KR101796874B1 - Method for Detecting or Determining Level of PIVKAII Based on Multiple Reaction Monitoring - Google Patents

Abstract

Disclosed in the present invention are a method for accurately quantifying DCP used as a marker for liver cancer using MRM technology based on liquid chromatography mass spectrometry, and a method for monitoring the early diagnosis and treatment prognosis of liver cancer using the same. The method according to the present invention can diagnose liver cancer rapidly, economically, and accurately compared to conventional immunoassay.

Description

(Method for Detecting or Determining Level of PIVKAII Based on Multiple Reaction Monitoring)

The present invention relates to a quantitative analysis method of PIVKAII for providing information necessary for diagnosis of liver cancer.

Hepatocellular carcinoma is the fifth most common cancer in the world and the third leading cause of cancer-related deaths. Hepatocellular carcinoma accounts for more than 90% of primary liver cancers (Ferrin, G .; Aguilar-Melero, P .; Rodriguez-Peralvarez, M .; Montero-Alvarez, JL; de la Mata, M. Hepatic medicine: evidence and research 2015, 7 , 1-10). Although studies on HCC continue and the symptoms are well known, it is still difficult to diagnose the disease at an early stage and the survival rate after diagnosis is very low (Xiao, JF et al., Journal of proteome research 2012, 11 , 5914-5923). Although surgery is necessary for complete removal of the tumor, the possibility of surgery is as low as 10-20%, so early diagnosis in patients with high risk factors for liver cancer is clinically important.

Diagnostic methods such as ultrasound, computed tomography, and magnetic resonance imaging are useful for diagnosis of liver cancer, but it is difficult to detect small - sized tumors and it is costly. Although alpha-fetoprotein (AFP) is a diagnostic marker used for the diagnosis of liver cancer, sensitivity is only about 50-60%, and even in positive liver diseases such as alcoholic hepatitis, viral hepatitis and liver cirrhosis, And the specificity is as low as 40-50%. PIVKAII is also a diagnostic marker used in the diagnosis of liver cancer.

Most of the existing methods for quantifying PIVKAII are based on antibodies and quantify using protein and antibody affinity at the protein level. A typical PIVKAII assay uses a monoclonal antibody produced by the MU-3 cell line and mainly detects PIVKAII with 9-10 Glu residues. However, PIVKAII levels are also elevated in patients with vitamin K deficiency and taking warfarin.

PIVKAII induced by vitamin K deficiency containing 2-9 Glu residues also partially reacts with the MU-3 antibody, leading to elevated PIVKAII in individuals who are not HCC. Thus, current analysis methods based on MU-3 antibodies may lead to unnecessary treatment or induce false therapy. In addition, antibody-based assays are less quantitative due to non-specifically binding anti-reagent antibodies, and when there is an analyte at a certain concentration or more, (high dose hook effect) occurs.

Korean Patent Laid-Open No. 10-2014-0057495 relates to a method for inhibiting a non-specific reaction in a measuring reagent, which comprises adding a specific divalent metal ion to a monoclonal antibody specifically binding to PIVKAII and prothrombin, Discloses a method of specifically measuring PIVKAII in a sample by inhibiting nonspecific aggregation reaction.

Korean Patent Laid-Open No. 10-2015-0041620 relates to a method for measuring PIVKA-II, a measuring reagent and a measurement kit, and a method for measuring PIVKA-II in a sample using prothrombin fragments F1 and F2 and anti-PIVKAII antibody .

However, these methods are based on antibody-based immunoassays. However, a method for quantifying PIVKAII that can complement the immunoassay method in terms of time and cost is needed.

The present invention provides a method for quantitative analysis of PIVKA-II using multiple simultaneous quantitation methods.

In one aspect, the present invention provides a method for diagnosing hepatocellular carcinoma, comprising the steps of: providing a sample requiring diagnosis; Treating the specimen with a protease to cleave the protein contained in the specimen; Mixing the internal standard material with the specimen; And performing an MRM (Multiple Reaction Monitoring) analysis on a subject peptide of the PIVKAII protein and an internal standard substance in the mixture, and normalizing the result of the MRM analysis of the subject peptide to the result of the analysis of the internal standard substance, Detecting the amount of PIVKAII in the specimen sample; And comparing the detection result with a result of the control sample.

The method according to the present invention selects an optimal target peptide for the PIVKAII protein for MRM analysis, in one embodiment the target peptide is ERECVEETCSY (indicated by amino acid single letter).

In the method according to the present invention, the internal standard material has the same amino acid sequence as the subject peptide, but the mass of one or more amino acids constituting the internal standard is different from the amino acid mass of the subject peptide.

The method according to the present invention can be carried out in particular using plasma, serum or whole blood, thus increasing convenience.

As the amount (concentration or level) of PIVKAII in the present invention increases with the progression to normal specimens, chronic hepatitis specimens and cirrhosis and liver cancer, a normal specimen, a chronic hepatitis specimen, and / or a cirrhotic specimen can be used as a control group, The method further comprises the step of discriminating the specimen as liver cancer when at least one of a specimen, a chronic hepatitis specimen, and a cirrhosis specimen can be used and the amount of PIVKAII of the specimen is increased as compared with the control specimen.

The step of determining the amount of PIVKAII in the method according to the present invention can be carried out in various ways, for example, using a standard concentration curve using a sample containing a known concentration of PIVKAII target peptide. The values obtained in the samples are normalized to the values obtained using internal standard materials for quantification.

For the MRM analysis according to the present invention, the values of the precursor peptide ion Q1 and the product peptide ion Q3 of the target peptide and the internal standard substance are monitored, and an optimum CE (collision energy) value for the Q1 / Q3 transition is used. Can be determined using the default CE.

The proteolytic enzymes that can be used in the method according to the present invention are neutral proteases which function at neutral pH and include, but are not limited to, one or more of, for example, chymotrypsin, trypsin, Lyc-C, Asp-N or Glu- It is not.

Prognostic measures in the method according to the present invention include determining whether the liver cancer has recovered after treatment.

The method according to the present invention can select the ERECVEETCSY as a target peptide of PIVKAII, which is a marker of liver cancer, and accurately quantify PIVKAII using MRM (Multiple Reaction Monitoring) based on liquid chromatography mass spectrometry.

The method according to the present invention can be used for diagnosing various diseases requiring the above-mentioned quantitation, shortens the time required for diagnosis, can screen multiple patients at the same time, and diagnoses liver cancer particularly with high discriminating power. This is advantageous in terms of time and cost because it can diagnose hepatocellular carcinoma rapidly, economically and accurately compared to conventional PIVKAII assay.

1 is a schematic diagram for determining the surrogate peptide required for quantification of PIVKAII in human serum according to one embodiment of the present invention. From all the peptides obtained in (A) in silico digestes, filtration was performed according to the criteria to select the surrogate peptides. One chymotrypsin digesting peptide representing the concentration of PIVKAII was selected. (B) The Gla domain is present at the N-terminal part of the prothrombin. In the Gla domain containing 10 glutamate residues (Glu, E), as many as possible mutated Glu were converted to Gla in HCC patients. Two trypsin digesting peptides (blue) and three chymotrypsin digesting peptides (red) were investigated experimentally using the MRM-MS assay.
Figures 2a and 2b show the reverse reaction curves for the subject PIVKAII peptide in pooled serum. The linearity of the MRM-MS analysis was determined using heavy peptides spiked into the serum matrix at various concentrations (6.10 - 100,000 fmol). Over the range of concentrations over 10 ⁵ times for satisfactory concentrations for linearity and precision were observed. Figure 2a shows the peak area calculated as the mean of 5 replicate experiments for all transition ions in a logarithmic plot. FIG. 2B is a calibrated or concentration standard curve in which the concentration of PIVKAII is calculated using b8 ions having a good correlation (= 0.9842) among the four transition ions. The LOD and LOQ of the b8 ion were calculated to be 0.30 nM and 0.63 nM, respectively. Individual concentration points were plated as an average of 5 replicates (shown in black); The error bar is 5 standard deviations (SD).
Figure 3 compares PIVKAII levels using the MRM-MS assay in a five-patient group, according to one embodiment of the present invention, wherein Figure 3A shows that PIVKAII levels were elevated based on disease progression, Supporting a statistical evaluation of data representing the expression of PIVKAII. The expression of PIVKAII in the HCC group versus normal group, chronic hepatitis group, and hepatocyte group was significantly different (P <0.0001). However, no significant difference was observed between the HCC and the recovery group (P = 0.3901). Box boundaries, 25% and 75% quartiles; Median horizon, mean; Beard, quartile boundary for numbers greater than 1.5 times the quadrant; Red Astris, outliers; Blue line, and average connecting line. Figure 3b shows the results of the AUCCs of participants in the HCC group versus the other groups (normal control, chronic hepatitis group, liver hardening group, and recovery group). The AUROC values of HCC group versus normal control group, chronic hepatitis group, liver hardening group and recovery group were calculated as 0.870, 0.766, 0.740 and 0.706, respectively. AUROC was also found to have a 95% confidence interval (CI).
Figure 4 shows a comparison of PIVKAII levels between an immunoassay and an MRM-MS assay according to one embodiment of the invention. Figure 4a shows that AUROC has a 95% confidence interval. The AUROC values of the immunoassay and MRM-MS analyzes were 0.764 and 0.706, respectively. Figure 4b is a Deming regression analysis of PIVKAII concentration in the HCC group and in the recovery group, as measured by MRM-MS (x-axis) versus immunoassay (y-axis). The solid and dashed lines represent the even line (y = x) and the 95% limit line, respectively. Figure 4c shows the Bland-Altman plot showing the Unity (gray solid line), the mean difference (black solid line), and the 95% limit agreement (black dotted lines) .
5 is a graphical representation of the principle of triple quadrupole mass spectrometry, in accordance with one embodiment of the present invention.
FIG. 6 shows the results of MRM analysis using hepatocellular carcinoma samples according to one embodiment of the present invention. The transition between the endogenous peptide and the heavy labeled peptide was converted to a peak area using Skyline software, area value and shows the result of analysis by normalization.
Figure 7 is a schematic representation of the procedures performed here including selection of the subject peptide, MRM-MS analysis optimization and comparison with existing immunization methods.

The present invention is based on the discovery that PIVKAII, known as a biomarker for the diagnosis of liver cancer, can be accurately quantified using liquid chromatography-based MRM (Multiple Reaction Monitoring Mass Spectrometry).

In one embodiment, the present invention provides a method for diagnosing liver disease, comprising the steps of: providing a sample requiring diagnosis of liver disease to provide information necessary for diagnosis or prognosis of liver disease; Treating the specimen with a protease to cleave the protein contained in the specimen; Mixing the internal standard material with the specimen; And performing an MRM (Multiple Reaction Monitoring) analysis on a subject peptide of the PIVKAII protein and an internal standard substance in the mixture, and normalizing the result of the MRM analysis of the subject peptide to the result of the analysis of the internal standard substance, Detecting the amount of PIVKAII in the specimen sample; And comparing the result of the detection with a result of a control sample, wherein the subject peptide is ERECVEETCSY, the internal standard substance has the same amino acid sequence as the subject peptide, but the mass of one or more amino acids constituting the internal standard substance Is different from the amino acid mass of the subject peptide, and the MRM assay monitors the progenitor peptide ion Q1 value, the product peptide ion Q3 value, and the CE value.

PIVKAII (protein induced by vitamin K absence / antagonist-II) is a prothrombin precursor abnormally produced during the synthesis of prothrombin, a coagulation protein, and is also called des-gamma carboxyprothrombin (DCP). In the steady state, 10 glutamic acid (Glu) at the amino terminal of the prothrombin precursor in the liver is converted to gamma-carboxyglutamic acid (Gla) by vitamin K-dependent carboxylase, but due to damage of gamma- glutamylcarboxylation, If all or a portion of the Glu are not converted to Gla, the abnormal prothrombin is secreted into the blood, which is called PIVKAII. As the number of Glu residues not converted to Gla varies, PIVKAII exists as a mixture with varying numbers of Glu residues ranging from 1 to 10 and can be distinguished from normal thrombin based on these amino acid residue differences.

In the method according to the present invention, the biomarker or the diagnosis marker distinguishes a patient suffering from liver cancer or liver disease from a normal group, a hepatitis group, a liver cirrhosis group, or a patient (or a recovery group) A sample derived from a patient suffering from liver cancer as compared with a control specimen, that is, a protein showing a concentration change in blood, or a peptide derived from the protein, and PIVKAII is used.

In the method according to the present invention, PIVKAII can be quantified or detected using mass spectrometry or mass spectrometry based techniques, for example (Kim, et al. 2010 J Proteome Res. 9: 689-99 ; Anderson, L et al . 2006. Mol Cell Proteomics 5: 573-88.

In one embodiment according to the present application, a Multiple Reaction Monitoring (MRM), which is a targeted MS technology, is used. Particularly, for example, a triple quadrupole (Q1, Q2, Q3) LC-MS / MS (for example, a triple quadrupole Multiple reaction monitoring (MRM) technology using QTRAP® system is used. MRM is a MS method capable of quantitatively and precisely measuring multiple markers such as a small amount of biomarkers existing in a biological sample, and uses a first mass filter (Q1) that passes through only a specific m / z peptide and acts as a filter , And selectively transfers the precursor ion or the parent ion among the ion fragments (peptides) generated in the ionization source to the collision tube (Q2). The precursor ions reaching the collision tube collide with the internal collision gas and are split to produce product ions or daughter ions of various lengths, which are then sent to the second mass filter Q3 where a specific m / z Is selected and converted to a digital signal at the detector to be seen as a peak chromatogram (Figure 5). In this way, it is a selective and sensitive analytical method that can detect only the information of a desired component. See, for example, Gillette et al., 2013, Nature Methods 10: 28-34.

In the method according to the present invention, the sample is treated with proteinase (protease, peptidase) for cleavage or degradation for PIVKAII analysis of the sample. As a result, full-length proteins can be cleaved and fragments of various lengths can be generated. The proteolytic enzyme used in the method according to the present invention is not particularly limited as long as it has an activity at pH 7-8.

A variety of known proteolytic enzymes can be used, for example, searching in the MEROPS database (http://merops.sanger.ac.uk/cgi-bin/pathway_index) and commercially available. Proteolytic enzymes can be classified on a variety of criteria and can be classified, for example, according to the appropriate pH with catalytic residues or activity.

The proteolytic enzyme that may be used in the method according to the invention in one embodiment according to the present application is a neutral proteolytic enzyme that acts at a neutral pH. Such proteolytic enzymes include, but are not limited to, chymotrypsin, trypsin, Lys-C, Asp-N and Glu-C. Chymotrypsin cleaves the carboxy terminal side of hydrophobic residues (tryptophan, tyrosine, phenyalalnine, leucine, methionine), and trypsin cleaves carboxy terminal side of lysine and arginine and has activity at pH 7-8. Lys-C cleaves the carboxy terminal side of the lysine residue, has activity at pH 7-9, Asp-N cleaves the amino terminal side of aspartic acid, and has activity at pH 4-9. Glu-C cleaves the carboxy terminal side of aspartic acid and glutamic acid, and has activity at pH 4-9.

In the method according to the invention, MRM analysis is performed on the subject peptide of PIVKAII in a sample of the sample.

The target peptide is a partial sequence used for the MRM analysis among the entire protein sequence to be analyzed. The target peptide is a condition in which the entire sequence of the protein is represented by a certain condition, for example, The optimal target peptide to be satisfactory can be selected. For example, a peptide having a length of 6 to 30 amino acids is selected. If the length is shorter than the above range, the selectivity is deteriorated. If the length is too long, the sensitivity is lowered. In addition, methionine, cysteine, and tryptophan are easily excluded because they are easy to chemically modify, such as oxidation.

In one embodiment according to the present invention, the entire sequence of prothrombin is cut into in silico with chymotrypsin using Skyline software as shown in Fig. 1B, and a peptide having 6 to 30 amino acid residues and no methionine residue is added to 1 The tea was selected. Of these, three peptides containing glutamic acid residues were selected, while the sequence was contained in the Gla region at the N-terminus, and all the peptide sequences of the Glu residues were transformed into Gla residues, , We can select peptides that are highly related to the known PIVKA II concentration.

May be used as the subject peptide of three chymotrypsin digesting peptides, EEVRKGNL, ERECVEETCSY, or ESSTATDVF PIVKAII in one embodiment according to the present application. In particular, in one embodiment of the present invention, when ERECVEETCSY is used as a target peptide of PIVKAII, it is possible to diagnose liver cancer with high accuracy and sensitivity.

In the method according to the invention, a heavy peptide is also used. The same amount of heavy peptide is added to all samples, so normalization of the whole assay results and verification of the target peptide to be detected are based on the actual analyte protein. The internal standard substance has the same amino acid sequence as the above-mentioned subject peptide, but the mass of at least one amino acid constituting the internal standard substance is different from the amino acid mass of the subject peptide, but since the sequences are the same, column (C18 resin) at the same retention time.

In one embodiment according to the present disclosure, the internal standard material may be labeled with one or more amino acids constituting it by a radioactive isotope such as ¹³ C or ¹⁵ N, or a mixture thereof.

In one embodiment according to the present target peptides ERECVEETCSY, EEVRKGNL, or ESSTATDVF, and the internal standard is this at least one amino acid in a peptide having the same sequence, in particular a carbon or nitrogen of the Y moiety of the C-term radioisotopes ¹³ C or ¹⁵ &lt; / RTI &gt;

The multiple simultaneous assays used in the method according to the present invention are based on mass spectrometry of the subject peptide of PIVKAII in a sample, detection of a specific peptide ion, using a standard internal substance, calculating the ratio of the relative concentration change between them, Or the amount of peptide can be quantitatively analyzed and has never been applied to PIVKAII until now.

In the method according to the present invention, the MRM analysis can be performed by monitoring the Q1 and Q3 values for the internal reference material and the target peptide, and the Q1 / Q3 transition and the optimal Collison Energy (CE) value representing the highest signal can be used . For high sensitivity in MRM-MS, it is important to fine-tune the parameters in a transition-specific manner. In one embodiment, the CE value applied in the peptide segmentation process is optimized, and the optimization method is described in detail in the description of the embodiments of the present invention Can be selected.

In one embodiment according to the present application, the Q1 and Q3 values for the internal reference material and the target peptide, and the optimized CE for each transition are shown in Table 1 below. Among the numerical values in Table 1, the detection limit is low and the linearity is the highest within a quantifiable range. In one embodiment of the invention, b8 ion is used.

[Table 1]

In Table 1, m / z represents the mass to charge ratio. Q3 (a) in bold is used for quantitation as an optimal transition value with the highest linearity within the quantifiable range, with no interference, no detection limit, and no interference in comparison with the internal standard material in analysis.

Next, in the method according to the present invention, the step of normalizing the result of the MRM analysis on the subject peptide with the result of the analysis of the internal standard material, and detecting the amount of PIVKAII in the specimen of the sample. As shown in FIG. 5, the analysis result can be calculated by deriving the peak area using software such as Skyline and dividing the peak area of the target peptide of the sample by the peak area of the peptide of the standard internal material. The method according to the present invention can be used for the diagnosis of liver disease.

The concentration of PIVKAII in the method according to the invention or The step of determining the amount can be determined using a standard concentration curve determined using a known concentration of the PIVKAII target peptide, for example, the method described in this Example can be referred to.

Liver diseases herein include chronic hepatitis, cirrhosis, cirrhosis and liver cancer (or liver cancer).

Hepatocellular carcinoma is a tumor in which hepatocytes lose their inherent function by continuous and various stimuli, transform into cancer cells, and develop permanent autoprolines, spreading to nearby or distant sites. There is a metastatic cancer that has been transferred to. Primary liver cancer includes hepatocellular carcinoma (HCC) and hepatocellular carcinoma (HCC), and hepatocellular carcinoma

In one embodiment according to the present application, the liver cancer is a primary malignant tumor originating in the tissue itself, and is hepatocellular carcinoma (HCC). Hepatocellular carcinoma occurs in patients with risk factors such as alcohol abuse, viral hepatitis and metabolic liver disease.

Hepatocellular carcinoma does not have fibrous stroma, and hemorrhage and cell necrosis occur, resulting in vascular infiltration into the portal vein system. In severe cases, it may lead to liver rupture and intraperitoneal hemorrhage. Such liver cancer is distinguished from chronic inflammation caused by liver, regenerated nodule and fibrosis of liver tissue, resulting in hepatocyte injury, scarring, reduction of liver function, ascites, outbreak disease, liver cirrhosis or liver cirrhosis showing symptoms of hepatic coma. Hepatoma patients usually have both hepatitis (inflammation) and cirrhosis (fibrosis). In hepatitis cases, most hepatocellular carcinomas disappear by medication, but 90% of liver cirrhosis patients are diagnosed with liver cancer. It is important to do.

The term diagnosis is used herein to determine susceptibility to a subject's disease for a particular disease or disorder, to determine whether a particular disease or disorder is presently present, to determine whether the subject has a transient nature The determination of the cancer status, determination of the stage or progress of the cancer, or determination of the cancer's responsiveness to treatment, or therametrics (e. G., Monitoring the status of the object to provide information about the therapeutic efficacy) .

The prognostic measures herein include diagnosing a liver disease, treating it, and determining whether it is recovering.

Samples which can be used as a control for the diagnosis of a patient with liver cancer in the method according to the present invention may be one or more samples from a normal person, hepatitis, a patient who has been cured after treatment with liver cirrhosis and / or liver cancer (liver cirrhosis). Compared with these control patients, the expression level of the marker PIVKAII according to the present invention in liver cancer patients is increased.

The marker PIVKAII used in the method according to the present invention has an increased expression level in patients with liver cancer as compared with a sample derived from a normal person as well as a patient suffering from hepatitis and cirrhosis.

Therefore, in the method according to the present invention, the diagnosis of liver cancer can be distinguished from that of liver cancer, and thus it is advantageous for early diagnosis because it can diagnose patients progressing from liver cirrhosis to liver cancer. In this case, a sample derived from a patient with liver cirrhosis can be included as a control group, and a sample derived from a cirrhotic patient includes a patient with liver cancer who maintains liver cirrhosis. It is also advantageous to distinguish patients recovered after liver cancer treatment. In this case, a liver cancer sample derived from the same patient can be used as a control group.

Herein, the sample or the sample includes tissues, cells, blood, whole blood, serum or plasma capable of detecting PIVKAII, and preferably includes whole blood, serum or plasma. In one embodiment of the invention, the sample is blood, particularly a serum sample. When blood is used as a sample, it can be used after decomposition with proteolytic enzymes (for example, chymotrypsin) after protein depletion and denaturation.

Samples or samples of the subject methods are obtained from mammals, especially humans. Human subjects include those suspected of having HCC, hepatitis, cirrhosis, or suspicious individuals who need to be diagnosed with HCC. In one embodiment, the method is used to determine whether a subject, such as a liver disease-related condition, such as abdominal pain, non-liver, ascites, jaundice, muscle weakness, hepatitis (e.g., HCV infection), or esophageal varices, May be performed on a subject having other symptoms.

In other embodiments, it is a subject that does not exhibit normal HCC symptoms in appearance. After obtaining the results using the markers, the results of comparison with the control group are used to determine whether or not the sample is liver cancer. As a control group or a comparative group, a negative control sample or a sample derived from a patient who was treated after hepatocellular carcinoma, a positive control sample, a patient-derived sample judged to be liver cancer by a method other than the marker according to the present invention, It may be a sample of the originating patient.

In one embodiment according to the present invention, a sample derived from a healthy person, a sample derived from a patient who has been treated with a liver cancer, is used as a control and used for diagnosis.

In another embodiment according to the present application, as a control group, a sample derived from a normal person, a sample derived from a hepatitis patient, a sample derived from a liver cirrhosis patient and / or a patient sample recovered after the treatment of liver cancer are used for diagnosis. For example, according to the method according to the present invention, the PIVKAII concentration in a sample derived from a healthy person, a sample derived from a hepatitis patient, a sample derived from a liver cirrhosis patient, and a sample of a liver cancer patient is determined by the degree of disease progression (normal group - hepatitis - cirrhosis ), It is possible to perform early diagnosis of liver cancer by comparing the values of one or more control samples with those of detection target samples.

In another embodiment, the method can be performed several times over a period of time, for example over a period of time, and can be used to monitor changes in the pattern of expression. An increase or decrease in the expression of the marker may be associated with liver disease status. Can be used to judge the onset, progression, exacerbation or alleviation of HCC in comparison with the previous test value for the same subject or the value of the control group. Prophylactic measures can be taken to prevent progression to liver cancer or severe liver cancer based on changes in PIVKAII over time.

For confirmation of liver disease, particularly liver cancer, the method according to the present invention may be used in conjunction with, for example, AFP testing, ultrasound, computerized axial tomography (CT scan) or magnetic resonance imaging (MRI) .

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Materials and Methods

Materials and reagents

HPLC grade acetonitrile (ACN), water and formic acid (FA) were purchased from Loughborough, U.K. Ammonium bicarbonate (ABC) solution was purchased from iNtRON Biotechnology (Sungnam, Korea). Dithiothreitol (DTT) was purchased from Amesco (Solon, OH, USA). Iodoacetamide (IAA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapigest surfactant (SF) was purchased from Waters (Milford, MA, USA). Sequencing grade chymotrypsin was purchased from Promega (Madison, Wis., USA).

Clinical sample

Human serum samples were approved by Seoul National University Hospital Clinical Trials Advisory Committee (approval No. 0506-150-005). The sample provider was recruited and the consent form was obtained. Clinical characteristics of the donor patient are listed in Table 2. A total of 250 patients were enrolled from January 2007 to April 2014. 50 healthy volunteers were recruited as normal controls. All subjects in the normal control group were confirmed to have not undergone any cancer diagnosis. The chronic hepatitis group was diagnosed to be infected with hepatitis B virus (HBV) if it was detected as positive for the hepatitis B surface antigen in the serum test. Fifty patients were diagnosed as cirrhosis by clinical examination of histologic examination or hepatic hypertrophy and appropriate imaging tests were performed on cirrhotic patients to rule out registered HCC within 6 months. Based on the histological results or the typical image characteristics defined in the guidelines of the Korean Liver Cancer Society, 50 patients were diagnosed as liver cancer.

All HCC patients who successfully received Locoregional therapy were also enrolled and their serum samples collected and classified as HCC (HCC) and post-treatment (recovery) groups.

PIVKAII  synthesis Peptides  design

Stable isotope-labeled internal standard (SIS) peptides (heavy peptides) for PIVKAII labeled with tyrosine (Tyr, Y) [( ¹³ C) ₉ H _{9 (} ¹⁵ N) O ₂ , Δmass = +10 amu] And non-labeled corresponding peptides (light peptides) were synthesized in a 21 ^st biochemical (Marlborough, MA). The purity measured by capillary zone electrophoresis was ≥ 98%; Thus, the peak area of the peptides was not accurate for measuring purity. The peptides were solubilized according to the manual and their expected monoisotopic mass was determined.

Serum sample preparation and MRM -MS analysis

Serum samples were subjected to reversed-phase liquid chromatography after statistical randomization and immunodepletion, denaturation, chymotrypsin digestion, and desalting. All MRM-MS analyzes were performed using a Agilent 6490 triple quadrupole (QqQ) mass spectrometer coupled to a 1260 Capillary LC system (Agilent Technologies, Santa Clara, Calif.) With two pumps and equipped with a Jetstream electrospray source. A reversed-phase guard column (12.5 mm x 2.1 mm id, 5.0 mm) containing mobile phase A [water 0.1% (v / v) formic acid] and B [acetonitrile 0.1% (v / v) formic acid] lt; / RTI &gt; particle size, Agilent Zorbax Eclipse plus-C18). An analytical column (35.0 mm x 0.5 mm id, 3.5 μm particle size) containing mobile phase A [water 0.1% (v / v) formic acid] and B [acetonitrile 0.1% (v / v) formic acid] , Agilent Zorbax SB-C18). Ten microliters of the chymotrypsin digest was injected into the column and separated using a binary gradient. All MRM-MS raw data files were imported and aligned using Skyline software (McCoss Lab, University of Washington, USA) to quantify features.

Data Analysis

The raw data was processed using a Skyline (McCoss Lab, University of Washington, USA) to visually inspect MRM-MS traces and calculate the peak area of transitions. The Savitzky-Golay method was applied to smoothen data points (smoothen). The relative concentration (abundance) of the subject peptides between saliva was compared using a normalized peak area for internal standard peptide (endogenous peptide / corresponding stable isotope-labeled synthesis (SIS) peptide).

To determine the predictive ability of the PIVKAII peptide, T-test and Area under the receiver operating characteristic (AUROC) values were generated. Give MedCalc. 12.7 (MedCalc, Mariakerke, Belgium) and Graph Pad Prism ver. 6.0 (Graph PAD, San Diego, USA).

Example 1. Selection of candidate surrogate peptides representing PIVKAII through modeling

To develop the optimal MRM-MS method for detection and quantification of PIVKAII, the first step was to define the surrogate peptides for quantitative analysis. In the steady state, 10 Glu residues at positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 32 of the N-terminal Gla domain were gamma-carboxylated by gamma-glutamylcarboxylase , Prothrombin is converted to the active form (Fig. 1A). However, in HCC patients, γ-glutamylcarboxylicase is damaged, so that 10 Glu residues can not be completely γ-carboxylated with Gla. It is known that PIVKAII mutations that are preferentially synthesized in HCC patients have less than 4 Gla, whereas PIVKAII mutations in positive epilepsy have more than 5 Gla (Naraki, T .; Kohno, N .; Saito, H. ; Fujimoto, Y .; Ohhira, M .; Morita, T .; Kohgo, Y. Biochimica et biophysica acta 2002, 1586 , 287-298). Here, we attempted to select the most representative surrogate peptide containing Glu residues in the Gla domain to diagnose HCC patients. First, the protein hydrolysis (proteolytic) chyeon digest was performed on the in silico (in silico) to import the entire sequence of prothrombin to Skyline. Skyline supports various enzymes and is analyzed using trypsin and chymotrypsin. As a result, two trypsin-degrading peptides, namely ANTFLEEVR and ECVEETCSYEEAFEALESSTATDVFWAK and three chymotrypsin digesting peptides, EEVRKGNL, ERECVEETCSY, and ESSTATDVF, which harbor the Glu residue in the Gla domain were selected and further analyzed as in the following examples 1b).

Example  2. PIVKAII  A representative object Peptides  (Surrogate Peptide)

In Example 1, three peptides containing Glu in the N-terminal Gla domain were obtained from chymotrypsin digestion digestion and two peptides from trypsin digested digestion were obtained (Fig. 1B). Against chymotrypsin and trypsin degradation decomposition digest five peptides selected from chyeon, gamma-carboxylation of the form was modeled in silico by using the Skyline program. As a result, for the trypsin-degrading peptide and the chymotrypsin-degrading peptide, 68 and 21 combinations were obtained in which each Gla was changed to Glu in the N-terminal Gla domain. MRM-MS analysis was performed targeting 89 potentially all possible peptide combinations that converted Gla to Glu using three pre-determined serum levels of PIVKAII from immunoassays, respectively (high, medium and low levels) Trypsin digestion and digestion of chymotrypsin digesta 68 and 21 combinations, respectively). As a result, none of the 68 modified trypsin degrading peptides showed noticeable intensity signals at the predicted mass values. This is because the four modeled peptides from the ANTFLEEVR sequence have ragged ends (-RR and -RK) that lower trypsin efficiency, whereas the 64 modeled peptides from the ECVEETCSYEEAFEALESSTATDVFWAK sequence have very long and hydrophobic sequences. Finally, only ERECVEETCSY, which is the only chymotrypsin digesting peptide from MRM-MS, was detected, and none of the Glu residues were converted to Gla (Fig. 1B). The intensity of the peptide with the three different levels of patient serum PIVKAII was found to be very consistent (data not shown). Taken together, the chymotrypsin digesting peptide (ERECVEETCSY) selected herein satisfies the following criteria: (1) the length is comprised between 6 and 30 residues; (2) the mass is greater than 800 m / z (to facilitate detection by electrospray ionization-MS); (3) No Met residue (to avoid chemical transformation). In addition, the sequence of the chymotrypsin-degrading peptide was found only in PIVKAII in the human protein database as a result of the Uniprot / Swiss-Prot database search. As a result, ERECVEETCSY of PIVKAII was determined as the target peptide for quantifying the level of PIVKAII in human blood.

As a result of the transition transition (Q1 / Q3) of the target peptide, it was confirmed that the Q1 value was 10.0 Da and the Q3 value was the same between the target peptide and the standard internal substance (peptide and heavy-labeled synthetic peptide. In order to select the CE (collision energy) value, the CE value which is the highest peak area value was selected by repeatedly analyzing the CE value of 11 points from the default CE value backward and backward by 2 points As a result of the analysis of the selected CE value, a comparison of the intensity of the PIVKAII with that of the default value was carried out. The MRM-MS analysis parameters for the determination of PIVKAII are as shown in Table 1 above.

Example 3 Determination of LOD and LOQ of Human Serum PIVKAII Peptide Calibration Curve

To confirm quantification, an experiment was conducted to confirm the linearity of the heavy-labeled synthetic peptide (internal standard) for the target peptide. First, the LOD (limit of detection) and the LOQ (limit of quantification) of the selected PIVKAII peptide were determined. For this purpose, reverse-calibrated curves were generated using digested pooled serum. (Light peptide, 7.81 nM) and a heavy peptide prepared by serial dilution with 0.08 nM to 1000.00 nM (10 ⁵ -fold difference) of a constant concentration of the target peptide determined in Example 2 (light peptide, 7.81 nM) To produce a reverse calibration curve.

LC-MRM analysis was performed as follows. Liquid chromatography (LC) was performed using Agilent's 1260 capillary LC system and peptide separation was performed with 2-Dimension (2D). Agilent Zorbax Eclipse plus-C18 (12.5 mm × 2.1 mm, 5 μm) was used as a guard column and Agilent Zorbax Eclipse plus-C18 (35.0 mm × 0.5 mm, 3.5 μm) was used as an analytical column. 10.0 μL of the peptide sample was injected into the analytical column through the guard column, and the flow rate was 40 L / min. Columns were eluted with SolB (0.1% formic acid in acetonitrile) for 5 min, 10% to 60%, and 90% gradient for 1 min. Mass spectrometry was monitored in MRM mode for the transition selected in Example 2 using a 6490-Triple quadrupole (QQQ) instrument from Agilent Technologies. Gas temperature was 250 ℃, gas flow was 15 L / min, nebulizer was 30 psi, sheath gas temperature was 350 ℃ and sheath gas flow was 12 L / min. The capillary voltage and the nozzle voltage were 2500 V and 2000 V, respectively. The resolution in Quadruple 1 (Q1) and Quadruple 3 (Q3) was set as a unit, and the unscheduled MRM method was used. The dwell time was set so that the total cycle time was about 2 sec. Based on this, we performed 3 repeated analyzes with scheduled MRM for window size 1 min.

All calibration points were analyzed in quintiles. To perform the linear regression analysis, the peak area ratio of the heavy peptide to the light peptide was plotted against the peptide concentration. All five assays showed good accuracy over a concentration of 10 ⁵ fold difference over the four transitions in Table 1, indicating that PIVKAII can be quantified without interference from endogenous proteins 2a).

In addition, we confirmed that the heavy-labeled synthetic peptide and the light-synthesized peptide, the internal standard, co-elution at the same time, and the intensity of six product ion types was also confirmed. In all four product ions, 0.24nM - -labeled synthetic peptide (R ² > 0.99) (Fig. 2B).

The LOD was obtained by multiplying the average value of the peak area ratio of the blank sample by three times the standard deviation. The LOD for four transitions was confirmed to be 304.21 - 360.16 fmol / mL. The LOQ was obtained by multiplying the average value of the peak area ratio of the blank by 10 times the standard deviation. The LOQ for 4 transitions was 632.41 - 820.78 fmol / mL. At this time, the measurement (n = 5) coefficient of variation (CVs) exceeding LOQ for the 5th iteration analysis was less than 20%. For quantification, b8 ions with LOD and LOQ of 0.30 nM and 0.63 nM, respectively, were selected (Fig. 2B). The b8 ion had no interference, comparable detection limit, and linearity (R ^ 2 = 0.9842) within the quantifiable range as compared with the internal reference material among the four Q3 ions.

Example  4. Clinical samples MRM -MS analysis

To verify the efficacy of the PIVKAII-based diagnosis of liver disease using the MRM-MS method of the present invention, the following analysis was performed on the clinical cohort. The purpose of this study was to select the normal group, hepatitis group, and cirrhosis group to be applied to the early diagnosis and to select 50 patients who were paired with liver cancer patients to confirm the reduction of PIVKAII level after treatment. (N = 50), chronic hepatitis (n = 50), liver cirrhosis (n = 50), liver cancer HCC (n = 50) and recovery A total of 250 serum samples were collected. The clinical characteristics of the samples are shown in Table 2 below.

[Table 2]

The experiment was carried out as follows. Six of the high-density proteins in the blood samples were depleted using the 6-multiple affinity removal system (MARS), Agilent). Serum protein was prepared by enriching the samples (Amicon, 3K) and the concentration was determined by biocinchoninic acid (BCA) analysis. Then, 0.1% Rapigest (Waters) / 10 mM DTT was treated and reacted at 60 ° C for 60 minutes. IAA (Iodoacetamine) was then treated to a final concentration of 20 mM and then reacted at room temperature for 30 min. After the treatment of chymotrypsin with 1: 100 (w / w), the reaction was carried out at 37 ° C for 4 hours. Then, formic acid was treated to a final concentration of 1.0%, centrifuged at 15,000 rpm for 1 hour, and the supernatant was taken. Then, the heavy-labeled peptide was added to 7.81 fmol / did.

The order of the MRM analysis of each of the samples was determined randomly for the five groups. Each sample was analyzed three times, and the data obtained through this analysis were imported into the skyline software and converted to the peak area value of the peptide transfer. The peak area value was normalized to an area value of a heavy-labeled peptide as an internal standard substance (see FIG. 6).

The concentration of PIVKAII was calculated by MRM-MS analysis using the calibration curve of FIG. 2B and the peak area ratio for each sample.

PIVKAII expression was significantly elevated in the HCC group when compared to the normal control (P <0.0001), the chronic hepatitis group (P <0.0001) and the cirrhotic group (P <0.0001) In order to confirm the significant difference in PIVKAII between groups, T-test results showed that there was a significant difference in p value <0.0001 when compared with HCC group. In the HCC group and the recovery group, p value <0.05 and HCC vs. The AUC of 0.706 in the recovery group was significantly higher than that in the recovery group and the liver cancer group. The results showed that the PIVKAII concentration was significantly increased as the disease progressed from the normal group to the chronic hepatitis and cirrhosis HCC group.

To determine the effectiveness of the diagnostic method, the receiver-operating characteristic (ROC) curve was drawn for individual samples analyzed to confirm the Area Under Curve (AUC) value. As a result, In normal control, the sensitivity was 74.0% and the specificity was 86.0%, confirming that the AUC value was 0.870. In the comparison of chronic hepatitis, the sensitivity was 66.0% and the specificity was 76.0%. In comparison of liver cirrhosis, sensitivity was 78.0% and specificity was 62.0%. In comparison of recovery, sensitivity was 66.0% and specificity was 72.0%, and the AUC value was 0.706 (Fig. 3B). In this cohort, PIVKAII was significantly elevated in HCC patients as compared with the early stage patients. These results indicate that MRM-MS analysis is useful for the determination of PIVKAII concentration in patient serum and diagnosis of liver disease based thereon.

Example  5. MRM - Comparison of MS and immunoassay

The present invention is for the first PIVKAII measurement without immunoassay for clinical cohort samples. The present inventors measured the PIVKAII concentration measured by MRM-MS using the same serum sample composed of the HCC group (n = 50) and the recovery group (n = 50) by the immunoassay to compare the MRM- - analysis. In an immunoassay with an AUROC of 0.764, PIVKAII showed 82.0% sensitivity and 64.0% specificity, and AUROC analysis (Figure 4A) showed 66.0% sensitivity and 72.0% specificity in MRM-MS analysis with AUROC of 0.706. The AUROC values obtained from both methods were not significantly different based on Delong'test ( P = 0.1681). A method comparative analysis was also conducted by constructing a Deming Regression Plat and a Bland-Altman Platto to determine the difference. Deming regression plots showed a slope and slope of 95% confidence limits, and a high correlation of 0.912 R ² between immunoassay and MRM-MS (FIG. 4B). The Bland-Altman plot showed an associated 95% confidence interval and a limit of agreement of +/- 1.96 SD, with a difference between the two corresponding measurements plated compared to the measured mean (FIG. 4C). Interestingly, the PIVKAII concentration measured by the MRM-MS method was almost 2.24 nM (log 2.24 = 0.35), which was much lower than that obtained by immunoassay.

In conclusion, 50 patients in the HCC group and 50 patients in the recovery group were examined by immunoassay for the relationship between the PIVKAII protein concentration and the PIVKAII peptide measured by MRM. In the case of the chymotryptic peptide ERECVEETCSY, the R ^ 2 value was found to be 0.912, indicating that there was a strong positive correlation between the immunoassay data and MRM data for the PIVKAII protein.

PIVKAII is a cancer-associated protein secreted by HCC and requires a robust assay that is sensitive to its utility in the diagnosis of HCC. PIVKAII measurement has been performed by immunoassay to date. The inventor first developed a sensitive and accurate platform based on the MRM-MS method to quantify PIVKAII at the nM level in human serum samples. In the analysis based on MRM-MS, PIVKAII is measured in the serum under denaturing conditions, whereas in the antigen-based assay, PIVKAII is measured based on antibody binding efficiency and epitope availability. It was possible to quantify PIVKAII at the nM level (LOD = 0.30 nM, LOQ = 0.63 nM). Furthermore, the correlation of PIVKAII concentrations between MRM-MS and immunoassay for 100 clinical samples (50 HCC and 50 recovery groups) was good (R = 0.912), suggesting that MRM-MS analysis is reliable It is to prove that it can be. Taken together, the MRM-MS method can serve as a complementary measure to PIVKAII. Furthermore, MRM-MS can be an alternative technique for antibody-based immunoassays, particularly where high-quality antibodies can not be obtained for sensitive immunoassays.

In sum, The method according to the invention is schematically shown in Fig. After digestion with chymotrypsin using in silico digestion method, target peptide was selected and it was confirmed that there is a correlation between the concentration of the clinical sample and the peak intensity. As a result of 5 times repeated analysis with optimal conditions developed by optmization of the parameters required for MRM-MS, it was confirmed that linearity was observed at 0.24nM to 1000.00nM in all four selected transitions, and CV (%) value Were all within 20%. This indicates that the method can quantify the PIVKAII concentration range in a clinical sample.

The AUC values of the HCC group and the other groups were compared with those of the normal group, chronic hepaititis, and liver cirrhosis, and the AUC value decreased as the disease progressed. P value < 0.0001). In contrast to the immunoassay results, the R ^ 2 value between the two assays was as high as 0.912, indicating that the MRM-MS assay according to the present invention can discriminate patients with liver cancer (AUC = 0.706) .

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (9)

In order to provide information necessary for diagnosis or prognosis of liver cancer,
Providing a sample requiring diagnosis of liver disease;
Treating the specimen with a protease to cleave the protein contained in the specimen;
Mixing the internal standard material with the specimen;
Performing multiple reaction monitoring (MRM) analysis on the subject peptide of the PIVKAII protein and an internal standard substance in the mixture,
Detecting the amount of PIVKAII in the test sample by normalizing the result of the MRM analysis of the target peptide with the result of analysis of the internal standard material; And
And comparing the detection result with a result of a control sample,
The subject peptide is ERECVEETCSY,
Wherein the internal standard material has the same amino acid sequence as the subject peptide but the mass of one or more amino acids constituting the internal standard material is different from the amino acid mass of the subject peptide.
The method according to claim 1,
Wherein the specimen is plasma, serum or whole blood.
The method according to claim 1,
The control group is a normal sample, a chronic hepatitis sample, or a liver cirrhosis sample,
Further comprising the step of determining the specimen as liver cancer when the amount of PIVKAII in the specimen is increased as compared with the control group.
The method according to claim 1,
Wherein the step of determining the amount is determined using a standard concentration curve with a sample containing a known concentration of PIVKAII subject peptide.
2. The method of claim 1, wherein the MRM assay monitors the value of the progenitor peptide ion Q1, the product peptide ion Q3, and the optimal collision energy (CE) value for the Q1 / Q3 is used.
6. The method of claim 5,
Wherein the Q1 and Q3 values of the MRM analysis for the subject material and the internal reference material, the default CE for the Q1 and Q3, and the optimum CE value are as shown in the following table.

The method according to claim 1,
Wherein said proteolytic enzyme is a neutral proteolytic enzyme that acts at neutral pH.
8. The method of claim 7,
Wherein said neutral protease comprises at least one of chymotrypsin, trypsin, Lyc-C, Asp-N or Glu-c.
The method according to claim 1,
Wherein the prognostic measure comprises determining whether the liver cancer has recovered after treatment.

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