U.S. patent application number 12/531564 was filed with the patent office on 2010-11-11 for plasma kallikrein fragments as diagnostic biomarkers for lung cancers.
Invention is credited to Je-Yeol Cho, Seung-Jin Lee, Jae-Yong Park.
Application Number | 20100285507 12/531564 |
Document ID | / |
Family ID | 39766010 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100285507 |
Kind Code |
A1 |
Cho; Je-Yeol ; et
al. |
November 11, 2010 |
PLASMA KALLIKREIN FRAGMENTS AS DIAGNOSTIC BIOMARKERS FOR LUNG
CANCERS
Abstract
Disclosed herein are diagnostic markers for lung cancer,
isolated from serum glycoproteins. The disclosed diagnostic markers
for lung cancer are specifically expressed only in the sera of lung
cancer patients at high levels, and thus will be very useful for
diagnosing lung cancer and estimating disease progression and
treatment.
Inventors: |
Cho; Je-Yeol; (Daegu,
KR) ; Park; Jae-Yong; (Daegu, KR) ; Lee;
Seung-Jin; (Daegu, KR) |
Correspondence
Address: |
LADAS & PARRY LLP
224 SOUTH MICHIGAN AVENUE, SUITE 1600
CHICAGO
IL
60604
US
|
Family ID: |
39766010 |
Appl. No.: |
12/531564 |
Filed: |
September 6, 2007 |
PCT Filed: |
September 6, 2007 |
PCT NO: |
PCT/KR2007/004322 |
371 Date: |
February 23, 2010 |
Current U.S.
Class: |
435/7.92 ;
435/196; 435/212; 435/219; 436/501; 530/350; 530/386; 530/387.1;
530/387.2; 536/24.3; 536/24.33 |
Current CPC
Class: |
G01N 33/57423
20130101 |
Class at
Publication: |
435/7.92 ;
435/196; 435/212; 435/219; 436/501; 530/350; 530/386; 530/387.1;
530/387.2; 536/24.3; 536/24.33 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12N 9/16 20060101 C12N009/16; C12N 9/48 20060101
C12N009/48; C12N 9/50 20060101 C12N009/50; G01N 33/566 20060101
G01N033/566; C07K 14/00 20060101 C07K014/00; C07K 16/00 20060101
C07K016/00; C07K 16/42 20060101 C07K016/42; C07H 21/04 20060101
C07H021/04; C07K 16/18 20060101 C07K016/18; C07K 16/40 20060101
C07K016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2007 |
KR |
10-2007-0027541 |
Claims
1. A diagnostic marker for lung cancer, which comprises a protein
selected from the group consisting of complement component c8 beta
chain precursor, protein s100-a9, C4b-binding protein alpha chain
precursor, isoform 2 of apolipoprotein-11 precursor, protein
s100-a8, proteins similar to cavia porcellus phosphatidic acid
phosphatase 2A mRNA, complement component c9 precursor,
inter-alpha-trypsin inhibitor heavy chain h3 precursor, F1j00385
protein (fragment), coagulation factor xii precursor, Ig alpha-2
chain c region, complement component c8 alpha chain precursor,
corticosteroid-binding globulin precursor, clusterin precursor,
gamma-g globin, serum amyloid p-component precursor, plasma
kallikrein (KLKB1) fragment, Igha1 protein, heparin cofactor 2
precursor, 16 kDa protein, Ig kappa chain v-iii region go1,
complement factor I precursor, hemoglobin subunit beta, proteins
similar to tripartite motif protein 49, isoform 1 of complement
factor b precursor (fragment), alpha-1-antitrypsin precursor,
carboxypeptidase n subunit 2 precursor, complement factor h-related
protein 3 precursor, plasma protease c1 inhibitor precursor, Ig
kappa chain c region, alpha 2 macroglobulin variant, dermcidin
precursor, complement c5 precursor and pregnancy zone protein
precursor.
2. The diagnostic marker of claim 1, which comprises
inter-alpha-trypsin inhibitor heavy chain h3 precursor.
3. The diagnostic marker of claim 1, which comprises plasma
kallikrein (KLKB1) fragment.
4. A composition for diagnosing lung cancer, which comprises an
antibody binding specifically to a protein selected from the group
consisting of complement component c8 beta chain precursor, protein
s100-a9, C4b-binding protein alpha chain precursor, isoform 2 of
apolipoprotein-11 precursor, protein s100-a8, proteins similar to
cavia porcellus phosphatidic acid phosphatase 2A mRNA, complement
component c9 precursor, inter-alpha-trypsin inhibitor heavy chain
h3 precursor, F1j00385 protein (fragment), coagulation factor xii
precursor, Ig alpha-2 chain c region, complement component c8 alpha
chain precursor, corticosteroid-binding globulin precursor,
clusterin precursor, gamma-g globin, serum amyloid p-component
precursor, plasma kallikrein (KLKB1) fragment, Igha1 protein,
heparin cofactor 2 precursor, 16 kDa protein, Ig kappa chain v-iii
region go1, complement factor I precursor, hemoglobin subunit beta,
proteins similar to tripartite motif protein 49, isoform 1 of
complement factor b precursor (fragment), alpha-1-antitrypsin
precursor, carboxypeptidase n subunit 2 precursor, complement
factor h-related protein 3 precursor, plasma protease c1 inhibitor
precursor, Ig kappa chain c region, alpha 2 macroglobulin variant,
dermcidin precursor, complement c5 precursor and pregnancy zone
protein precursor.
5. The composition of claim 4, which comprises an antibody binding
specifically to inter-alpha-trypsin inhibitor heavy chain h3
precursor.
6. The composition of claim 4, which comprises an antibody binding
specifically to plasma kallikrein (KLKB1) fragment.
7. A kit for diagnosing lung cancer, which comprises an antibody
binding specifically to inter-alpha-trypsin inhibitor heavy chain
h3 precursor.
8. A protein chip for diagnosing lung cancer, which has attached
thereto an antibody binding specifically to inter-alpha-trypsin
inhibitor heavy chain h3 precursor.
9. A kit for diagnosing lung cancer, which comprises an antibody
binding specifically to plasma kallikrein (KLKB1) fragment.
10. A protein chip for diagnosing lung cancer, which has attached
thereto an antibody binding specifically to plasma kallikrein
(KLKB1) fragment.
11. A composition for diagnosing lung cancer, which comprises a
primer or probe specific for a nucleic acid encoding a protein
selected from the group consisting of complement component c8 beta
chain precursor, protein s100-a9, C4b-binding protein alpha chain
precursor, isoform 2 of apolipoprotein-11 precursor, protein
s100-a8, proteins similar to cavia porcellus phosphatidic acid
phosphatase 2A mRNA, complement component c9 precursor,
inter-alpha-trypsin inhibitor heavy chain h3 precursor, F1j00385
protein (fragment), coagulation factor xii precursor, Ig alpha-2
chain c region, complement component c8 alpha chain precursor,
corticosteroid-binding globulin precursor, clusterin precursor,
gamma-g globin, serum amyloid p-component precursor, plasma
kallikrein (KLKB1) fragment, Igha1 protein, heparin cofactor 2
precursor, 16 kDa protein, Ig kappa chain v-iii region go1,
complement factor I precursor, hemoglobin subunit beta, proteins
similar to tripartite motif protein 49, isoform 1 of complement
factor b precursor (fragment), alpha-1-antitrypsin precursor,
carboxypeptidase n subunit 2 precursor, complement factor h-related
protein 3 precursor, plasma protease c1 inhibitor precursor, Ig
kappa chain c region, alpha 2 macroglobulin variant, dermcidin
precursor, complement c5 precursor and pregnancy zone protein
precursor.
12. The composition of claim 11, which comprises a primer or probe
specific for a nucleic acid encoding inter-alpha-trypsin inhibitor
heavy chain h3 precursor (ITI-H3).
13. The composition of claim 11, which comprises a primer or probe
specific for a nucleic acid encoding plasma kallikrein
fragment.
14. A kit for diagnosing lung cancer, which comprises a primer or
probe specific for a nucleic acid encoding inter-alpha-trypsin
inhibitor heavy chain h3 precursor.
15. A DNA chip for diagnosing lung cancer, which has attached
thereto a primer or probe specific for a nucleic acid encoding
inter-alpha-trypsin inhibitor heavy chain h3 precursor.
16. A kit for diagnosing lung cancer, which comprises a primer or
probe specific for a nucleic acid encoding plasma kallikrein
fragment.
17. A DNA chip for diagnosing lung cancer, which has attached
thereto a primer or probe specific for a nucleic acid encoding
plasma kallikrein fragment.
18. A method for diagnosing lung cancer, which comprises the steps
of: (a) bringing a biological sample into contact with an antibody
binding specifically to the diagnostic marker of claim 1; and (b)
detecting the formation of an antigen-antibody complex.
19. method of claim 18, wherein the biological sample in the step
(a) is serum.
20. method of claim 18, wherein the detection of the formation of
the antigen-antibody in the step (b) is carried out using a method
selected from the group consisting of RIA (radioimmunoassay), RIPA
(radioimmunoprecipitation assay), IFA (immunoflourescence assay),
EILSA (enzyme-linked immunosorbent assay) and Western blot.
21. A method for diagnosing lung cancer, which comprises the steps
of: (a) removing a plasma kallikrein fragment and the heavy chain
precursor form thereof from the serum of a subject; (b) bringing
the serum, from which the plasma kallikrein fragment and the heavy
chain precursor form thereof had been removed in the step (a), into
contact with an antibody specific for a region comprising the H4
domain of the plasma kallikrein fragment; and (c) detecting the
formation of an antigen-antibody complex.
22. The method of claim 21, wherein the removal of the plasma
kallikrein fragment or the heavy chain precursor form thereof in
the step (a) is carried out by bringing the serum into contact with
an antibody binding specifically to the plasma kallikrein fragment
or is carried out using a molecular size separation column.
23. method of claim 21, wherein the detection of the
antigen-antibody complex in the step (c) is carried out using a
method selected from the group consisting of RIA
(radioimmunoassay), RIPA (radioimmunoprecipitation assay), IFA
(immunoflourescence assay), EILSA (enzyme-linked immunosorbent
assay) and Western blot.
24. method of claim 21, wherein, if a fragment of 15-20 kDa is
detected in the step (c), the subject is diagnosed to have lung
cancer.
25. A method for identifying diagnostic markers for lung caner,
which comprises the steps of: (a) separating glycoproteins from a
serum sample using a multi-lectin affinity column; (b)
concentrating the separated glycoproteins of step (a) by acetone
precipitation; (c) deglycosylating the concentrated glycoproteins
of step (b) by enzymatic treatment; (d) subjecting the
deglycosylated proteins of step (c) to SDS-PAGE; (e) obtaining
peptides from the proteins by in-gel trypsin digestion; and (f)
analyzing the peptides of step (e) by LC-MS/MS to identify proteins
which are specifically expressed only in the sera of lung cancer
patients compared to normal sera.
Description
TECHNICAL FIELD
[0001] The present invention relates to the use of a plasma
kallikrein fragment as a diagnostic biomarker for lung cancer.
BACKGROUND ART
[0002] Lung cancer is the most common form of cancer in the world,
which is estimated to account for 12.3% of all cancers and 17.8% of
cancer-related deaths (Parkin, D. M., Lancet Oncol 2001, 2,
533-43). Also, the incidence of lung cancer and the resulting
mortality rate in Korea are estimated to continue to increase.
Despite the recent development of cancer therapy, the survival rate
of lung cancer patients is very low. This is because the cancer is
diagnosed at a late stage in most cases. Thus, there is an urgent
need to develop markers which can diagnose lung cancer at an early
state to increase the survival rate of lung cancer patients.
[0003] Meanwhile, human body fluids, such as blood and urine, are
useful for recognizing the pathological conditions (conditions
associated with tumors, immune responses and vascular diseases) of
the body, because they can be easily collected for diagnosis and
include secretory proteins which are expressed differently in
abnormal and normal conditions. In addition, due to abundant serum
proteins (such as albumin, IgG and transferrin), it is difficult to
detect low abundant proteins which can be used as new biomarkers. A
number of studies have attempted various approaches to reduce
abundant serum proteins. Methods for removing the abundant serum
proteins were introduced by some researchers and are generally
classified into two categories. One of them is the use of an
immunoaffinity HPLC column to reduce albumin, IgA, IgG, transferin,
haptoglobin (HP) and antitrypsin (Okano, T. et al., Proteomics
2006, 6, 3938-48; Yu, K. H. et al., J Proteome Res 2005, 4,
1742-51). Another is the isolation of serum glycoproteins using
hydrazide chemistry (Liu, T. et al., J Proteome Res 2005, 4,
2070-80) or lectin affinity (Yang, Z. et al., J Chromatogr A 2004,
1053, 79-88; Yang, Z. et al., Proteomics 2005, 5, 3353-66;
Vosseller, K. et al., Mol Cell Proteomics 2006, 5, 923-34; Zhang,
H. et al., Mol Cell Proteomics 2005, 4, 144-55).
[0004] Two recent studies reported proteins expressed differently
between healthy person's serum and lung cancer patient's serum
using 2-DE and MALDI-TOF (Maciel, C. et al., J Exp Ther Oncol 2005,
5, 31-8) or 2-DE and LC-MS/MS (Okano, T. et al., Proteomics 2006,
6, 3938-48), and NSE, CEA and CYFRA 21-1 are currently known as
serum markers for lung cancer. However, the sensitivity and
specificity thereof as lung cancer markers are not sufficient
(Tarro, G. et al., J Cell Physiol 2005, 203, 1-5). For this reason,
the development of novel biomarkers specific for lung cancer is
urgently needed.
DISCLOSURE OF INVENTION
Technical Problem
[0005] Accordingly, the present inventors have studied to develop
novel diagnostic markers capable of diagnosing lung cancer and, as
a result, have identified proteins, which are specifically
expressed only in the sera of lung cancer patients, and thus can be
used as biomarkers for diagnosing lung cancer, by separating
glycoproteins from the sera of lung cancer patients using a
multi-lectin affinity column, removing the glycans of the
glycoproteins by enzymatic treatment, collecting peptides from the
proteins by in-gel trypsin digestion, and then analyzing the
peptides by LC-MS/MS, thereby completing the present invention.
[0006] It is therefore an object of the present invention to
provide diagnostic markers for lung cancer, separated from serum
glycoproteins.
Technical Solution
[0007] To achieve the above object, in one aspect, the present
invention provides diagnostic markers for lung caner, separated
from serum glycoproteins.
[0008] In another aspect, the present invention provides a
composition for diagnosing lung cancer, which comprises an antibody
binding specifically to the diagnostic marker.
[0009] In still another aspect, the present invention provides a
composition for diagnosing lung cancer, which comprises a primer or
probe specific for a nucleic acid encoding the diagnostic
marker.
[0010] In still another aspect, the present invention provides a
method of diagnosing lung cancer using the diagnostic marker.
[0011] In yet another aspect, the present invention provides a
method for identifying the diagnostic markers.
[0012] Hereinafter, the present invention will be described in
detail.
[0013] As used herein, the term "diagnosis" refers to the
determination of the presence or properties of pathological
conditions. For the purpose of the present invention, the term
"diagnosis" means determining the incidence of lung cancer.
[0014] As used herein, the term "lung cancer" means a malignant
tumor occurring in the lungs.
[0015] As used herein, the term "diagnostic marker" is intended to
indicate a substance capable of diagnosing lung cancer by
distinguishing lung cancer cells from normal cells, and includes
organic biomolecules, which increase in lung cancer cells compared
to normal cells or in cancer subjects compared to normal subjects.
Examples of the organic biomolecules include, but are not limited
to, polypeptides, proteins, nucleic acids, lipids, glycolipids,
glycoproteins and sugars. In the present invention, the organic
biomolecules preferably refers to glycoproteins.
[0016] In order to discover novel diagnostic markers for lung
cancer, the present inventors have identified glycoproteins which
are expressed differently between the sera of lung cancer patients
and the sera of normal persons. For this purpose, a multi-lectin
affinity column was used to glycoprotein from serum, the
glycoprotein was deglycosylated by enzymatic treatment, and then
treated with trypsin, thus obtaining a peptide. The obtained
peptide was separated by liquid chromatography, while it was
analyzed with an ion trap mass spectrometer (LC-MS/MS). The
LC-MS/MS data were searched against the IPI human protein database
using computer program, thus confirming glycoproteins present in
the sera of lung cancer patients. The glycoprotein was compared
with a glycoprotein derived from the sera of normal persons to
confirm that it is present specifically in the sera of lung cancer
patients, and thus can be used as a diagnostic marker for lung
cancer. An experimental method for identifying a diagnostic marker
in the sera of lung cancer patients according to a preferred
embodiment of the present invention is shown in FIG. 1.
[0017] In one Example of the present invention, a total of six
blood samples were collected from lung cancer patients (three
samples) and normal persons (three samples) and centrifuged to
obtain serum samples (see Example <1-1>). Glycoproteins were
isolated from the obtained serum samples using a multi-lectin
affinity column and concentrated by acetone precipitation (see
Example <1-2>).
[0018] In one Example of the present invention, the eluate
(concentrated glycoprotein) and flow-through fractions, obtained by
the multi-lectin affinity column, were subjected to Coomassie
brilliant straining and GelCode glycoprotein staining to determine
the efficiency of the multi-lectin affinity column for glycoprotein
separation (see Example <1-3>) and, as a result, it could be
seen that, by the multi-lectin affinity column, serum glycoproteins
were concentrated and albumin was removed (see FIG. 2).
[0019] Accordingly, in the present invention, the above-obtained
concentrated serum glycoproteins were treated with
peptide-N-glycosidase F (PNGase F) to remove the sugar moieties and
subjected to 1D-SDS PAGE (see Example <2-1>), and the
resulting materials were subjected to in-gel digestion, thus
obtaining peptide mixtures (see Example <2-2>). The obtained
peptide mixtures were analyzed with LC-MS/MS, thus identifying 38
proteins, the expression of which was increased in the sera of lung
cancer patients compared to the sera of normal persons (see Example
3).
[0020] Some of the 38 identified proteins were reported that they
can be used as lung cancer markers in the serum or plasma of lung
cancer patients. Examples thereof include heptoglobin (HP),
inter-alpha-trypsin inhibitor H4 (ITI-H4), complement C3 precursor,
leucin-rich alpha 2 glycoprotein and the like. The proteins other
than them are not yet known as lung cancer markers or
biomarkers.
[0021] Accordingly, the present invention provides a diagnostic
marker for lung cancer, which comprises a protein selected from the
group consisting of complement component c8 beta chain precursor,
protein s100-a9, C4b-binding protein alpha chain precursor, isoform
2 of apolipoprotein-11 precursor, protein s100-a8, proteins similar
to cavia porcellus phosphatidic acid phosphatase 2A mRNA,
complement component c9 precursor, inter-alpha-trypsin inhibitor
heavy chain h3 precursor, F1j00385 protein (fragment), coagulation
factor xii precursor, Ig alpha-2 chain c region), complement
component c8 alpha chain precursor, corticosteroid-binding globulin
precursor, clusterin precursor, gamma-g globin, serum amyloid
p-component precursor, plasma kallikrein fragment (KLKB1 fragment),
Igha1 protein, heparin cofactor 2 precursor, 16 kDa protein, Ig
kappa chain v-iii region go1, complement factor I precursor,
hemoglobin subunit beta, proteins similar to tripartite motif
protein 49, isoform 1 of complement factor b precursor (fragment),
alpha-1-antitrypsin precursor, carboxypeptidase n subunit 2
precursor, complement factor h-related protein 3 precursor, plasma
protease c1 inhibitor precursor, Ig kappa chain c region, alpha 2
macroglobulin variant, dermcidin precursor, complement c5 precursor
and pregnancy zone protein precursor, which are shown in Table 4
below.
[0022] Preferably, the present invention provides a diagnostic
marker, which contains inter-alpha-trypsin inhibitor heavy chain h3
precursor or plasma kallikrein fragment (KLKB1 fragment).
[0023] The diagnostic marker is specifically expressed only in the
sera of lung cancer patients.
[0024] In one Example of the present invention, whether
inter-alpha-trypsin inhibitor heavy chain h3 precursor (ITI-H3) is
specifically expressed only in the sera of lung cancer patients was
analyzed by Western blotting (see Example 4). As a result, it could
be seen that the protein was expressed highly in the sera of lung
cancer patients compared to in the sera of normal persons (see FIG.
4).
[0025] In another Example of the present invention, whether plasma
kallikrein fragment (KLKB1 fragment) is specifically expressed only
in the sera of lung caner patients was analyzed by Western blotting
(see Example 6). As a result, it could be seen that a band having a
size of 15-20 kDa was detected only in the sample of lung cancer
patients at a high level (see FIG. 5). This was thought to be
because the heavy chains of KLKB1 were partially cleaved.
[0026] In other Examples of the present invention, a Western blot
was performed on 8 normal persons and 28 patients using an antibody
specific for the plasma kallikrein fragment (see Examples 7 and 8).
It could be seen that a 18-kDa fragment was weakly detected in one
person of the 8 normal persons and was detected in 25 patients of
the 28 patients at high levels (see FIGS. 6 and 7). Also, a Western
blot was additionally performed on 24 lung cancer patients (see
FIG. 9). As a result, in the normal persons, the KLKB1 fragment was
not almost detected or was detected at low levels, but in the lung
cancer patients, the KLKB1 fragment was detected in 20 patients of
the 24 patients at high levels (see FIGS. 9 to 12).
[0027] From the results of Example 7 to Example 9, it could be seen
that the 18 kDa fragment was detected in 45 patients of the 52 lung
cancer patients, and thus had a sensitivity of 87% and a
specificity of 67%. On the other hand, the levels of the 18 kDa
fragment in the normal persons were always lower than that in the
lung cancer patients.
[0028] Meanwhile, it is known that the plasma kallikrein fragment
consists of a total of 619 amino acids except for 19 signal
peptides among 638 amino acids (see SEQ ID NO: 1) and is originally
synthesized in the liver. It is a protein known to be involved in
blood coagulation, fibrinolysis and kinin formation. It consists of
4 heavy chains (amino acid residues 20 to 390) and 1 light chain
(amino acid residues 391-638) (see FIG. 8), in which the heavy
chains are classified into H1, H3 and H4 domains.
[0029] The antibody specific for the plasma kallikrein fragment,
used in the Western blot in Example of the present invention, was
prepared using an epitope of amino acid residues of 361-400 of
plasma kallikrein, which corresponded to the latter part of the H4
domain. Thus, the fragment detected in Example of the present
invention is believed to be a fragment containing the H4 domain of
KLKB1.
[0030] The detection of the inventive diagnostic marker in
biological samples can be performed either using an antibody
binding specifically to the diagnostic marker or using a primer or
probe specific for a nucleic acid encoding the diagnostic
marker.
[0031] Accordingly, the present invention provides a composition
for diagnosing lung cancer, which contains an antibody bonding
specifically to the diagnostic marker of the present invention.
[0032] Preferably, the present invention provides a composition for
diagnosing lung cancer, which contains an antibody specific for
inter-alpha-trypsin inhibitor heavy chain h3 precursor (ITI-H3) or
plasma kallikrein fragment.
[0033] As used herein, the term "antibody" refers to a protein
molecule which is directed specifically to an antigenic site.
Examples of antibodies for use in the present invention include
monoclonal or polyclonal antibodies, immunologically active
fragment (e.g., Fab or (Fab).sub.2 fragments), antibody heavy
chains, humanized antibodies, antibody light chains, genetically
engineered single chain Fv molecules, and chimeric antibodies.
[0034] Because the proteins known in Table 4 are known proteins,
the antibodies that are used in the present invention can be
prepared using the known proteins as antigens according to any
conventional method widely known in the immunological field. The
protein that is used as an antigen for the antibody according to
the present invention can be extracted naturally or synthesized.
Alternatively, it can be prepared by a recombinant method based on
DNA sequences. When the gene recombinant technology is used, the
antigen protein can be prepared by inserting into a suitable
expression vector a nucleic acid encoding the protein, culturing
host cells transformed with the recombinant expression vector so as
to express the target protein, and then collecting the target
protein from the cultured cells.
[0035] For example, polyclonal antibodies can be produced by
injecting an antigen into an animal and collecting blood from the
animal to obtain an antibody-containing serum. Such antibodies can
be prepared using various warm-blooded animals, such as horses,
cattle, goats, sheep, dogs, fowls, turkeys, rabbits, mice or
rats.
[0036] Monoclonal antibodies may be prepared in accordance with a
fusion method (Kohler and Milstein, European Journal of Immunol.,
6:511-519(1976)), a recombinant DNA method (U.S. Pat. No. 4,816,56)
or a phage antibody library (Clackson et al, Nature,
352:624-628(1991); and Marks et al, J. Mol. Biol., 222:58,
1-597(1991)).
[0037] The diagnostic composition of the present invention may
comprise, in addition to the antibody specific for the protein,
reagents which are used for immunological assays. The immunological
assays may include methods capable of measuring the binding of an
antigen to the antibody of the present invention. These methods are
known in the art and include, for example, immunocytochemical
assays, immunohistochemical assays, radioimmunoassays, ELISA
(enzyme linked immunoabsorbent assay), immunoblotting, Farr assays,
precipitin reaction, turbidimetry, immunodiffusion, counter-current
electrophoresis, single radical immunodiffusion and
immunofluorescence.
[0038] Reagents which are used in the immunological assays include
a labeling substance capable of emitting detectable signals, a
solubilizer and a washing agent. Furthermore, if the labeling
substance is enzyme, a substrate capable of measuring enzymatic
activity and a reaction stopping agent may be used. The labeling
substance capable of emitting detectable signals enabling
quantitative or qualitative measurement of the formation of
antigen-antibody complexes include enzymes, and examples thereof
include enzymes, fluorescent substances, ligands, luminescent
substances, microparticles, redox molecules and radioactive
isotopes. The enzymes may include .beta.-glucuronidase,
.beta.-D-glucosidase, .beta.-D-galactosidase, urease, peroxidase,
alkaline phosphatase, acetylcholine esterase, glucose oxidase,
hexokinase, malate dehydrogenase, glucose-6-phosphate
dehydrogenase, invertase and the like. The fluorescent substances
include fluorescein, isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, fluorescein, isothiocyanate, and the
like. The ligands include biotin derivatives, and the luminescent
substances include acridinium ester, luciferin, and luciferase
acrydinium ester. The microparticles include colloidal gold, and
colored latex, and the redox molecules include ruthenium complexes,
viologen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone,
hydroquinone, and the like. The radioactive isotopes include
.sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36Cl, .sup.51Cr,
.sup.57Co, .sup.58Co, .sup.59Fe, .sup.90Y, .sup.125I, .sup.131I,
.sup.186Re and the like. However, in addition to the
above-exemplified substances, any substance can be used, as long as
it can be immunological assays.
[0039] Also, to detect the presence of the marker protein of the
present invention, a peptide binding specifically to the marker
protein of the present invention may be used. Accordingly, the
present invention provides a composition for diagnosing lung
cancer, which contains a peptide binding specifically to one
selected from among the proteins shown in Table 4. Preferably, the
peptide may consist of 7-35 amino acids derived from the marker
protein of the present invention.
[0040] Also, the diagnostic composition of the present invention
can be immobilized on a suitable carrier or support in order to
enhance the rapidness and convenience of diagnosis (Antibodies: A
Laboratory Manual, Harlow & Lane; Cold Spring Harbor, 1988).
Examples of suitable carriers or supports include agarose,
cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes,
carboxymethyl cellulose, polyacrylamides, polystyrene, gabbros,
filter paper, magnetite, ion-exchange resin, plastic film, plastic
tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer,
amino acid copolymer, ethylene-maleic acid copolymer, nylon, cups
and flat packs. In addition, other solid substrates include cell
culture plates, ELISA plates, tubes and polymeric membranes. The
support material may have any possible configuration including
spherical (e.g., bead), cylindrical (e.g., inside surface of a test
tube or well, or flat (e.g., sheet, test strip).
[0041] Preferably, the inventive composition for diagnosing lung
cancer can be provided in the form of a diagnostic kit or a protein
chip.
[0042] The diagnostic kit can be provided in the form of a lateral
flow assay kit based on immunochromatography to detect a specific
protein in a sample. The lateral flow assay kit comprises: a sample
pad to which a sample is applied; a releasing pad which is coated
with an antibody for detection; a developing membrane (e.g.,
nitrocellulose) or strip in which the sample is transferred and
separated and an antigen-antibody reaction occurs; and an
absorption pad. Also, the diagnostic kit of the present invention
may also be in the form of, but is not limited to, a rapid kit or
an ELISA kit.
[0043] In the protein chip, either an antibody specific for the
inventive marker protein or a peptide binding specifically to the
inventive marker protein is generally attached to a slide glass
surface treated with a specific reagent, such that the protein
binding specifically to the antigen or the peptide can be detected
by antigen-antibody reactions or protein-protein interactions. It
is used to detect various different kinds of antibodies or peptides
and may comprise kallikrein fragment peptides or antibodies.
[0044] In another aspect, the present invention provides a
composition for diagnosing lung cancer, which contains a primer or
probe specific for a nucleic acid encoding the diagnostic marker
protein of the present invention.
[0045] Preferably, the present invention provides a composition for
diagnosing lung cancer, which contains a primer or probe specific
for a nucleic acid encoding either inter-alpha-trypsin inhibitor
heavy chain h3 precursor (ITI-H3) or plasma kallikrein fragment
(KLKB1 fragment).
[0046] The detection of a specific nucleic acid using a primer can
be performed by amplifying the sequence of a target gene using an
amplification method such as PCR, and then analyzing the
amplification of the gene using a method known in the art. Also,
the detection of a specific nucleic acid using a probe can be
performed by bringing a sample nucleic acid into contact with the
probe in suitable conditions, and then analyzing the presence of a
hybridized nucleic acid.
[0047] The term "primer", as used herein, refers to a short nucleic
acid sequence having a free hydroxyl group, which is able to
undergo base-pairing interaction with a complementary template and
serves as a starting point for replicating the template strand. For
example, the primer of the present invention can be chemically
synthesized using a method known in the art, such as the
phosphoramidite solid support method.
[0048] As used herein, the term "probe" refers to a nucleic acid
fragment of RNA or DNA consisting of a few or a few hundreds bases,
which can bind specifically to mRNA. It is labeled such that the
presence of a specific mRNA can be detected. The probe can be
prepared in the form of oligonucleotide probes, single-stranded DNA
probes, double-stranded DNA probes and RNA probes and may be
labeled with biotin, FITC, rhodamine, DIG or radioactive
isotopes.
[0049] Also, the probe may be labeled with a detectable label, for
example, a radioactive label which provides a suitable signal and
has a sufficient half life. The labeled probe can be hybridized to
a nucleic acid on a solid support as described in the literature
(Sambook et al., Molecular Cloning, A Laboratory Mannual,
1989).
[0050] Examples of the methods of detecting a specific nucleic acid
using said probe or primer include, but are not limited to,
polymerase chain reaction (PCR), DNA sequencing, RT-PCR, primer
extension method (Nikiforeov et al., Nucl Acids Res 22, 4167-4175,
1994), oligonucleotide ligation analysis (Nickerson et al., Pro Nat
Acad Sci USA, 87, 8923-8927, 1990), allele-specific PCR (Rust et
al., Nucl Acids Res, 6, 3623-3629, 1993), RNase mismatch cleavage
(Myers et al., Science, 230, 1242-1246, 1985), single strand
conformation polymorphism (Orita et al., Pro Nat Acad Sci USA, 86,
2766-2770, 1989), simultaneous analysis of SSCP and heteroduplex
(Lee et al., Mol Cells, 5:668-672, 1995), denaturation gradient gel
electrophoresis (DGGE, Cariello et al., Am J Hum Genet, 42,
726-734, 1988), denaturing high performance liquid chromatography
(Underhill et al., Genome Res, 7, 996-1005, 1997), hybridization
reactions and DNA chips. Examples of the hybridization reactions
include Northern hybridization (Maniatis T. et al., Molecular
Cloning, Cold Spring Habor Laboratory, NY, 1982), in situ
hybridization (Jacquemier et al., Bull Cancer, 90:31-8, 2003) and
microarrays (Macgregor, Expert Rev Mol Diagn 3:185-200, 2003).
[0051] The inventive composition for diagnosing lung cancer may
additionally comprise reagents which are generally used in the
methods for detecting nucleic acids. For example, the composition
may comprise deoxynucleotide triphosphate (dNTP), heat-resistant
polymerase and metal ion salts such as magnesium sulfate, which are
required in PCR reactions, as well as dNTP and sequenase, which are
required in sequencing.
[0052] Preferably, the inventive composition for diagnosing lung
cancer may be provided in the form of a diagnostic kit or
microarray.
[0053] Examples of the diagnostic kit or microarray include, but
are not limited to, RT-PCR kits containing each of primers specific
for the inventive marker gene, and DNA chips comprising a substrate
having attached thereto the cDNA or oligonucleotide of the
inventive marker gene.
[0054] In another aspect, the present invention provides a method
of diagnosing lung cancer using the diagnostic marker of the
present invention.
[0055] Preferably, the diagnostic method of the present invention
comprises the steps of: (a) bringing a biological samples into
contact with an antibody binding specifically to the diagnostic
marker of the present invention; and detecting the formation of an
antigen-antibody complex.
[0056] The term "biological sample" or "sample" in the step (a)
refers to blood or other liquid samples of biological origin.
Preferably, the biological sample or sample may be whole blood,
plasma or serum. The sample is collected from animals, preferably
mammals, and most preferably humans. The sample can be pretreated
before use in detection. For example, the pretreatment can involve
filtration, distillation, extraction, concentration, inactivation
of interfering components, the addition of reagents, and the like.
In addition, nucleic acids and proteins may be isolated from the
samples and used in detection.
[0057] The term "antigen-antibody complex in the step (b) refers to
a combination of a specific protein in a biological sample with an
antibody binding specifically to the specific protein.
[0058] The detection of the formation of the antigen-antibody
complex in the step (b) can be performed using a method known in
the art. Examples of the detection method include, but are not
limited to, rapid diagnostic kits, RIA (radioimmunoassay), RIPA
(radioimmunoprecipitation assay), IFA (immunoflourescence assay),
EILSA (enzyme-linked immunosorbent assay) and Western blot.
[0059] Preferably, the inventive method for diagnosing lung cancer
may comprise the steps of: (a) removing a plasma kallikrein
fragment and the heavy chain precursor form thereof from the serum
of a subject; (b) bringing the serum, from which the plasma
kallikrein fragment and the heavy chain precursor form thereof had
been removed in the step (a), into contact with an antibody
specific for a region comprising the H4 domain of the plasma
kallikrein fragment; and (c) detecting the formation of an
antigen-antibody complex.
[0060] The removal of the plasma kallikrein fragment and the heavy
chain precursor form thereof in the step (a) can be performed by
bringing the serum into contact with an antibody specific for the
total plasma kallikrein fragment or by using molecular size
separation columns.
[0061] The antibody specific for the total plasma kallikrein
fragment may be a polyclonal or monoclonal antibody prepared using,
as an antigen, the total plasma kallikrein fragment having an amino
acid sequence represented by SEQ ID NO: 1.
[0062] The antibody specific for the region comprising the H4
domain of the plasma kallikrein fragment in the step (b) may be
prepared using an epitope of amino acid residues 361-400 of the
amino acid represented by SEQ ID NO: 1.
[0063] Meanwhile, the detection of the formation of the
antigen-antibody complex in the step (c) can be performed according
to the above-described method, and if a fragment having a size of
about 15-20 kDa, and preferably about 18 kDa, is detected in a
sample, the sample would be diagnosed as lung cancer.
[0064] In another aspect, the present invention provides a method
for identifying a diagnostic marker for lung cancer, the method
comprising the steps of: (a) isolating glycoproteins from a serum
sample using a multi-lectin affinity column; (b) concentrating the
isolated glycoproteins of step (a) by acetone precipitation; (c)
deglycosylating the concentrated glycoproteins of step (b) by
enzymatic treatment; (d) subjecting the decosylated proteins of
step (c) to SDS-PAGE; (e) collecting peptides from the proteins by
in-gel trypsin digestion; and (f) analyzing the peptides of step
(e) by LC-MS/MS to identify proteins which are specifically
expressed only in the sera of lung cancer patients compared to the
sera of normal persons.
Advantageous Effects
[0065] The diagnostic markers for lung cancer according to the
present invention are highly expressed specifically in the sera of
lung cancer patients at high levels, and thus are very useful for
diagnosing lung cancer and estimating disease progression and
treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 is a schematic diagram showing an ultra high speed
method for analyzing a human serum protein according to the method
of the present invention.
[0067] FIG. 2 shows Coomassie brilliant straining results (A) and
GelCode staining results
[0068] (B) for a glycoprotein isolated from human serum using a
multi-lectin affinity column, and Coomassie brilliant straining
results (C) for a deglycosylated serum glycoprotein. In FIGS. 2A
and 2B, lane 1: size marker; lanes 2, 4 and 6: normal flow-through
samples; lanes 3, 5 and 7: normal eluate samples; lanes 8, 10 and
12: cancer flow-through samples; lanes 9, 11 and 13: cancer eluate
samples; and in FIG. 2C, lane 1: size marker; lanes 2 to 4: normal
samples; and lanes 5 to 7: cancer samples.
[0069] FIG. 3 shows classification according to the intracellular
location (A), molecular function (B) and physiological process of
the inventive diagnostic marker for lung cancer.
[0070] FIG. 4 shows the results of Western blotting of inter-alpha
trypsin inhibitor H3 in lung cancer patients and normal
persons.
[0071] FIG. 5 shows the results of Western blotting of a plasma
kallikrein fragment in the sera of lung cancer patients and normal
persons or in glycoproteins concentrated from the sera.
[0072] FIG. 6 shows the results of Western blotting of a plasma
kallikrein fragment in the whole sera of normal persons or lung
cancer patients 50-60 years old.
[0073] FIG. 7 shows the results of Western blotting of a plasma
kallikrein fragment in the whole sera of normal persons or lung
cancer patients more than 65 years old.
[0074] FIG. 8 shows the amino acid sequence of a plasma kallikrein
fragment. In FIG. 8, green: signal peptide; blue: heavy chain; and
bold: H4 domain.
[0075] FIG. 9 shows the results of Western blotting of a plasma
kallikrein fragment in the whole sera of normal persons and lung
cancer patients.
[0076] FIG. 10 shows the results of Western blotting of a plasma
kallikrein fragment in the whole sera of normal persons and lung
cancer patients.
[0077] FIG. 11 shows the results of Western blotting of a plasma
kallikrein fragment in the whole sera of normal persons and lung
cancer patients.
[0078] FIG. 12 shows the results of Western blotting of a plasma
kallikrein fragment in the whole sera of normal persons and lung
cancer patients.
BEST MODE FOR CARRYING OUT THE INVENTION
[0079] Hereinafter, the present invention will be described in
further detail. It is to be understood, however, that these
examples are illustrative only, and the scope of the present
invention is not limited thereto.
Example 1
Isolation and Deglycosylation of Glycoprotein from Human Serum
[0080] <1-1> Serum Sample
[0081] 6 blood samples were collected from 3 lung adenocarcinoma
patients and 3 healthy normal persons among 65-year-old smoking men
under consent in Kyungpook National University Hospital, Korea
(Table 1). The blood samples were centrifuged at 4.degree. C. at
12000 rpm for 10 minutes to collect sera, which were then stored at
-70.degree. C. before use in experiments.
TABLE-US-00001 TABLE 1 Features of lung cancer patients and normal
patients Lung cancer group Normal group Number of subjects 3 3
Clinical history Lung cancer Healthy Sex Men Men Age 64 64 Smoking
Smokers Smokers
[0082] <1-2> Isolation and Concentration of Glycoproteins
Using Multi-Lectin Affinity Column
[0083] A multi-lectin affinity column was used to isolate and
concentrate glycoproteins from the serum samples obtained in
Example <1-1>. The multi-lectin affinity column (Qiagen, USA)
was packed with ConA (concanavalinA), LCH (lentil lectin), GNA
(snowdrop lectin), WGA (wheat germ agglutinin), SNA (elderberry
lectin), MAL (maackia amurensis lectin), AIL (jacalin) and PNA
(peanut agglutinin). ConA, LCH and GNA could capture N-glycosylated
proteins, and WGA, SNA and MAL could bind to sialic acid modified
proteins. Also, AIL and PNA could isolate O-glycosylated proteins.
After centrifugation at 500 rpm for 2 minutes, the spin column was
supplemented with 500 .quadrature. of binding buffer,
50.quadrature. of each serum obtained in Example <1-1> was
added and diluted in 500 .quadrature. of protease inhibitor
solution-containing binding buffer, and the dilution was loaded
into the spin column and incubated at room temperature for 1
minute.
[0084] To measure the efficiency of the column, the column was
centrifuged at 500 rpm for 2 minutes, and the flow-through fraction
was collected. An elution buffer was added to the column to collect
the eluate, which was then concentrated and desalted by acetone
precipitation.
[0085] <1-3> Coomassie Brilliant Staining and Gelcode
Glycoprotein Staining
[0086] In order to measure the efficiency of the multi-lectin
affinity column for glycoprotein separation, the eluate
(concentrated glycoprotein) and flow-through fractions, obtained by
the multi-lectin affinity column in Example <1-2>, were
subjected to Coomassie brilliant straining and GelCode sugar
protein staining.
[0087] The Coomassie brilliant straining was performed in the
following manner. Each of the eluate and flow-through fractions,
obtained in Example <1-2>, was electrophoresed on gel, and
then the gel was washed three times with ddH.sub.2O for 5 minutes
and stained with Bio-safe Coomassie G250 stain (Bio-Rad) at room
temperature for 1 hour with gentle stirring. After incubation, the
gel was desalted with ddH.sub.2O and supplemented three times with
ddH.sub.2O one time every 10 minutes.
[0088] The GelCod glycoprotein staining (PIERCE) was performed
according to the manufacturer's instruction. Specifically, the gel
was immersed and fixed in 50% methanol for 30 minutes, and then
washed twice with 100 ml of 3% acetic acid for 10 minutes. The
washed gel was incubated in oxidizing solution for 15 minutes and
washed with 3% acetic acid for 5 minutes, and the incubation and
washing processes were repeated three times. A GelCod glycoprotein
staining reagent was added to the gel and stirred slowly for 15
minutes. Finally, the stained gel was washed sequentially with 3%
acetic acid and distilled water.
[0089] In the experimental results, some of nonstained proteins in
the flow-through fractions derived from the lung cancer patients
and the normal persons, particularly proteins having sizes of more
than about 40 kDa and 100 kDa, were detected in the eluate of the
multi-lectin affinity column. A serum albumin band was observed in
all the flow-through fractions, but was weakly stained by the
Coomassie staining. Such experimental results revealed that low
abundant proteins were concentrated in the eluate of the
multi-lectin affinity column (FIG. 2A). Also, it was shown that the
proteins in the eluate were more clearly stained by the GelCod
glycoprotein staining compared to those in the flow-through
fractions (FIG. 2B). Accordingly, it could be seen that the
multi-lectin affinity column was an efficient tool for the
concentration of serum glycoprotein and the removal of albumin.
MODE FOR THE INVENTION
Example 2
Deglycosylation of Glycoproteins and Preparation of Peptides
[0090] <2-1> Deglycosylation of Glycoproteins
[0091] The serum glycoproteins concentrated in Example 1 were
treated with peptide-N-glycosidase F (PNGase F) to remove the sugar
moieties and were subjected to 1D-SDS PAGE.
[0092] 15 .quadrature. of the concentrated glycoproteins were
denatured in a buffer, containing 100 mM mercaptoethanol and 2%
octyl beta-D-glucopyranoside, at 100.degree. C. for 10 minutes, and
were cooled at room temperature. Then, the proteins were incubated
in a reaction buffer, containing 5 .quadrature. of PNGase F (Sigma,
Germany), at 37.degree. C. for 3 hours. The deglycosylated proteins
were loaded onto SDS-PAGE to separate the samples and were
subjected to Coomassie brilliant straining. The SDS-PAGE results
are shown in FIG. 2C.
[0093] <2-2> In-Gel Digestion
[0094] The gel Coomassie-stained in Example <2-1> was
cleaved, and the resulting protein bands were desalted by treatment
with 75 mM ammonium bicarbonate/40% ethanol (1:1). After desalting,
a sufficient amount of DTT solution (5 mM dithiothreitol/25 mM
ammonium bicarbonate) was added to the tube and incubated at
60.degree. C. for 3 minutes. After removal of the liquid from the
gel, the gel pieces were cooled at room temperature. For alkylation
of the proteins, the gel was incubated in 55 mM iodoaceto amide at
room temperature for 30 minutes, and then the gel pieces were
dewatered with 100% acetonitrile and dried. The gel pieces were
expanded in 10 .quadrature. of 25 mM ammonium bicarbonate buffer,
containing 20 .quadrature./ml of modified sequencing grade trypsin
(Roche Applied Science), and were incubated at 37.degree. C.
overnight for trypsin degradation. The peptide mixture produced by
trypsin was eluted with 0.1% formic acid for LC-MS/MS analysis.
Example 3
LC-ESI-MS/MS Analysis
[0095] <3-1> LC-ESI-MS/MS
[0096] The peptide mixture produced in Example 2 was analyzed in
the following manner using LC-MS/MS. The LC-MS/MS was performed
using a Thermo Finnigan's ProteomeX workstation LTQ linear ion trap
MS (Thermo Electron, San Jose, Calif., USA) equipped with nonospray
ionization sources (NSI sources, San Jose, Calif.). 12 .quadrature.
of the peptide mixture was injected and loaded into a peptide trap
cartridge (Agilent, Palo Alto, Calif.). The trapped peptide was
eluted using a 10-cm reversed-phase PicoFrit column (5
.quadrature., 300 .ANG. diameter C18) packed in a housing and was
separated by gradient elution in a reverse phase column (RP
column). As the mobile phase, each of solutions A (H.sub.2O) and B
(acetonitrile, ACN) was used, and the solutions all contained 0.1%
(v/v) formic acid. The flow rate was maintained at 200 nL/min.
[0097] The gradient elution started with 2% mobile phase, and
linear gradient elution was performed such that the mobile phase
reached 60% within 50 minutes. Then, the mobile phase B reached 80%
within 5 minutes, and then the mobile phase A reached 100% within
15 minutes.
[0098] The data-dependent acquisition mode was enabled (m/z
300-1800), and each full MS scan was followed by five MS/MS scans
with the 30 s dynamic exclusion option on. The spray voltage and
ion transfer tube temperature were set at 1.8 kV and 160 C,
respectively. The normalized collision energy was set at 35%. The
LC-MS/MS analysis was performed independently twice of each
sample.
[0099] <3-2> Data Analysis
[0100] The MS/MS data base obtained in Example <3-1> were
searched against the IPI human protein database using the SEQUEST
algorithm (Thermo Electron, San Jose, Calif.) contained in the
BioWorks software (version 3.2). The database searching permitted
modification of cysteine (carboxyamidomethylation, 57 Da), variable
modification of methionine (oxidation, 16 Da), peptide mass
tolerance of 1.5 Da, and fragment mass tolerance of 1 Da.
[0101] The SEQUEST results were filtered by Xcorr versus charge
state X. Xcorr values of 1.9 for singly charged ions, 2.2 for
doubly charged ions, and 3.75 for triply charged ions were
considered a match. The present inventors set Delta Cn=0.1, Rsp=4
and the probability limit 0.001. Proteins were identified based on
the identity of the corresponding peptide(s). Additional filtering
was carried out for proteins that scored more than ten, and
proteins for which there were more than two corresponding
peptides.
[0102] The present inventors used NetNGlyc 1.0 and NetOGlyc 3.1 for
statistical analysis of N- and O-linked glycosylation sites,
respectively, and an in-house informatics tool, ProtAn, was used
for subtractive proteome analysis.
[0103] In the experimental results, 74, 121 and 80 proteins were
identified in two repeated experiments for three normal samples,
respectively, and 99, 115 and 101 proteins were identified in two
repeated experiments for three cancer samples. The proteins
identified by the LC-MS/MS analysis were 65.6-76.1% in the cancer
samples and the normal samples (see Table 2).
TABLE-US-00002 TABLE 2 Number of proteins identified by LC-MS/MS
analysis First protein Second Common Reproducibility Samples ID
protein ID protein (%) Normal sample 1 (N1) 95 86 74 69.2 Normal
sample 2 (N2) 133 147 121 76.1 Normal sample 3 (N3) 95 107 80 65.6
Cancer sample 1 (C1) 121 120 99 69.7 Cancer sample 2 (C2) 127 141
115 75.2 Cancer sample 3 (C3) 112 128 101 72.7
[0104] Also, to examine the efficiency of concentration of
glycoproteins, the glycosylation sites of the glycoproteins were
predicted with the NetNGlyc 1.0 and NetOGlyc 3.1 programs. The
detected proteins were predicted to be about 90% of the total
proteins identified by matching an NXS/T sequence for N-linked
glycosylation and an S/T sequence for O-linked glycosylation (Table
3). Thus, it could be seen that more than about 90% of the
identified proteins contained one or more glycosylation sites. A
reproducibility of about 71.4% and an efficiency of glyprotein
concentration of 90% in protein detection show that the profiling
of serum glycoprotein-linked LC-MS/MS is very useful for the
discovery of serum biomarkers.
TABLE-US-00003 TABLE 3 Proteins identified by LC-MS/MS analysis,
number of glycosylated proteins and glycosylation sites Proteins
containing N- Proteins containing Protein linked glycosylation
O-linked glycosylation Percentage Samples ID sites sites Total
glycoproteins of glycoproteins Normal 132 97 89 119 90.2% sample
Cancer 148 106 101 133 89.9% sample
[0105] <3-3> Comparison of Expression of Glycoproteins in
Cancer Samples and Normal Samples
[0106] The glycoproteins identified in cancer samples were analyzed
comparatively with the glycoproteins identified in normal samples.
99 proteins were commonly detected in the sera of three cancer
patients, and among them, 38 proteins were expressed highly in the
sera of cancer patients compared to those in normal persons.
Meanwhile, some of glycoproteins identified in the normal serum 2
(N2) were different from the other normal sera with respect to MS
scores and the number of hit peptides. Thus, the N2 data were
excluded. It was shown that the number of the hit peptides of the
38 proteins were about 1.5-fold higher than that in normal persons.
In particular, the number of hit peptides of 21 proteins among the
cancer glycoproteins was two-fold higher than that of the normal
glycoproteins (Table 4). About 60% of the 38 proteins belong to
proteins which are not highly expressed in human serum. Also, it
was reported to some of these proteins can be used as lung cancer
markers in the sera or plasmas of lung cancer patients. Examples
thereof include heptoglobin (HP), inter-alpha-trypsin inhibitor H4
(ITI-H4), complement C3 precursor, and leucin-rich alpha 2
glycoprotein. Proteins other than these proteins are not yet known
as lung cancer markers or biomarkers.
[0107] The 38 proteins, which increased in the sera of lung cancer
patients, consisted of 6 IGgs (15.8%), 8 high abundant protein
(21.1%), 1 hemoglobulin (2.6%) and 23 non-high abundant proteins
(60.5%). Such results indicate that the concentration and isolation
of serum proteins are useful tools for the identification of low
abundant proteins.
TABLE-US-00004 TABLE 4 Lung cancer biomarkers identified by the
present invention Scores Peptides (hit) IPN No. Protein names
Cancer Normal Cancer Normal Fold IPI00294395.1 Complement component
c8 beta chain 17 0.0 4 0.0 -- precursor IPI00027462.1 Protein
s100-a9 17 0.0 3 0.0 -- IPI00021727.1 C4b-binding protein alpha
chain 40 13.3 15 2.7 5.75 precursor IPI00186903.3 Isoform 2 of
apolipoprotein-II 23 3.3 4 0.7 5.50 precursor IPI00007047.1 Protein
s100-a8 20 6.7 6 1.3 4.50 IPI00250430 Similar to cavia porcellus
phosphatidic 10 3.3 3 0.7 4.50 acid phosphatase 2A mRNA
IPI00022395.1 Complement component c9 precursor 43 26.7 21 5.0 4.20
IPI00028413.4 Inter-alpha-trypsin inhibitor heavy 17 3.3 3 0.7 4.00
chain h3 precursor IPI00168728.1 FIj00385 protein (fragment) 60
20.0 28 7.7 3.65 IPI00019581.1 Coagulation factor xii precursor 13
3.3 4 1.3 3.00 IPI00384948.3 Ig alpha-2 chain c region 10 6.7 5 1.7
3.00 IPI00011252.1 Complement component c8 alpha chain 10 6.7 5 1.7
2.80 precursor IPI00027482.1 Corticosteriod-binding globulin 23 6.7
4 1.7 2.60 precursor IPI00291262.3 Clusterin precursor 47 56.7 36
14.0 2.60 IPI00030809 Gamma-g globin 13 6.7 17 7.0 2.48
IPI00022391.1 Serum amyloid p-component precursor 33 16.7 11 4.7
2.36 IPI00008558.1 Plasma kallikrein precursor 40 26.7 13 5.7 2.29
IPI00166866.3 Ighal protein 23 16.7 13 6.7 2.00 IPI00292950.4
Heparin cofactor 2 precursor 27 16.7 7 3.3 2.00 IPI00334432.3 16
kDa protein 33 10.0 12 6.0 2.00 IPI00385252.1 Ig kappa chain v-iii
region gol 10 3.3 3 1.3 2.00 IPI00291867.3 Complement factor I
precursor 57 50.0 16 8.3 1.92 IPI00382950.1 Hemoglobin subunit beta
40 23.3 41 22.0 1.88 IPI00164623.3 Complement 3 precursor fragment
293 116.7 142 77.7 1.83 IPI00431645 Hp protein 130 110.0 529 291.0
1.82 IPI00060731.5 Similar to tripartite motif protein 49 10 6.7 3
1.7 1.80 IPI00019591.1 Isoform 1 of complement b precursor 53 36.7
37 21.0 1.78 (fragment) IPI00553177.1 Alpha-1-antitrypsin precursor
93 86.7 211 119.9 1.76 IPI00218192.1 Isoform 2 of
inter-alpha-trypsin 143 92.7 91 51.7 1.75 inhibitor heavy chain h4
precursor IPI00022417.4 Leucine-rich alpha-2-glycoprotein 27 10.0 7
4.0 1.75 precursor IPI00479116.1 Carboxypeptidase n subunit
precursor 20 6.7 4 2.3 1.71 IPI00027507.1 Complement factor
h-related protein 3 20 16.7 3 2.0 1.67 precursor IPI00291866.4
Plasma protease C1 inhibitor precursor 113 86.7 145 87.0 1.67
IPI00550315.1 Ig kappa chain c region 10 10.0 25 15.0 1.67
IPI00550315.2 Alpha 2 macroglobulin variant 213 126.7 173 106.3
1.63 IPI00027547.2 Dermcidin precursor 13 6.7 4 2.3 1.57
IPI0003229.1 Complement c5 precursor 63 40.0 10 6.7 1.55
IPI00025426.1 Pregnancy zone protein precursor 40 26.7 59 39.3
1.51
[0108] The 38 selected proteins shown in Table 4 were classified,
according to intracellular distribution, into extracellular
proteins and membrane proteins (FIG. 3A). Also, such proteins were
classified according to molecular functions (FIG. 3B) and
biological processes (FIG. 3). In a viewpoint of molecular
functions, 20% of the 38 proteins had endopeptidase inhibitory
activity, 15% had antigen binding activity, and 11% had trypsin
activity. In a viewpoint of biological processes, most of the 38
proteins were associated with transport and immune-inflammatory
response proteins.
Example 4
Western Blotting of Inter-Alpha-Trypsin Inhibitor Heavy Chain h3
Precursor
[0109] Whether Inter-alpha-trypsin inhibitor heavy chain h3
precursor (ITI-H3) among the 38 proteins selected in Example 3 is
specifically expressed only in the sera of lung cancer patients was
analyzed by Western blotting.
[0110] 20 .quadrature. of the serum glycoproteins, obtained from
each of the cancer patients and the normal persons in Example 2,
were loaded onto 15% SDS-PAGE. After electrophoresis, the gel was
transferred to a nitrocellulose membrane (Whatman, Germany). The
proteins were diluted with monoclonal goat anti-ITI-H3 antibody
(Santa cruz, Calif., USA) at 1:500 dilution and incubated at
4.degree. C. overnight. Then, the proteins were diluted with
anti-goat IgG antibody (Santa cruz, Calif., USA) at 1:2,000
dilution and incubated at room temperature for 1 hour. To detect
signals, the ECL advanced Western blotting system system was
used.
[0111] In the experimental results, in the LC-MS/MS analysis of the
normal sera, ITI-H3 was not identified, but in the Western
blotting, it was detected even in the normal sera. However, it was
shown that ITI-H3 was expressed highly in the lung cancer sera
compared to the normal sera (FIG. 4A). Accordingly, it can be seen
that ITI-H3 can be used as a biomarker for lung cancer.
Example 5
Analysis of Peptide Number of Plasma Kallikrein
[0112] Peptides of plasma kallikrein (KLKB1) were detected from 3
normal persons (N1, N2 and N3) and 3 lung cancer patients (C1, C2
and C3) using a mass spectrometer. In the experimental results, it
could be seen that the number of the peptides detected in the lung
cancer patients was larger than in the normal persons.
Particularly, the peptide number of the H4 domain was about 3-fold
larger in the cancer patients than in the normal persons (Table
5).
TABLE-US-00005 TABLE 5 Peptide number of plasma kallikrein N1 N2 N3
C1 C2 C3 H1 0 6 2 5 8 3 H3 0 1 0 1 0 0 H4 0 2 2 3 6 3 L 0 8 0 2 9 0
H1, H3 and H4: heavy chain domains L: light chain domain
Example 6
Western Blotting 1 of Plasma Kallikrein Fragment
[0113] On the basis of the results of Example 5, whether the H4
domain of KLKB1 is specifically expressed only in the sera of lung
cancer patients was analyzed by Western blotting using an epitope
antibody.
[0114] The sera, obtain from the lung cancer patients (C1, C2 and
C3), or the glycoproteins, obtained from the sera using the lectin
column, were concentrated, and then analyzed by Western blotting.
100 of each of the sera or 20 .quadrature. of each of the
glycoproteins was loaded onto 15% SDS-PAGE. After electrophoresis,
the gel was transferred to a nitrocellulose membrane (Whatman,
Germany). The sera were diluted with anti-KLKB1 antibody (Santa
Cruz, prepared using an epitope of amino acid residues 361-400 of
plasma kallikrein) at 1:1,200 dilution and incubated at 4.degree.
C. overnight. Then, the sera were diluted with anti-goat IgG
antibody (Santa cruz, Calif., USA) at 1:2,000 dilution and
incubated at room temperature for 1 hour.
[0115] To detect signals, the ECL advanced Western blotting
analysis system (Amersham Biosciences, UK) was used. In the results
of Western blot of the concentrated glycoproteins, a band having a
size of 15-20 kDa was detected only in the samples of lung cancer
patients at high levels. Also, in the experimental results obtained
using the sera, a band of 15-20 kDa was detected in the sera of
lung cancer patients at a level higher than in the normal persons
(FIG. 5).
Example 7
Western Blotting 2 of Plasma Kallikrein Fragment
[0116] 100 .quadrature. of each of the whole sera of normal persons
or lung cancer patients 50-60 years old was loaded onto 15%
SDS-PAGE (2.5 .quadrature. sera), and then subjected to Western
blot in the same manner as in Example 6.
[0117] The sera were treated with anti-KLKB1 antibody (1:200
dilution) and incubated at 4.degree. C. overnight, followed by
incubation at room temperature for 1 hour. In the experimental
results, a KLKB1 fragment of 18 kDa was detected in 13 patients of
the 14 lung cancer patients. However, it was not substantially
detected in the normal persons (FIG. 6).
Example 8
Western Blotting 3 of Plasma Kallikrein Fragment
[0118] The whole sera of normal persons or lung cancer patients
more than 65-year-old were collected, and whether KLKB1 is
specifically expressed only in the sera of the lung cancer patients
was analyzed by Western blotting in the same manner as in Example
7.
[0119] In the experimental results, a KLKB1 of 18 kDa was detected
in 12 patients of the 14 lung cancer patients at high levels.
However, it was weakly detected in only one person of the normal
persons (FIG. 7).
Example 9
Western Blotting 4 of Plasma Kallikrein Fragment
[0120] The whole sera of 34 normal persons and 24 lung cancer
patients 50-70 years old were additionally collected, and whether
the KLKB1 fragment is specifically expressed in the sera of the
lung cancer patients was analyzed by Western blot in the same
manner as in Example 7.
[0121] In the experimental results, in the normal persons, the
KLKB1 fragment was not substantially detected or was detected at
low levels, but in the lung cancer patients, the KLKB1 fragment was
detected in 20 of the 24 patients at high levels (see FIGS. 9 to
12).
[0122] In Examples 7 to 9, a total of 42 normal persons and 52
patients were subjected to Western blot and, as a result, it could
be seen that the 18 kDa fragment was weakly detected in 17 persons
of the 42 normal persons, very weakly detected in 11 persons and
was not detected in 14 persons, and it was detected in 45 patients
of the 52 lung cancer patients at high levels. From these results,
it could be seen that the 18 kDa fragment was detected in 45 of the
52 lung cancer patients, and thus it had a sensitivity of 87% and a
specificity of 67%. Meanwhile, the level of the 18 kDa fragment in
the normal persons was lower than that in the lung cancer
patients.
INDUSTRIAL APPLICABILITY
[0123] As described above, the inventive diagnostic markers for
lung cancer are specifically expressed only in the sera of lung
cancer patients at high levels, and thus will be very useful for
diagnosing lung cancer and estimating disease progression and
treatment.
Sequence CWU 1
1
11638PRTHomo sapiens 1Met Ile Leu Phe Lys Gln Ala Thr Tyr Phe Ile
Ser Leu Phe Ala Thr1 5 10 15Val Ser Cys Gly Cys Leu Thr Gln Leu Tyr
Glu Asn Ala Phe Phe Arg 20 25 30Gly Gly Asp Val Ala Ser Met Tyr Thr
Pro Asn Ala Gln Tyr Cys Gln 35 40 45Met Arg Cys Thr Phe His Pro Arg
Cys Leu Leu Phe Ser Phe Leu Pro 50 55 60Ala Ser Ser Ile Asn Asp Met
Glu Lys Arg Phe Gly Cys Phe Leu Lys65 70 75 80Asp Ser Val Thr Gly
Thr Leu Pro Lys Val His Arg Thr Gly Ala Val 85 90 95Ser Gly His Ser
Leu Lys Gln Cys Gly His Gln Ile Ser Ala Cys His 100 105 110Arg Asp
Ile Tyr Lys Gly Val Asp Met Arg Gly Val Asn Phe Asn Val 115 120
125Ser Lys Val Ser Ser Val Glu Glu Cys Gln Lys Arg Cys Thr Asn Asn
130 135 140Ile Arg Cys Gln Phe Phe Ser Tyr Ala Thr Gln Thr Phe His
Lys Ala145 150 155 160Glu Tyr Arg Asn Asn Cys Leu Leu Lys Tyr Ser
Pro Gly Gly Thr Pro 165 170 175Thr Ala Ile Lys Val Leu Ser Asn Val
Glu Ser Gly Phe Ser Leu Lys 180 185 190Pro Cys Ala Leu Ser Glu Ile
Gly Cys His Met Asn Ile Phe Gln His 195 200 205Leu Ala Phe Ser Asp
Val Asp Val Ala Arg Val Leu Thr Pro Asp Ala 210 215 220Phe Val Cys
Arg Thr Ile Cys Thr Tyr His Pro Asn Cys Leu Phe Phe225 230 235
240Thr Phe Tyr Thr Asn Val Trp Lys Ile Glu Ser Gln Arg Asn Val Cys
245 250 255Leu Leu Lys Thr Ser Glu Ser Gly Thr Pro Ser Ser Ser Thr
Pro Gln 260 265 270Glu Asn Thr Ile Ser Gly Tyr Ser Leu Leu Thr Cys
Lys Arg Thr Leu 275 280 285Pro Glu Pro Cys His Ser Lys Ile Tyr Pro
Gly Val Asp Phe Gly Gly 290 295 300Glu Glu Leu Asn Val Thr Phe Val
Lys Gly Val Asn Val Cys Gln Glu305 310 315 320Thr Cys Thr Lys Met
Ile Arg Cys Gln Phe Phe Thr Tyr Ser Leu Leu 325 330 335Pro Glu Asp
Cys Lys Glu Glu Lys Cys Lys Cys Phe Leu Arg Leu Ser 340 345 350Met
Asp Gly Ser Pro Thr Arg Ile Ala Tyr Gly Thr Gln Gly Ser Ser 355 360
365Gly Tyr Ser Leu Arg Leu Cys Asn Thr Gly Asp Asn Ser Val Cys Thr
370 375 380Thr Lys Thr Ser Thr Arg Ile Val Gly Gly Thr Asn Ser Ser
Trp Gly385 390 395 400Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys
Leu Thr Ala Gln Arg 405 410 415His Leu Cys Gly Gly Ser Leu Ile Gly
His Gln Trp Val Leu Thr Ala 420 425 430Ala His Cys Phe Asp Gly Leu
Pro Leu Gln Asp Val Trp Arg Ile Tyr 435 440 445Ser Gly Ile Leu Asn
Leu Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser 450 455 460Gln Ile Lys
Glu Ile Ile Ile His Gln Asn Tyr Lys Val Ser Glu Gly465 470 475
480Asn His Asp Ile Ala Leu Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr
485 490 495Glu Phe Gln Lys Pro Ile Cys Leu Pro Ser Lys Gly Asp Thr
Ser Thr 500 505 510Ile Tyr Thr Asn Cys Trp Val Thr Gly Trp Gly Phe
Ser Lys Glu Lys 515 520 525Gly Glu Ile Gln Asn Ile Leu Gln Lys Val
Asn Ile Pro Leu Val Thr 530 535 540Asn Glu Glu Cys Gln Lys Arg Tyr
Gln Asp Tyr Lys Ile Thr Gln Arg545 550 555 560Met Val Cys Ala Gly
Tyr Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly 565 570 575Asp Ser Gly
Gly Pro Leu Val Cys Lys His Asn Gly Met Trp Arg Leu 580 585 590Val
Gly Ile Thr Ser Trp Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro 595 600
605Gly Val Tyr Thr Lys Val Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys
610 615 620Thr Gln Ser Ser Asp Gly Lys Ala Gln Met Gln Ser Pro
Ala625 630 635
* * * * *