMBH. The applicant listed for this patent is BAYER INTELLECTUAL PROPERTY GMBH. Invention is credited to Odile Carrette, Denis F. Hochstrasser, Gerhard Konig, Jean-Charles Sanchez, Ozkan Yalkinoglu.
Application Number | 20140194318 14/217716 |
Document ID | / |
Family ID | 31197803 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140194318 |
Kind Code |
A1 |
Yalkinoglu; Ozkan ; et
al. |
July 10, 2014 |
BIOMARKERS FOR DIAGNOSING ALZHEIMER'S DISEASE
Abstract
A method for assessing the state of Alzheimer's disease in
patients is disclosed. A method for monitoring the progression of
Alzheimer's disease in patients is also disclosed. The method
applies detection of specific peptide markers, e.g., using mass
spectrometric analysis.
Inventors: |
Yalkinoglu; Ozkan;
(Wuppertal, DE) ; Konig; Gerhard; (Newton, MA)
; Hochstrasser; Denis F.; (Collonge-Bellerive, CH)
; Sanchez; Jean-Charles; (Geneva, CH) ; Carrette;
Odile; (Roubaix, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER INTELLECTUAL PROPERTY GMBH |
MONHEIM |
|
DE |
|
|
Assignee: |
BAYER INTELLECTUAL PROPERTY
GMBH
MONHEIM
DE
|
Family ID: |
31197803 |
Appl. No.: |
14/217716 |
Filed: |
March 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13099110 |
May 2, 2011 |
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14217716 |
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10525633 |
Apr 10, 2006 |
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PCT/EP2003/008879 |
Aug 11, 2003 |
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13099110 |
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Current U.S.
Class: |
506/9 ; 506/12;
530/326 |
Current CPC
Class: |
G01N 33/6896 20130101;
C07K 14/805 20130101; C07K 14/8139 20130101; C07K 7/08 20130101;
C07K 14/70539 20130101; G01N 2800/2821 20130101 |
Class at
Publication: |
506/9 ; 506/12;
530/326 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 7/08 20060101 C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 2002 |
EP |
02018283.8 |
Nov 29, 2002 |
EP |
02026643.3 |
Claims
1. (canceled)
2. An isolated polypeptide having the amino acid sequence of SEQ ID
NO: 17.
3. A method for assessing the state of Alzheimer's disease in a
subject, wherein the method comprises detecting in a sample from
the subject at least one polypeptide from each of the following
groups: a) a polypeptide having an amino acid sequence selected
from the group consisting of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13,
14, 15, and 16; b) a polypeptide having the amino acid sequence
selected from the group consisting of SEQ ID NO: 3, 4, 5, and 6;
and c) a polypeptide having the amino acid sequence of SEQ ID NO:
17.
4. The method of claim 3, wherein the method further comprises
contacting the sample with an antibody that specifically binds one
of the polypeptides.
5. The method of claim 3, wherein at least one polypeptide is
detected by SELDI-TOF MS.
6. The method of claim 3, wherein the method is performed on at
least two samples from the subject.
7. The method of claim 3, wherein the sample is CSF, blood, serum,
plasma, urine, seminal plasma, nipple fluid, or cell extract.
8. The method of claim 3, wherein the polypeptides are detected in
one sample from the subject.
9. A method for assessing the state of Alzheimer's disease in a
subject, wherein the method comprises detecting in a sample from
the subject at least three distinct polypeptides, wherein a first
polypeptide is selected from the group consisting of SEQ ID NO: 3,
4, 5, 6, 7 and 17; and wherein a second polypeptide and a third
polypeptide are selected from the group consisting of: a) a
polypeptide having the amino acid sequence selected from the group
consisting SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, or 17; b) a human cystatin C; c) a human
beta-2-microglobulin; d) a 7.7 kD homologue of human myoglobin; e)
neurosecretory protein VGF; f) a fragment of at least 5 amino acids
of human cystatin C; g) a fragment of at least 5 amino acids of
human beta-2-microglobulin; h) a fragment of at least 5 amino acids
of the 7.7 kD homologue of human myoglobin; and i) a fragment of at
least 5 amino acids of neurosecretory protein VGF.
10. The method of claim 9, wherein the method further comprises
contacting the sample with an antibody that specifically binds one
of the polypeptides.
11. The method of claim 9, wherein at least one polypeptide is
detected by SELDI-TOF MS.
12. The method of claim 9, wherein the method is performed on at
least two samples from the subject.
13. The method of claim 9, wherein the sample is CSF, blood, serum,
plasma, urine, seminal plasma, nipple fluid, or cell extract.
14. The method of claim 9, wherein the polypeptides are detected in
one sample from the subject.
15. The method of claim 9, wherein the method comprises detecting
at least four distinct polypeptides, wherein a fourth polypeptide
is selected from the group consisting of: a) a polypeptide having
the amino acid sequence selected from the group consisting SEQ ID
NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17;
b) a human cystatin C; c) a human beta-2-microglobulin; d) a 7.7 kD
homologue of human myoglobin; e) neurosecretory protein VGF; f) a
fragment of at least 5 amino acids of human cystatin C; g) a
fragment of at least 5 amino acids of human beta-2-microglobulin;
h) a fragment of at least 5 amino acids of the 7.7 kD homologue of
human myoglobin; and i) a fragment of at least 5 amino acids of
neurosecretory protein VGF.
16. The method of claim 15, wherein the polypeptides are detected
in one sample from the subject.
17. The method of claim 15, wherein the method comprises detecting
at least five distinct polypeptides, wherein a fifth polypeptide is
selected from the group consisting of: a) a polypeptide having the
amino acid sequence selected from the group consisting SEQ ID NO:
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; b) a
human cystatin C; c) a human beta-2-microglobulin; d) a 7.7 kD
homologue of human myoglobin; e) neurosecretory protein VGF; f) a
fragment of at least 5 amino acids of human cystatin C; g) a
fragment of at least 5 amino acids of human beta-2-microglobulin;
h) a fragment of at least 5 amino acids of the 7.7 kD homologue of
human myoglobin; and i) a fragment of at least 5 amino acids of
neurosecretory protein VGF.
18. The method of claim 17, wherein the polypeptides are detected
in one sample from the subject.
Description
FIELD OF THE INVENTION
[0001] The invention is in the field of diagnostics. More
specifically, the invention is in the field of assessing the state
of Alzheimer's disease in subjects by detection of Alzheimer's
disease-specific marker polypeptides.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease
[0003] Alzheimer's disease is an increasingly prevalent form of
neurodegeneration that accounts for approximately 50-60% of the
overall cases of dementia among people over 65 years of age.
Pathologically, Alzheimer's disease neurodegeneration is
characterised by prominent atrophy of corticolimbic structures with
neuronal death and loss of neuronal synapses, neurofibrillary
tangle (NFT) formation, and the formation of senile plaques
containing deposits of amyloid .beta.1-42 (A.beta.42) aggregates in
the brain [Francis PT 1999]. The duration of the progressive
cognitive decline is approximately 7 years from the occurrence of
first signs until death. It is assumed that the clinical phase is
preceded by a 15-30 years preclinical period of continuous
deposition of amyloid plaques and neurofibrillary tangles. Age of
onset and progression of the disease are largely determined by
causative gene mutations and by genetic susceptibility factors.
Several environmental risk factors may add to the individual
genetic risk factors. Genetic factors known to be involved in the
familial form of Alzheimer's disease with early onset of the
disease are: mutations in presenilin 1 (PS1), presenilin 2 (PS2),
and amyloid precursor protein (APP) genes, and the presence of the
apolipoprotein E4 allele. However, the majority (95%) of
Alzheimer's disease cases is sporadic and heterogeneous.
[0004] Currently, clinical diagnosis of Alzheimer's disease can
only be established at later stages of the disease, when cognitive
performance is significantly decreased and paralleled by structural
alterations of the brain. The clinical diagnostic work up requires
a careful medical history; physical and neurological examination;
blood, urine and cerebrospinal fluid (CSF) examinations to exclude
metabolic and medical disease states that might masquerade
Alzheimer's disease; detailed psychometric examinations to assess
mental status and cognitive performance, and imaging techniques
such as computed tomographic scan or magnetic resonance imaging of
the brain. Diagnostic evaluations at expert centres reach an
accuracy of about 80-85%. Due to the fact that these tests are
expensive and time consuming, and are particularly inconvenient to
patients, there is an increasing need for easy-accessible specific
diagnostic biomolecule markers, which can be measured in body
fluids, such as CSF, blood or urine, and which have a high positive
predictive value for diagnosis of Alzheimer's disease, or would
help to distinguish Alzheimer's disease from other forms of
dementia. Furthermore, reliable markers sensitive to disease
progression may constitute surrogate parameters, a major
prerequisite for the evaluation and development of new causal
oriented and disease modifying therapeutic strategies in
Alzheimer's disease.
[0005] Since CSF directly surrounds the brain, changes in its
protein composition may most accurately reflect pathologic
conditions that are associated with specific alterations of the
protein expression patterns. Over the last decade, a number of
biological abnormalities have been reported in the cerebrospinal
fluid (CSF) of Alzheimer's disease patients, in particular altered
levels of the A.beta.1-42 fragment of the amyloid precursor
protein, and altered levels of the hyperphosphorylated tan protein.
The sensitivity and specificity of these markers, however, is low
or only modest [The Ronald and Nancy Reagan Research Institue of
the Alzheimer's Association and the National Institute on Aging
Working Group, 1998, Robles A 1998, Termissen C E et al.,
2002].
[0006] Hence, there is a need for novel biomarkers with sufficient
sensitivity and specificity for (i) detecting Alzheimer's disease
as early as possible, and (ii) to allow disease differentiation
from other types of dementia or neurodegenerative diseases, and
(iii) monitoring therapeutic efficacy as surrogate parameter, e.g.
in clinical drug development, and to initiate pharmacotherapy as
early as possible and postpone loss of memory and disease
progression.
[0007] Protein Chip Technology
[0008] A Protein chip technology called Surface Enhanced Laser
Desorption/Ionisation time of flight mass spectrometry (SELDI-TOF
MS) has recently been developed to facilitate protein profiling of
complex biological mixtures [Davies H A 2000, Fung ET 2001,
Merchant M 2000].
[0009] Protein chip mass spectrometry has already been used by
several groups to detect potentially novel biomarkers of prostate
and bladder [Adam B L 2001] or breast cancer [Wulfkuhle I D 2001]
in serum, seminal plasma, nipple fluid, urine or cell extracts. For
a review on biomarker search using SELDI-TOF MS, see [Issaq H J
2002].
[0010] Cystatin C
[0011] Initially described in 1961 in cerebrospinal fluid (CSF),
cystatin C (y trace or post-.gamma. globulin, Ace. No. P01034) is a
small cystein proteinase inhibitor present in all human body fluids
at physiologically relevant concentrations. The physiological role
of cystatin C is likely to regulate extracellular cysteine protease
activity, which results from microbial invasion or release of
lysosomal proteinases from dying or diseased cells. Cystatin C
colocalises with .beta.-amyloid (A.beta.) within the arteriolar
walls in Alzheimer's disease brains and cerebral amyloid angiopathy
[Levy E 2001]. There are two common haplotypes of the CST3 gene
coding for cystatin C (A and B) that differ from each other at
three sites: two single base pair changes in the promoter region
and one in the signal peptide domain that causes an amino acid
substitution (alanine to threonine). Recently, case control studies
found associations of CST3 with increased risk for late onset
Alzheimer's disease [Crawford F C 2000, Finckh U 2000, Beyer K
2001].
[0012] Hereditary cerebral hemorrhage with amyloidosis, Icelandic
type (HCHWA-I), also called hereditary cystatin C amyloid
angiopathy (HCCAA), is an autosomal dominant form of cerebral
amyloid angiopathy (CAA). The amyloid deposited in the brain
vessel's walls is composed mainly of a variant of cystatin C
characterised by the presence of the Leu68-Gln substitution [Cohen
1983, Ghiso 1986]. This pathology is also coupled to a decreased
concentration of this major cystein proteinase inhibitor in
cerebrospinal fluid and leads to its amyloid deposition in the
brain [Grubb A O 1984].
[0013] Leung-Tack et al have also purified two N-terminal truncated
isoforms of cystatin C in urine from one patient who had received
renal transplant. According to their data, (des1-4) cystatin C has
an inhibiting effect on two functions of human peripheral
mononuclear cells (PMN): O.sub.2.sup.- release and phagocytosis,
which may be due to the N-terminal sequence `KPPR`. Their data
support a potentially important role for cystatin C as a possible
immunomodulator during inflammation. Accumulating evidence
indicates that increased free radical mediated damage to cellular
function contributes to the ageing process and age-related
neurodegenerative disorders. Oxidative stress may play a role in
Alzheimer's disease, Parlcinson's disease, amyotrophic lateral
sclerosis (ALS). Although free-radical damage to neurons may not be
the primary event initiating these diseases, it appears that
free-radical damage is involved in the pathogenetic cascade of
these disorders.
[0014] Beta-2-microglobulin
[0015] Beta-2-microglobulin (Ace. No. P01884) constitutes the small
constant component of the class I major histocompatibility complex
(CMH) and its presence in biological fluids represents the balance
between membrane protein turnover and elimination. Since this
peptide seems to be increased in some diseases characterised by an
elevation of the immune response, its quantification in body fluids
has become a useful index of immunological state in vivo [Hoelcman
et al 1985]. The function of this protein is unclear, but it seems
to be implicated in diseases, which involve glial cell destruction
[Ernerudh et al 1987].
[0016] The technical problem which is solved by the present
invention is the provision of improved methods for diagnosing
Alzheimer's disease and/or monitoring the progression of
Alzheimer's disease in a subject.
[0017] Neurosecretary Protein (VGF)
[0018] VGF (human VGF, Acc.-No.: 015240) is a secretory peptide
precursor that is expressed and processed by neuronal cells [Canu.
et al. 1997]. In situ hybridization studies in the adult rat
central nervous system have revealed that the VGF mRNA is widely
distributed throughout the brain with prominent expression in the
hippocampus, entorhinal cortex, and neocortex. Furthermore, it has
been shown that VGF transcription and secretion is selectively
upregulated by neurotrophins like NGF and BDNF, and by
depolarization in vitro. Increased BDNF expression can be observed
in dentate gyrus and CA3 regions of the hippocampus, which are
tissues that appear to die early in Alzheimer Disease
pathogenesis.
DESCRIPTION OF THE INVENTION
[0019] The invention is based on the surprising finding that
specific polypeptides are differentially expressed in subjects
having Alzheimer's disease when compared to a healthy control
group. These differentially expressed polypeptides can be, e.g.,
detected in samples of cerebrospinal fluid of the subject in which
Alzheimer's disease is to be diagnosed. The individual polypeptides
of the invention can be detected and/or quantified alone or in
combination with other polypeptides of the invention.
[0020] The polypeptide markers of the invention are defined by
their respective molecular weight. Five markers identified by the
SAX2 method as described in the Examples show the following
molecular masses: [0021] Marker 1 (M1): 4824.+-.20 Da; [0022]
Marker 2 (M2): 7691.+-.20 Da; [0023] Marker 3 (M3): 11787.+-.20 Da;
[0024] Marker 4 (M4): 11988.+-.20 Da; [0025] Marker 5 (M5):
13416.+-.20 Da.
[0026] Table one shows the observed molecular weight of polypeptide
markers M1 to M5 as determined by SELDI-TOF MS, the amino acid
sequences of observed fragments of polypeptide markers M1 to M5,
and the protein from which the polypeptide markers M1 to M5
originate.
TABLE-US-00001 TABLE 1 SEMI ob- Marker served MW Amino Acid
Sequence Protein Name M1 4823.5 VGEEDEEAAEAEAEAEEAER VGF4.8 Da .+-.
1.7 M2 7691.4 XXAD(L/I)AGHG(Q/K)EV(L/I)(L/I) human myoglobin Da
.+-. 4.9 R new variant HGTVV(L/I)TA(L/I)KGG(L/I)(L/I) M3 11786.9
VNHVTLSQPK human beta-2- Da .+-. 7.6 VEHSDLSFSK micro-globulin M4
11988.4 IEKVEHSDLSFSK Da .+-. 5.9 SNFLNCYVSGFHPSDIEVDLLK M5 13416.4
ASNDMYHSR human Cystatin C Da .+-. 9.4 ALDFAVGEYNK RALDFAVGEYNK
LVGGPMDASVEEEGVR QIVAGVNYFLDVELGR LVGGPMDASVEEEGVRR
KQIVAGVNYFLDVELGR TQPNLDNCPFHDQPPHLK TQPNLDNCPFHDQPPHLKR
SSPGKPPRLVGGPMDASVEEEGVR
[0027] The differentially expressed polypeptides of the invention
can be, e.g., detected in samples of cerebrospinal fluid of the
subject in which Alzheimer's disease is to be diagnosed. In
addition, depending on the specific embodiment, the source of
samples to measure the abundance of the polypeptide markers of the
invention, can also be blood, serum, or urine, but is not limited
to these body compartments.
[0028] As shown in FIG. 1, compared to a negative diagnosis
(healthy controls), M1 is under-expressed in the CSF of Alzheimer's
disease patients (p<0.05), while the markers M2 to M5 are
over-expressed in CSF of Alzheimer's disease patients
(p<0.05).
[0029] An altered level of one or several polypeptides of the
invention, compared to the level of polypeptides of the invention
in healthy control subjects, will allow assessing the state of
and/or monitoring the progression of Alzheimer's disease in a
subject, will allow monitoring the effectiveness of Alzheimer's
disease treatment, and will be useful information for drug
development. Furthermore, these biomolecule markers are useful for
differentiating Alzheimer's disease from other forms of dementia
and neurodegenerative disorders.
[0030] Preferred subjects in which Alzheimer's disease is to be
diagnosed or monitored are human subjects. However, diagnosis of
Alzheimer's disease according to the invention is also possible
with other mammals. If necessary, orthologues of the peptide
markers of the invention can be used.
[0031] The invention also relates to the use of mass spectrometry
(MS) for detecting Alzheimer's disease in human subjects and for
assessing the progression of Alzheimer's disease in human subjects
by detecting and/or quantifying the amount of specific polypeptides
in samples drawn from the subject's body fluids. In a preferred
embodiment of the invention, the sample is drawn from the subject's
cerebrospinal fluid (CSF).
[0032] Detection and/or quantification of the polypeptides of the
invention is preferably achieved by quantifying the signal which is
detected by MS at specific molecular mass to charge ratios (M/z)
which correspond to the M/z ratios of the polypeptides of the
invention. Preferably, M/z ratios close to the one of the
polypeptides of the invention are also measured.
[0033] Detection and/or quantification of the polypeptides of the
invention can also be achieved by using an immunoassay using
specific antibodies raised against the specified marker(s) or
polypeptide fragments thereof. Antibodies can be prepared by using
the purified marker(s) or fragments thereof, or using synthetic or
recombinantly expressed polypeptide(s) consisting of the specific
amino acid sequence of the marker(s) using any suitable method
known in the art [Coligan 1991]. Such techniques include, but are
not limited to, antibody preparation by selection of antibodies
from libraries of recombinant antibodies in phage or appropriate
vectors, as well as preparation of polyclonal and monoclonal
antibodies by immunising rabbits or mice [Huse 1989, Ward 1989].
After the antibody is provided, a marker can be detected and/or
quantified using any of a number of standard immunological binding
assays [U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and
4,837,168]. Useful assays include, but are not limited to, for
example, an enzyme immune assay (ETA) such as enzyme-linked
immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western
blot assay, or a slot or dot blot assay. For a review of the
general immunoassays see [Coligan 1991]. Generally, a sample
obtained from a subject can be contacted with the antibody that
specifically binds the marker. A powerful technique to capture the
specified marker(s) from a complex body fluid sample is to use the
antibody fixed to solid supports, such as glass or plastic, e.g.
microtiter plate, a stick, a bead, or microbead. Alternatively,
marker(s) can also be captured from the body fluid sample by the
specific antibody immobilised to a probe substrate or a
ProteinChip.TM. array, as described for the SELDI-based immunoassay
[Xiao 2001]. After incubating the sample with antibodies, the
non-bound material is washed under specified conditions and the
antibody-marker complex formed can be detected, using appropriate
detection reagents. In an embodiment using the SELDI
ProteinChip.TM. array technique the marker(s) selectively enriched
by the immobilised antibody can be detected and quantified by
matrix-assisted laser desorption ionisation mass spectrometry.
[0034] The invention specifically relates to [0035] 1. a method of
assessing the state of Alzheimer's disease in a subject comprising
detection of at least one polypeptide comprised in a group of
polypeptides consisting of [0036] i) a polyp eptide having a
molecular mass of 4824.+-.20 Da, [0037] ii) a polypeptide having a
molecular mass of 7691.+-.20 Da, [0038] iii) a polypeptide having a
molecular mass of 11787.+-.20 Da, [0039] iv) a polypeptide having a
molecular mass of 11988.+-.20 Da, and [0040] v) a polypeptide
having a molecular mass of 13416.+-.20 Da. [0041] The invention
further relates to a method of assessing the state of Alzheimer's
disease in a subject comprising detection of at least one
polypeptide comprised in a group of polypeptides having,
respectively, molecular masses of 4824.+-.20 Da, of 7691.+-.20 Da,
of 11787.+-.20 Da, of 11988.+-.20 Da, of 13416.+-.20 Da, of
4769.+-.20 Da, of 6958.+-.20 Da, of 6991.+-.20 Da, of 13412.+-.20
Da, of 13787.+-.20 Da, of 17276.+-.20 Da, of 40437.+-.20 Da, of
6895.+-.20 Da, of 6928.+-.20 Da, of 7691.+-.20 Da, of 7769.+-.20
Da, of 7934.+-.20 Da, of 5082.+-.20 Da, of 6267.+-.20 Da, of
6518.+-.20 Da, of 7274.+-.20 Da, and of 8209.+-.20 Da. [0042]
Whereas detection of one such polyp eptide is in most cases
sufficient to reliably diagnose Alzheimer's disease, detection of
two or more polypeptides of the invention can increase the
sensitivity and robustness of the method. Preferably, 1, 2, 3, 4,
5, 10, and, most preferred, all of said polypeptides will be
detected from the same sample. The detection can also be carried
out simultaneously with the detection of other polypeptides which
are preferably also differentially expressed in subjects having
Alzheimer's disease as compared to healthy subjects. "Assessing the
state of Alzheimer's disease" shall be understood as diagnosing the
presence of Alzheimer's disease in a subject or a patient, as
assessing the progression of the disease in a subject or a patient,
and/or as assessing the proneness of a subject to develop
Alzheimer's disease. [0043] 2. The invention further relates to the
method of point 1 in which 2, or 3, or 4, or 5 polypeptides of said
group of peptides are detected. The invention further relates to a
method of point 1 in which 2, or 3, or 4, or 5, or 10 or all
polypeptides of said group of peptides are detected. [0044] 3. The
invention further relates to a method of assessing the state of
Alzheimer's disease in a subject comprising detection of at least
one polypeptide comprising the sequence of SEQ ID NO:1, SEQ ID
NO:2, SEQ II) NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and/or SEQ 1D
NO:16. The invention further relates to a method of assessing the
state of Alzheimer's disease in a subject comprising detection of
at least one polypeptide comprising the sequence of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, and/or SEQ ID NO:17. Whereas detection of one such
polypeptide is in most cases sufficient to reliably diagnose
Alzheimer's disease, detection of two or more polypeptides of the
invention can increase the sensitivity and robustness of the
method. Preferably, 1, 2, 3, 4, 5, 10, or all of said polypeptides
will be detected from the same sample. The detection can also be
carried out simultaneously with the detection of other polypeptides
which are preferably also differentially expressed in subjects
having Alzheimer's disease as compared to healthy subjects. [0045]
4. The invention further relates to a method of assessing the state
of Alzheimer's disease in a subject comprising detection of at
least one polypeptide comprised. in a group of polypeptides
consisting of [0046] i) human cystatin C, [0047] ii) human
beta-2-microglobulin, [0048] iii) human myoglobin (new variant),
[0049] iv) a fragment of at least 5, 8, 10, or 20 amino acids of
human cystatin C, [0050] v) a fragment of at least 5, 8, 10, or 20
amino acids of human beta-2-microglobulin, and [0051] vi) a
fragment of at least 5, 8, 10, or 20 amino acids of human myoglobin
(new variant). [0052] The invention further relates to a method of
assessing the state of Alzheimer's disease in a subject comprising
detection of at least one polypeptide comprised in a group of
polypeptides consisting of [0053] i) human cystatin C, [0054] ii)
human beta-2-microglobulin, [0055] iii) human myoglobin (new
variant), [0056] iv) human neurosecretory protein VGF, [0057] v) a
fragment of at least 5, 8, 10, or 20 amino acids of human cystatin
C, [0058] vi) a fragment of at least 5, 8, 10, or 20 amino acids of
human beta-2-microglobulin, [0059] vii) a fragment of at least 5,
8, 10, or 20 amino acids of human myoglobin (new variant), and
[0060] viii) a fragment of at least 5, 8, 10, or 20 amino acids of
neurosecretory protein VGF. [0061] Whereas detection of one such
polypeptide is in most cases sufficient to reliably diagnose
Alzheimer's disease, detection of two or more of said polypeptides
can increase the sensitivity and robustness of the method.
Preferably, 1, 2, 3, 4, 5, or 6 of said polypeptides will be
detected from the same sample. More preferably, 1, 2, 3, 4, 5, 6 or
all of said polypeptides will be detected from the same sample. The
detection can also be carried out simultaneously with the detection
of other polypeptides which are preferably also differentially
expressed in subjects having Alzheimer's disease as compared to
healthy subjects. [0062] 5. The invention further relates to a
method of investigating the progression of Alzheimer's disease in a
subject characterised in that a method of any of points 1. to 4 is
performed with at least two distinct samples drawn from the same
subject. For this purpose, samples drawn from a subject at
different points in time will be analysed. Changes in the amount of
the respective polypepfide(s) will allow to draw conclusions on the
progression of Alzheimer's disease in the subject. [0063] 6. The
invention further relates to a method of any of points 1 to 5,
wherein detection of said polypeptide(s) is by SELDI-TOF MS. Other
suitable mass spectrometric methods and other methods of detection
can alternatively be used. More specifically, the invention relates
to a method of any of points 1 to 5, wherein detection of said
polypepticle(s) is by SELDI-TOF MS in which the hydrophobic H50,
the WCX2, or the IMAC surface is used as a support upon ionisation.
Different supports for ionisation yield different sensitivity for
specific proteins of interest. [0064] 7. The invention further
relates to a method of any of points 1 to 5, wherein specific
antibodies or antibodies recognising said polypeptide(s) are used
for detection of said polyp eptide(s). [0065] 8. The invention
further relates to a method of any of points 1 to 7, wherein
detection is in a sample comprising CSF of said patient. A sample
drawn from a subject can be processed immediately after it has been
taken, or it can first be frozen and be analysed later. Samples may
also consist of or contain other body fluids such as blood, serum,
plasma, urine, seminal plasma, nipple fluid, or cell extracts.
[0066] 9. The invention further relates to a kit comprising a
polypeptide having a molecular mass of 4824.+-.20 Da, a polypeptide
having a molecular mass of 7691.+-.20 Da, a polypeptide having a
molecular mass of 11787.+-.20 Da, a polypeptide having a molecular
mass of 11988.+-.20 Da, and/or a polypeptide having a molecular
mass of 13416.+-.20 Da. The invention further relates to a kit
comprising a polypeptide having a molecular mass of 4824.+-.20 Da,
a polypeptide having a molecular mass of 7691.+-.20 Da, a
polypeptide having a molecular mass of 11787.+-.20 Da, a
polypeptide having a molecular mass of 11988.+-.20 Da, and a
polypeptide having a molecular mass of 13416.+-.20 Da. The
invention further relates to a kit comprising polypeptides having a
molecular mass of 4824.+-..+-.20 Da, of 7691.+-.20 Da, of
11787.+-.20 Da, of 11988.+-.20 Da, of 13416.+-.20 Da, of 4769.+-.20
Da, of 6958.+-.20 Da, of 6991.+-.20 Da, of 13412.+-.20 Da, of
13787.+-.20 Da, of 17276.+-.20 Da, of 40437.+-.20 Da, of 6895.+-.20
Da, of 6928.+-.20 Da, of 7691.+-.20 Da, of 7769.+-.20 Da, of
7934.+-.20 Da, of 5082.+-.20 Da, of 6267.+-.20 Da, of 6518.+-.20
Da, of 7274.+-.20 Da, and/or of 8209.+-.20 Da. Such a kit can be
applied for various purposes, e.g., for use as a standard in one of
the above mentioned methods. Said kit can comprise 2, 5, 10, or all
of the above polypeptides. [0067] 10. The invention further relates
to a kit comprising a fragment of at least 5 amino acids of human
cystatin C, a fragment of at least 5 amino acids of human
beta-2-microglobulin, and a fragment of at least 5 amino acids of
human myoglobin. This kit can be applied for various purposes,
e.g., for use as a standard in one of the above mentioned methods.
The invention further relates to a kit comprising a fragment of at
least 5, 10 or 20 amino acids of human cystatin C, a fragment of at
least 5, 10 or 20 amino acids of human beta-2-microglobulin, a
fragment of at least 5, 10 or 20 amino acids of human myoglobin,
and a fragment of at least 5, 10 or 20 amino acids of
neurosecretory protein VGF. These kits can be applied for various
purposes, e.g., for use as a standard in one of the above mentioned
methods.
BRIEF DESCRIPTION OF THE FIGURES
[0068] FIG. 1: Average intensities of the five marker peptides of
Table 1, which are differentially expressed in the diseased group
when compared to the control groups.
EXAMPLES
[0069] The invention is further described by one or several of the
following examples. These examples are not to be understood as
restricting the scope of the invention to the examples by any
means.
Example 1
Patients Evaluation and CSF Sampling
[0070] Diagnosis of Alzheimer's disease in human subjects was made
according to criteria of the National Institute of Neurologic and
Communicative Disorders and Stroke-Alzheimer's disease and Related
Disorders Association (NINCDS-ADRDA). The Alzheimer's disease group
consisted of 9 patients aged 75.+-.7 years, six men and three
women. The group of healthy control subjects consisted of 10
individuals aged 78.+-.14 years, two men and eight women with no
history, symptoms or signs of psychiatric or neurological
disease.
[0071] Informed consent was given by each patient and the patients'
caregivers before the investigation. The study was approved by the
local ethics committee. After lumbar puncture, CSF samples were
frozen on dry ice immediately upon withdrawal at the bedside in 0.5
ml aliquots and stored at -80.degree. C. until analyses.
Example 2
ProteinChip SELDI Analysis of CSF on SAX2 Chip
[0072] SAX 2 Proteinchip array (Ciphergen Biosystems, Fremont,
Calif., USA) were equilibrated for 5 min with 5 .mu.l of binding
buffer (100 mM Na Acetate pH=4.0). The buffer was carefully removed
with an handkerchief and 2.5 .mu.l of binding buffer was added to
the wells. Crude CSF samples (2.5 .mu.l ) were added to the wells
and incubated for 20 min at room temperature in a humidity chamber
on a rocking platform. CSF was removed and the wells were
individually washed with 10 .mu.l of binding buffer for 5 min. The
arrays were then placed in a 15 ml conical Eppendorf and washed
twice with the binding buffer for 5 min. Finally, the chip was
rinsed twice with distilled water, Excess of H.sub.2O was removed
and while the surface was still moist, two additions per well of
0.5 .mu.l of sinapinic acid. (SPA) (2 mg/ml) in 50% (vol/vol)
acetonitrile and 0.5% (vol/vol) trifluoroacetic acid was perfothied
and dried. The arrays were then read in a ProteinChip reader
system, PBS II series (Ciphergen Biosystems). The laser beam was
focused on the sample in vacuo. This caused the proteins absorbed
to the matrix to become ionised and, simultaneously to be desorbed
from the Proteinchip array surface. The ionised proteins were
detected and their molecular masses were determined according to
their time-of-flight (TOF). TOF mass spectra, collected in the
positive ion mode were generated using an average of 65 laser shots
throughout the spot at a laser power set slightly above threshold
(10-15% higher than the threshold) High mass to acquire was set at
40 kDa, optimised from 1 to 15 kDa. Spectra were collected and
analysed using the Ciphergen Proteinchip (version 3.0) software.
External calibration of the reader was performed using the
"all-in-1" peptide molecular weight standards (Ciphergen
biosystems, Inc.) diluted in the SPA matrix (1:1, vol/vol) and
directly applied onto a well. Protein profile comparison was
performed after normalisation on total ion current of all the
spectra included in the same experiment. The reproducibility was
tested by analysing different aliquots of the same CSF sample on 4
different wells of the same proteinchip array (intraassay intrachip
reproducibility), on two different chips (intraassay interchip
reproducibility) processed in parallel, and reproduced in an other
experiment (interassay reproducibility).
[0073] Analysis of CSF samples from 9 patients diagnosed with
Alzheimer's disease relative to 10 controls revealed that 5 peaks
were significantly differentially expressed between the two groups
(p<0.05). The approximate average SELDI mass associated with the
five differentially expressed proteins was 4.82 kDa, 7.7 kDa, 11.8
kDa and 12.0 kDa and 13.4 kDa (p<0.05) (see Table 1, FIG.
1).
Example 3
Strong Anionic Exchange Chromatography (SAX) Purification
[0074] In order to identify the proteins corresponding to these
peaks, a fractionation of crude CSF on a SAX spin column was
performed. The elated fractions were analysed by SELDI-TOF MS.
[0075] SAX spin column, lot number SAX2-001116-01, (Ciphergen
Biosystems, Fremont, Calif., USA) was rehydrated overnight at
4.degree. C. in the equilibration buffer (20 mM
tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 5 mM
NaCl, pH 9.0). The column was warmed up at room temperature and air
bubbles were removed. The equilibration buffer was let flow through
column matrix by gravity. Equilibration buffer (0.5 ml) was added
to the column and passed through the resin twice. Two ml of control
CSF was diluted in the equilibration buffer (1:1, vol/vol). Protein
sample was loaded to the column by fraction of 0.8 ml and allowed
to run through the column by gravity until no drops came out of the
column. The column was then centrifuged at 150.times.g for 1 min.
The resin was then washed with an equivalent volume of
equilibration buffer. This step was repeated several times in order
to load the whole sample onto the resin. Elution of the bound
proteins was performed by decreasing the pH. Elution buffer A
consisted of 20 mM Tris-HCl, 5 mM NaCl pH 8.0; elution buffer B=20
mM sodium phosphate pH 7.0; elution buffer C=20 mM sodium phosphate
pH 6.0; elution buffer D=20 mM sodium phosphate and citrate pH 5.0;
elution buffer E=20 mM sodium phosphate and citrate pH 4.0; elution
buffer F=20 mM sodium phosphate and citrate pH 3.4; elution buffer
G=30% acetonitrile in elution buffer F. Elution was performed by
applying 2.times.75 .mu.l of the elution buffer and centrifugation
at 150.times.g for 1 min. Each collected fraction (150 .mu.l) was
concentrated on a speed-vac to a volume of 10 .mu.l. Protein
profiles were analysed on SELDI-TOF MS using SAX 2 Proteinchip
arrays. The chip was equilibrated with a binding buffer consisting
of 20 mM Tris-HCl, 5 nM NaCl, pH=9.0. An aliquot of 0.5 .mu.l of
each concentrated fraction was applied directly onto 2.5 .mu.l of
binding buffer per spot and processed as previously described. The
rest of the fractions were loaded onto a Tris tricine gel as
described below.
[0076] The differentially expressed peak of 13.4 kDa was eluted
with buffer A (20 mM Tris-HCl 5 mM NaCl pH 8.0) and B (20 mM sodium
phosphate pH 7.0). The differentially expressed peaks of 11.8 kDa
and 12.0 kDa were found in the fraction eluted with buffer C (20 mM
sodium phosphate pH 6.0) and D (20 mM sodium phosphate and citrate
pH 5.0). The cluster of 7.7 kDa was eluted with buffer D (20 mM
sodium phosphate and citrate pH 5.0) and E (20 mM sodium phosphate
and citrate pH 4.0).
[0077] Each eluted fraction was loaded on a 16.5% Tris Tricine
sodium dodecyl sulfate polyacrylamide gel and electrophoresed (SDS
PAGE). After coloration with coomassie blue, the bands seen on the
gel confirmed the results obtained by SELDI analysis. The band
corresponding to the cluster of 7.7 kDa, 11.8 kDa and 12.0 kDa were
cut out. Proteins were extracted as described in Example 6 and
identified by Q-TOF. The 7.7 kDa peak MS analysis did not match
with any known human protein, however, may indicate to a new
variant or homologue of myoglobin. Peptide sequences were the
following
TABLE-US-00002 XXAD(L/I)AGHG(Q/K)EV(L/I)(L/I)R and
HGTVV(L/I)TA(L/I)GG(L/I)(L/I)K.
[0078] The MS analysis of the 11.8 kDa and 12.0 kDa peaks
identified beta-2-microglobulin for both of them.
[0079] Since the cluster of 13.4 kDa could not be seen on the Tris
Tricine gel, a Tris glycine SDS-PAGE electrophoresis was performed
on crude CSF samples. The band corresponding to the
beta-2-microglobulin could be easily found on this stained get We
concluded that the protein migrating just above the
beta-2-microglobulin could correspond to the next abundant protein
seen on the SELDI profile, namely the 13.4 kDa peak. The band was
excised from the gel and digested by trypsin before MALDI analysis.
The peptide mass fingerprint analysis allowed to identify the
Cystatin C. The sequence coverage provided by the analysis was
60%.
Example 4
Monodimensional Electrophoresis/Tris Glycine Gels
[0080] Twenty .mu.l of CSF were mixed with 10 .mu.l of denaturing
Laemmli buffer [Laemmli 1970]. The samples were heated to
95.degree. C. for 5 min, and loaded on a 15% T (T=total acrylamide
concentration) SDS-polyacrylamide gel according to the method of
Laemmli. Gels were stained in a solution containing Coomassie
Brilliant Blue R-250 (0.1% w/v) and methanol (50% v/v) for 30 min.
Destaining was clone in a solution containing methanol (40% v/v)
and acetic acid (10% v/v).
Example 5
Monodimensional Electrophoresis: Tris Tricine Gels
[0081] Tris tricine SDS-PAGE electrophoresis was performed
according to Schagger and von Jagow [1987] using precast 16.5% T
gels (Biorad, Hercules, Calif.). The anode buffer consisted of 0.2M
Tris-HCl, pH 8.9 and the cathode buffer consisted of 0.1M Tris-HCl,
0.1M Tricine, 0.1% SDS, pH 8.25. Samples were diluted in 10 .mu.l
of 50 mM of bromophenol blue, pH 6.8. After denaturation at
95.degree. C. for 5 min, samples were loaded onto the gel. Gels
were run at 80V for 3 hours. After electrophoresis, gels were fixed
in 40% methanol, 10% acetic acid for 30min. Gels were then stained
with Colloidal blue coomassie G250 overnight and destained in 30%
methanol. Bands to be identified were immediately cut, placed in an
eppendorf and kept at 4.degree. C. until further analysis. The
apparent molecular masses were determined by running polypeptide
molecular weight (MW) standards: Triosephophate isomerase MW
26,625; Myoglobin MW 16,950; .alpha.-lactalbumin MW 14,437;
Aprotinin MW 6,512; Insulin b chain, oxidised MW 3,496 and
Bacitracin MW 1,423 (Biorad).
Example 6
Protein Digestion and Peptide Extraction [Bienvenu 1999]
[0082] Fragments of gels containing proteins of interest were cut
out for digestion of the proteins with trypsin using previous
published procedures [Shevchenko 1996, Hellman 1994, Rosenfeld
1992] and modified as described below. The piece of gel was first
destained with 100 .mu.l of 50 mM ammonium bicarbonate, 30% (v/v)
acetonitrile during 15 min at room temperature. Destaining solution
was removed and replaced by 25 .mu.l of 10 mM DL-dithiothreitol
(DTT) in 50 mM ammonium bicarbonate and incubated 35 min at
56.degree. C. DTT solution was then replaced by 25 .mu.l of 55 mM
iodoacetamide in 50 mM ammonium bicarbonate and incubated during 45
min at room temperature in the dark. Gel pieces were washed for 10
min with 100 .mu.l of 50 mM ammonium bicarbonate and for 10 min
with 100 .mu.l of 50 mM ammonium bicarbonate and 30% (v/v)
acetonitrile.
[0083] Gel pieces were then dried for 30 min in a Hetovac vacuum
centrifuge (HETO, Allerod, Denmark). Dried pieces of gel were
rehydrated for 45 min at 4.degree. C. in 5-20 .mu.l of a solution
of 50 mM ammonium bicarbonate containing trypsin at 6.25 ng/.mu.l.
After an over-night incubation at 37.degree. C., gel pieces were
dried under high vacuum centrifuge before being rehydrated by the
addition of 20 .mu.l of distilled water and finally dried again in
a speed-vac for 30 min. Extraction of the peptides was performed
with 20 .mu.l of 0.1% (v/v) trifluoroacetic acid (TEA) for 20 min
at room temperature with occasional shaking. The TFA solution
containing the peptides was transferred to a polypropylene tube. A
second elution was performed with 20 .mu.l of 0.1% (v/v) TFA in 50%
(v/v) acetonitrile for 20 min at room temperature with occasional
shaking The second TFA solution was pooled with the first one. The
volume of the pooled extracts was reduced to 1-2 .mu.l by
evaporation under vacuum. Control extractions (blanks) were
performed using pieces of gels devoid of proteins.
Example 7
Protein Identification by Peptide Mass Fingerprinting Analysis
[0084] 1.5 .mu.l of sample was placed on a MALDI 100-well target
plate. Same volumes of matrix (10 mg/ml cc-Cyano-4-hydroxycinnamic
acid in 50% (v/v) acetonitrile, 0.1% (v/v) TFA) were added to the
previously loaded digest. Samples were dried as quickly as possible
using a vacuum container. Mass measurement from liquid solution
were conducted with a MALDI-TOF mass spectrometer Voyager.TM. Elite
and Super STR (PerSeptive Biosystems, Framingham Mass., USA)
equipped with a 337 nm nitrogen laser. The analyser was used in the
reflectron mode at an accelerating voltage of 20 kV, a delayed
extraction parameter of 100-140 ns and a low mass gate of 850 Da.
Laser power was set slightly above threshold (10-15% higher than
the threshold) for molecular ion production. Spectra were obtained
by summation of 10 to 256 consecutive laser shots. Masses of the 60
highest peaks were extracted from the spectra and used for protein
identification using the SmartIdent peptide mass fingerprint tool
[Gras 1999]. The research was conducted against SWISS-PROT and
TrEMBL databases. The query was made for the human, the minimum
number of matched masses was 4, the maximal tolerance for masses
was 50 ppm after an internal calibration using autolysis product of
trypsin, at most one missed cleavage for tryptic peptides was
allowed, and the modifications accepted were carboxymethylation
with iodoacetamide of cysteines and ariefactual oxidation of
methionines.
Example 8
Protein Identification by Peptide Fragmentation Analysis
[0085] Prior to nanoLC (LC=liquid chromatography) separation, the
volumes of peptide containing solutions were adjusted to 7 .mu.l by
addition of a 0.1% (v/v) formic acid solution. Samples were settled
in a Triathlon autosampler (Spach, Emmen, Holland) For each
experiment, 5 .mu.l of peptide containing solution were injected on
a C18 reverse phase column of 75 .mu.m inner diameter
(YMS-ODS-AQ200, Michrom Bioresource, Auburn, Calif.). Peptides were
eluted with an acetonitrile (ACN) gradient in the presence of 0.1%
(v/v) formic acid, using SunFlow pumps (SunChrom, Friderichsdorf,
Germany). A flow splitter was used in order to decrease the flow
rate after the pumps from 200 to 0.4 .mu.l/min. Peptides were
analysed with a quadrupole time-of-flight (Q-TOF) mass spectrometer
(Micromass, Wythenshawe, England). A 2700 V tension was applied on
the nanoelectrospray capillary (New Objective, Woburn, Mass., USA).
Argon was used as collision gas. The collision energy was settled
as a function of the precursor ion mass. MS/MS spectra were
acquired by automatic switching between MS and MS/MS mode. Acquired
MS/MS data were converted in a compatible format (DTA files) by
ProteinLynx software (Micromass, Wythenshawe, England) and analysed
using conventional search engines against SWISS-PROT, TrEMBL,
NCBTInr and EST databases. In cases of manual inter-pretation of
MS/MS data, identification was performed by sequence only
search.
[0086] It was found that marker M5 was a fragment of Cystatin C,
markers M3 and M4 were isoforms of beta-2-microglobulin and M2 was
a new variant or homologue of myoglobin. Marker M1 was found to be
a fragment of the neurosecretory protein VGF.
Example 9
Statistical Analysis
[0087] P-values were calculated using standard statistical methods
known to the person skilled in the art. P-values smaller than 0.05
were considered to be statistically significant.
Example 10
Isolation of the 4.8 kDa Fragment (Marker M1)
[0088] Some CSF samples from control patients were fractionated by
Centricon 30 filtration device (Millipore Corp., Bedford, Mass.) in
order to remove the protein with a molecular weight higher than 30
kDa. The salt and polypeptide with a molecular weight lower than 3
kDa were removed using a Centricon 3 (Millipore Corp., Bedford,
Mass.). The Centricon 3 was then washed with ultrapure distilled
water. In that wash fraction, the 4.82 kDa was found to be the
major component. This liquid fraction was first reduced with a 10
mM solution of 1,4-Dithioerythritol for 1 h at 56.degree. C., then
alkylated with 54 mM iodoacetamide for 45 min at room temperature.
Finally, the polypeptide was digested with 6 mg/l trypsin overnight
at 37.degree. C. This liquid fraction was analysed by nanoLC and
Q-TOF as previously described.
Example 11
Different Surface Materials for SELDI-TOF Analysis
[0089] Using SELDI-TOF, an analysis of 10 CSF samples from AD
patients and 10 controls was performed on three different surfaces:
the hydrophobic H50, the WCX2, and the IMAC surface (Ciphergen
Biosystems, Fremont, Calif., USA, resp.). Seven differentially
expressed peaks were found on the 1150, five markers on the WCX2,
and five markers on the MAC surface. A diagnostic test using the
markers on the H50 chip revealed a specificity and sensitivity of
100% and 70%, respectively. The combination of the markers found on
H50 and WCX2 gave a specificity and sensitivity of 100% and 80%.
Finally, the combination of the markers found on H50, WCX2 and LMAC
gave a specificity and sensitivity of 100% and 90%.
[0090] The average masses of the differentially expressed
polypeptides as determined by SELDI-TOF using different surface
materials were as follows:
[0091] Surface hydrophobic H50: 7 peaks
[0092] Marker 1: 4769.+-.s.d. Da
[0093] Marker 2: 6958/s.d. Da
[0094] Marker 3: 6991/s.d. Da
[0095] Marker 4: 13412.+-.s.d. Da
[0096] Marker 5: 13787.+-.s.d. Da
[0097] Marker 6: 17276.+-.s.d. Da
[0098] Marker 7: 40437.+-.s.d. Da
[0099] Surface IMAC Cu: 5 peaks
[0100] Marker 1: 6895.+-.s.d. Da
[0101] Marker 2: 6928.+-.s.d.Da
[0102] Marker 3: 7691.+-.s.d. Da
[0103] Marker 4: 7769.+-.s.d. Da
[0104] Marker 5: 7934.+-.s.d. Da
[0105] Surface WCX2: 5 peaks
[0106] Marker 1: 5082.+-.s.d. Da
[0107] Marker 2: 6267.+-.s.d. Da
[0108] Marker 3: 6518.+-.s.d. Da
[0109] Marker 4: 7274.+-.s.d. Da
[0110] Marker 5: 8209.+-.s.d. Da
[0111] The standard deviation (s.d.) is 20 Da for each marker
above. However, the standard deviation can also be 40 Da, or 10 Da,
or 5 Da for each marker above.
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Sequence CWU 1
1
17115PRTHomo sapiensMISC_FEATURE(1)..(2)Xaa = any amino acid 1Xaa
Xaa Ala Asp Xaa Ala Gly His Gly Xaa Glu Val Xaa Xaa Arg 1 5 10 15
214PRTHomo sapiensMISC_FEATURE(1)..(14)Xaa = L or I 2His Gly Thr
Val Val Xaa Thr Ala Xaa Gly Gly Xaa Xaa Lys 1 5 10 310PRTHomo
sapiens 3Val Asn His Val Thr Leu Ser Gln Pro Lys 1 5 10 410PRTHomo
sapiens 4Val Glu His Ser Asp Leu Ser Phe Ser Lys 1 5 10 513PRTHomo
sapiens 5Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys 1 5 10
622PRTHomo sapiens 6Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His
Pro Ser Asp Ile 1 5 10 15 Glu Val Asp Leu Leu Lys 20 79PRTHomo
sapiens 7Ala Ser Asn Asp Met Tyr His Ser Arg 1 5 811PRTHomo sapiens
8Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys 1 5 10 912PRTHomo
sapiens 9Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys 1 5 10
1016PRTHomo sapiens 10Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu
Glu Glu Gly Val Arg 1 5 10 15 1116PRTHomo sapiens 11Gln Ile Val Ala
Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg 1 5 10 15
1217PRTHomo sapiens 12Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu
Glu Glu Gly Val Arg 1 5 10 15 Arg 1317PRTHomo sapiens 13Lys Gln Ile
Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly 1 5 10 15 Arg
1418PRTHomo sapiens 14Thr Gln Pro Asn Leu Asp Asn Cys Pro Phe His
Asp Gln Pro Pro His 1 5 10 15 Leu Lys 1519PRTHomo sapiens 15Thr Gln
Pro Asn Leu Asp Asn Cys Pro Phe His Asp Gln Pro Pro His 1 5 10 15
Leu Lys Arg 1624PRTHomo sapiens 16Ser Ser Pro Gly Lys Pro Pro Arg
Leu Val Gly Gly Pro Met Asp Ala 1 5 10 15 Ser Val Glu Glu Glu Gly
Val Arg 20 1720PRTHomo sapiens 17Val Gly Glu Glu Asp Glu Glu Ala
Ala Glu Ala Glu Ala Glu Ala Glu 1 5 10 15 Glu Ala Glu Arg 20
* * * * *