JP2009210355A - Lung adenocarcinoma convalescence diagnostic marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of estimating a recurrence risk during the convalescence of lung adenocarcinoma by detecting protein related to the recurrence of the lung adenocarcinoma, and a kit used for this estimation method. <P>SOLUTION: The method of estimating the recurrence of lung adenocarcinoma includes: a step (step A) of measuring the expressed amount of a modified body of at least one protein selected from a group comprising delta 3,5-delta 2,4-dienoyl-CoA isomerase, annexin A2, aldehyde dehydrogenase, peptidyl-prolylcis-trans isomerase A, inorganic pyrophosphatase 2, tropomyosin beta chain, proteasome subunit beta type 2, and an elongation factor 2, as to a bio-specimen deriving from an inspected person; a step (step B) of comparing the expressed amount of the protein as to the bio-specimen deriving from the inspected person with a reference value; and a step (step C) of determining that the inspected person has a high recurrence risk of lung adenocarcinoma when variations are found through the comparison of the expressed amount. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for predicting recurrence of lung adenocarcinoma. More specifically, the present invention relates to a method for predicting recurrence of lung adenocarcinoma including a step of detecting a protein associated with recurrence of lung adenocarcinoma, and a kit for predicting recurrence of lung adenocarcinoma containing an antibody of the protein.

  About 50,000 lung cancers die annually in Japan, and the number of cancer deaths is the first in men and second in women after stomach cancer, and is a very difficult disease to cure completely. Lung cancer is roughly classified into small cell cancer and non-small cell cancer, and the latter is further classified into tissue types such as adenocarcinoma (hereinafter referred to as lung adenocarcinoma), squamous cell carcinoma, large cell carcinoma, and adenosquamous carcinoma. Is done. Of these, the most common case is lung adenocarcinoma, but in general this type of cancer is particularly recurrent, and even if surgical treatment is performed in the early stage I, the lung or other It is characterized by metastasis to organs and death in many cases. Therefore, in order to improve the treatment results of lung cancer, it is very important to determine good or bad prognosis in lung adenocarcinoma patients.

  Diagnosis of lung adenocarcinoma in the clinic is mainly for biopsy by collecting lesion tissue in bronchoscopy for lung cancer discovered by methods centered on diagnostic imaging (chest X-ray examination, CT examination, PET examination, etc.) Or it is common to examine a tissue type etc. in detail by puncture aspiration cytodiagnosis. Moreover, CEA, SLX, CA-125 and the like are currently used clinically as tumor markers for in vitro diagnostic agents using serum as a sample. However, these markers are not sufficient for specificity and sensitivity to lung cancer. In addition, even though it can be diagnosed clinically as lung adenocarcinoma, it is impossible to obtain information predicting the prognosis after surgical resection, which is most important for formulating a treatment strategy.

With the completion of decoding of the human genome sequence, research on disease proteomics has been actively conducted based on the database revealed here. In this study, for example, by producing expression profiles of many proteins produced in the human body and comparing a plurality of profiles, it is possible to find a protein whose expression varies specifically in a certain state. By using this proteomic analysis, it is considered possible to search for a marker that varies specifically depending on a disease and a disease-related factor that is a target of drug discovery. Therefore, in addition to using immunoassay methods such as Western blotting and immunoassay as protein profiling methods (see Patent Document 1), two-dimensional electrophoresis, SELDI, liquid chromatography and mass spectrometry are combined. An LC-MS method and the like have been developed and are currently used for disease analysis (see Patent Document 2).
WO01 / 011372 pamphlet JP 2006-53113 A

  However, in previous studies, for example, using serum or tissue as a sample, searching for tumor markers comparing healthy individuals with cancer patients, or after processing the sample protein with a protease in advance to make a digested fragment peptide However, most of the reports have been analyzed by high-sensitivity LC-MS method, and there are few reports on proteomic analysis of proteins including post-translational modifications and search for proteins related to clinical prognosis. No effective candidate protein has been found.

  An object of the present invention is to provide a method for predicting a recurrence risk in prognosis of lung adenocarcinoma by detecting a protein involved in recurrence of lung adenocarcinoma, and a kit used for the prediction method.

  As a result of repeated studies to solve the above-mentioned problems, the present inventors have used a fluorescence difference gel two-dimensional electrophoresis method (2D-DIGE method) capable of producing a protein profile including post-translational modification. By analyzing samples derived from patients with early lung adenocarcinoma and a good or bad prognosis, multiple prognostic marker proteins whose expression levels fluctuate depending on whether the lung adenocarcinoma is good or bad The headings and many of these proteins have been found together with specific modifications that have been post-translationally modified, and the present invention has been completed.

That is, the present invention
(1) Delta 3,5-delta 2,4-dienoyl-CoA isomerase (Annexin A2), aldehyde dehydrogenase in biological samples derived from subjects (Aldehyde dehydrogenase), Peptidyl-prolyl cis-trans isomerase A, Inorganic pyrophosphatase 2, Trotropyosin beta chain, Proteasome subunit beta type 2 (Proteasome subunit beta type 2) and a step of measuring the expression level of a modified form of at least one protein selected from the group consisting of elongation factor 2 (step A), a biological sample derived from the subject A step of comparing the expression level of the protein with a reference value (step B), and A method for predicting recurrence of lung adenocarcinoma, comprising a step of determining that the risk of recurrence of lung adenocarcinoma is high when the variation is observed by comparison (step C),
(2) A kit for predicting recurrence of lung adenocarcinoma, comprising an antibody against a modified form of the protein,
About.

  By detecting the prognostic marker protein in a sample by the method of the present invention, the level of recurrence risk of lung adenocarcinoma of the sample provider can be determined. Based on this risk, the sample provider can follow up accurately after surgery, and improvement in clinical outcomes of lung adenocarcinoma can be expected.

  The present invention predicts recurrence of lung adenocarcinoma including a step of measuring the expression level of a protein (step A) and a step of comparing the measured expression level between a control subject and a subject (step B). The method is characterized in that the protein is a specific modified protein. Such a protein was found by analyzing the difference in the expression level of patients diagnosed with early lung adenocarcinoma and having a good prognosis and a poor prognosis. By using the found protein, it becomes possible to predict the recurrence risk of lung adenocarcinoma. In the present specification, “good prognosis” means a state in which no recurrence cancer originated from lung adenocarcinoma is observed for more than 4 years after excision of cancer tissue, and “prognosis is poor”. This refers to a state in which recurrent cancer originating from lung adenocarcinoma is observed within 4 years after excision of cancer tissue.

<Prognostic marker protein associated with recurrence of lung adenocarcinoma>
Prognostic marker proteins related to recurrence of lung adenocarcinoma used in the present invention include Annexin A2 (Annexin A2), Aldehyde dehydrogenase, Peptidyl-prolyl cis-trans isomerase A (Peptidyl-prolyl cis-) shown in Table 1. trans isomerase A), Delta 3,5-delta 2,4-dienoyl-CoA isomerase, Inorganic pyrophosphatase 2, Trotropyosin beta chain (Tropomyosin) It is a modified form of a protein selected from beta chain), proteasome subunit beta type 2 (Proteasome subunit beta type 2), and elongation factor 2 (Elongation factor 2). These proteins were found to be associated with the prognosis of lung adenocarcinoma, not the unmodified form, and the expression level of the modified form. Individual identification of such modified proteins can be performed according to a known protein identification method using a MALDI (matrix-assisted laser desorption ionization) mass spectrometer, an ESI (electrospray ionization) mass spectrometer, or the like. By referring to known databases such as NCBI and SWISS Prot, information such as the molecular weight, isoelectric point, and constituent amino acids of the protein can be obtained. Among these proteins, delta 3,5-delta 2,4-dienoyl-CoA isomerase or annexin A2 is particularly preferred as a marker. Moreover, as a marker, although it does not specifically limit among the protein groups shown in Table 1, a several protein can also be selected by the combination which optimizes the accuracy of prognosis prediction of lung adenocarcinoma.

  The mode of modification in the modified protein in the present invention is not particularly limited, but post-translational modifications such as phosphorylation, glycosylation, acetylation, hydroxylation, amidation, etc. are preferred. It is preferable that the isoelectric point deviates from the theoretical value as compared with the protein. The degree of isoelectric point divergence is preferably shifted 0.1 or more to the acidic side or basic side, more preferably 0.5 or more.

  The protein shown in Table 2 is different from Table 1 in that the expression level of the protein that is not assumed to be a modified form is related to the prognosis of lung adenocarcinoma, in which the molecular weight and isoelectric point both agree with the theoretical value of the unmodified form. It has been found. Among these proteins, mitochondrial inner membrane protein is particularly preferable as a marker.

  In Tables 1 and 2, “Protein name” means a general protein name. “Change” is a change in a poor prognosis case with respect to the corresponding protein expression level in a good prognosis case. The magnification of the change is shown in the “Ratio” column. The “Swiss Prot” and “UniProt” columns in the “Accession No.” column are registered IDs registered in the Swiss Prot and UniProt databases. In addition, “Acidic form” and “Basic form” in “Detection on 2D-DIGE gel” in Table 1 mean that the respective isoelectric points are shifted to the acidic side and the basic side. “MW form” and “Low MW form” mean that the respective molecular weights are shifted to high molecular weight and low molecular weight. These are changes resulting from post-translational modification due to the addition of some modifying group or the disappearance of a specific amino acid sequence.

  In the present invention, the recurrence of the lung adenocarcinoma of the subject is predicted using the expression level of the modified protein, which is the prognostic marker protein, as an index. That is, the method for predicting recurrence of lung adenocarcinoma of the present invention comprises a step of measuring the expression level of at least one modified protein shown in Table 1 in a biological sample derived from a subject (step A), A step of comparing the expression level of the protein in the biological sample with the expression level in the control person (step B), and when the variation is recognized by the comparison of the expression level, the subject is determined to have a high risk of recurrence of lung adenocarcinoma The process (process C) to perform is included.

<Method for measuring protein expression level associated with recurrence of lung adenocarcinoma>
First, a biological sample derived from a subject is collected, and a cell extract is extracted from the sample. The biological sample is not particularly limited, but is preferably one selected from the group consisting of lung tissue, cells, body fluids, urine, and other biological protein extracts. In addition, the collection method of a biological sample and the cell extraction method are not particularly limited, and can be performed according to a known method.

  Next, the expression level of the modified protein in the obtained cell extract is measured. The method for measuring the protein expression level is not particularly limited as long as the protein to be measured is shown in Table 1. For example, an antibody capable of specifically recognizing the protein to be measured can be used. Such antibodies can be prepared by methods well known to those skilled in the art. However, since many modified proteins can be detected with high sensitivity using different fluorescent labels, it is preferable to use the 2D-DIGE method. In addition, the protein to be measured may be at least one of the proteins shown in Table 1, and since it is highly likely to be present in a body fluid, delta 3,5-delta 2,4-dienoyl- CoA isomerase or annexin A2 is preferred.

  The 2D-DIGE method is not particularly limited as long as it is a method that separates from two physical properties of a protein such as isoelectric point and molecular weight. Generally, first, a capillary gel or a commercially available strip is used. Isoelectric focusing is performed using a gel as a separation medium, and the gel after electrophoresis is electrophoresed in a direction perpendicular to the development direction of isoelectric focusing by a second planar SDS-polyacrylamide gel. It is a method to do. Since the isoelectric point and molecular weight of the protein are clarified by such a method, it becomes clear that post-translational modifications such as phosphorylation, sugar chain addition, acetylation, hydroxylation, amidation and the like have been performed.

<Comparison of protein expression level related to recurrence of lung adenocarcinoma / Determination of recurrence risk>
With respect to the expression level of the protein to be measured in the sample obtained as described above, the value calculated from the expression level of the protein to be measured in the biological sample of the control person is set as a “reference value”, and the protein in the biological sample of the subject Compared to the expression level. When a change is recognized by comparison of the expression levels, that is, when it is higher or lower than the reference value, it is determined that the risk of recurrence of lung adenocarcinoma is high. In this case, the “control person” is a healthy person, as well as a patient who has been diagnosed with early lung adenocarcinoma (stage is stage 1A or 1B) and whose lung adenocarcinoma tissue has been removed, and has a good prognosis. Some patients are also included.

<Kit for predicting recurrence of lung adenocarcinoma>
In another aspect of the present invention, a kit for predicting recurrence of lung adenocarcinoma is provided.

  The kit of the present invention includes all kits that can detect the expression level of the modified protein shown in Table 1. Specifically, a kit containing an antibody capable of recognizing the modified protein shown in Table 1 can be mentioned. If the detection method uses an antibody when detecting a protein in a sample (for example, ELISA, etc.), Kits can be used. In addition, if the modified protein shown in Table 1 is detected, proteins other than the modified protein shown in Table 1 may be simultaneously detected by the antibody. It can also be used for primary screening for predicting recurrence of lung adenocarcinoma.

  Using the kit of the present invention, the expression level of the protein to be measured related to the recurrence of lung adenocarcinoma present in the sample of the control subject and the subject was measured, and a significant difference occurred between the expression levels of the two In some cases, the future risk of recurrence of lung adenocarcinoma in the subject can be predicted, and diagnosis of recurrence of lung adenocarcinoma in those who are suspected of recurrence of lung adenocarcinoma can be performed.

EXAMPLES The present invention will be described below with reference to examples. However, the examples are provided for better understanding of the present invention, and the scope of the present invention is intended to be limited to these examples. It is not a thing.

<Sample>
Samples were lung adenocarcinoma surgically excised for treatment from 16 patients diagnosed as early stage lung adenocarcinoma (stage is stage 1A or stage 1B) by diagnostic imaging and histopathology The tissue of was used. As for the groupings of good prognosis and bad prognosis, cases in which no recurrence cancer originated from lung adenocarcinoma was observed after a clinical course follow-up for 4 years or more after cancer tissue resection were considered good prognosis. By this grouping, 16 patients had 12 cases with good prognosis and 4 cases with poor prognosis. In addition, the excised tissue was frozen immediately after collecting a portion including a lesion for analysis, and stored at −80 ° C. until analysis was performed.

<Sample preparation>
About 5 mg of frozen lung adenocarcinoma tissue is collected in a frozen state and washed with tissue washing solution (protease inhibitor cocktail Complete Mini (Roche Diagnostics) 1 tablet / 10 mL (1 × concentration)) and Pefabloc SC. Thawed in 200 μL of 10 mmol / L sodium phosphate buffer pH 7.4 (hereinafter referred to as PBS) containing 5 μL / mL protector (Roche Diagnostics). The tissue pieces in the solution were cut into small pieces while cooling on an ice bath using surgical scissors, then stirred for 1 minute with a vortex mixer, centrifuged at 14,000 × g for 3 minutes at 4 ° C., and the supernatant was removed. . To the precipitated minced tissue, 200 μL of the tissue washing solution was further added, stirred for 30 seconds with a vortex mixer, centrifuged at 4 ° C. and 14,000 × g for 3 minutes, and the supernatant was removed. In this way, the precipitate of minced tissue from which contaminants such as serum components contained in the tissue have been removed is added to the lysis buffer (30 mmol / L Tris-HCl, 2 mol / L thiourea, 7 mol / L urea, 4% CHAPS, 1 × Concentration Complete Mini, 5 μL / mL Pefabloc SC protector, pH 8.0) 200 μL was added, and processing was performed according to the same instructions using Sample Grinding Kit (manufactured by GE Healthcare Bioscience) to prepare a cell extract. . The concentration of the protein contained in the cell extract was quantified by the Bradford method using Advanced protein assay reagent (Cytoskeleton). At this time, Protein Assay Kit I (Bio-Rad Laboratories) using bovine gamma-globulin as a standard protein was used.

<Two-dimensional electrophoresis>
The expression profile of the protein contained in the extract was performed using a fluorescence difference gel electrophoresis apparatus (2D-DIGE method, manufactured by GE Healthcare Biosciences) using two-dimensional electrophoresis. The protein fluorescent labeling method used was a saturated fluorescent labeling method in which the fluorescent molecule Cy3 or Cy5 was introduced into all reduced cysteine residues. The labeling method and 2D-DIGE method are described in the literature (Jun XY et al., Proteomics 2, 1682-1698 (2002), Joanne S. et al., Proteomics 3, 1181-1195, (2003)). Although the details are described, a schematic diagram of the experimental design of the embodiment of the present invention is shown in FIG. 2, and the details are described below.

  The fluorescent labeling of the protein by the saturated fluorescent labeling method was carried out by designing an experiment so that each of the Cy3 and Cy5 fluorescent labeling samples would be an equal mixture of 5 μg per one two-dimensional electrophoresis gel. About the tissue extraction sample prepared as described above, a volume containing 5 μg of protein was dispensed into a 1.5 mL microtube, and lysis buffer was added thereto to adjust the volume to 9 μL. To this solution, add 1 μL of 2 mmol / L Tris (2-carboxyethyl) -phosphine hydrochloride (PIERCE) solution, perform reduction treatment under light-shielding conditions at 37 ° C. for 1 hour, and then prepare in advance with dimethylformamide (DMF) 2 μL of 2 nmol / μL of Cy3 or Cy5 reagent (GE Healthcare Bioscience) was added and mixed, and a coupling reaction was performed at 37 ° C. for 30 minutes. After that, 2x sample buffer (2% IPG buffer pH3-10NL (GE Healthcare Bioscience), 1x sample buffer containing 130mmol / L dithiothreitol (DTT) (2mol / L thiourea, 7mol / L urea) , 4% CHAPS)) 12 μL was added to stop the reaction, and this was used as a fluorescently labeled sample solution. In the analysis of the 2D-DIGE method, Cy3 labeled protein is used as an internal standard sample. Therefore, the tissue extract of all samples to be analyzed is mixed in the same amount as the protein amount, and Cy5 labeled protein is used for each patient. Each of the derived analysis samples was prepared.

  Samples containing 5 μg of Cy3 and Cy5 labeled protein were mixed, adjusted to a final volume of 450 μL with a swelling buffer (1% sample buffer containing 1% IPG buffer pH3-10NL, 13 mmol / L DTT), and then IPG Pour the same solution into the strip holder, then set Immobiline DryStrip, pH 3-10NL, 24cm (GE Healthcare Bioscience), then use IPGphor (GE Healthcare Bioscience) to Isoelectric focusing (Rehydration: 20 ° C., 12 hours, 500 V for 1 hour, 1,000 V for 1 hour, 6,000 V 90,000 Vhr) was performed under light-shielding conditions.

  Immobiline DryStrip after isoelectric focusing was performed using equilibration buffer (6 mol / L urea, 0.1 mol / L Tris-HCl, 30% glycerol, 2% Sodium Dodecyl Sulfate (SDS), 0.5% DTT, pH 8.0) After soaking in 10 mL and gently stirring for 15 minutes under light-shielding conditions, it was placed on top of a previously prepared 24 × 21 cm SDS-polyacrylamide gel. This gel is a gel consisting only of a 12.5% separated gel monolayer prepared according to the Laemmli method (Laemmli UK, Nature 227, 680-685 (1970)), and the separated fluorescent protein is directly detected as a gel support. It was produced using a low fluorescent glass plate so that it was possible. Immobiline DryStrip was fixed with a 0.75% agarose solution (containing bromophenol blue (BPB) dye), and then electrophoresed in an electrophoresis tank Ettan DALT twelve (GE Healthcare Biosciences). At this time, as a buffer for electrophoresis, 1 × buffer (25 mmol / L Tris hydrochloride, 192 mmol / L glycine, 0.1% SDS) is used for the cathode, and double concentration of the same buffer for the anode. 2 × buffer was used. After setting up to 12 gels for two-dimensional electrophoresis to which samples were applied, electrophoresis was performed at 1.5 ° C / gel for about 16 hours at 20 ° C, and BPB dye was eluted from the lower part (anode side) of the gel By the way, electrophoresis was stopped. The gel after two-dimensional electrophoresis after the gel was taken out from the electrophoresis tank was prepared using Cy3 (excitation wavelength 532 nm / detection wavelength 580 BP30), Cy5 (633 nm / 670 BP30) using a fluorescent scanner Typhoon 9200 (manufactured by GE Healthcare Bioscience). ) Each electrophoresis image was detected. In the analysis by the 2D-DIGE method, four two-dimensional electrophoresis images (4 × 16 samples, total of 64 gel images) were obtained for each sample, and the following analysis was performed.

<Processing of two-dimensional electrophoresis images>
The detected two-dimensional electrophoresis image in the 2D-DIGE method was first subjected to background noise removal processing with ImageQuant TL (GE Healthcare Bioscience) software. Here, the electrophoretic images of all 64 sheets are checked in detail, and the analysis target is 3 sheets for each sample (n = 3, total) except for the gel image of the 4 sheets of each sample, which is the worst one. 48). Next, for all the gel images, the acidic and basic portions in a common range were selected and cut out (Fig. 3), and these images were obtained by using DeCyder (version 6.5, GE Healthcare Bio), software exclusively for 2D-DIG analysis. Forwarded to Science). The removal of the acidic high molecular weight portion of the gel image means that the spots of the cell constituent proteins having a high expression amount that are present in a relatively large amount are effectively removed.

  In the DeCyder software, first, an operation of detecting protein spots from the entire gel image was performed, and for each sample, 2,000 or more spots to be analyzed were obtained by combining the acidic and basic regions. Next, the Cy3 fluorescence detection image of the internal standard sample obtained here was confirmed, and one gel with relatively good separation was selected from these images and set as a master gel. Position correction (matching) was performed with the spots in the Cy3 fluorescence detection image of all gels, using the detection spots in the gel image of the Cy3 fluorescence labeled internal standard sample detected with this master gel as a reference. After matching is completed in all gels, the fluorescence intensity of the spot indicating the concentration of the detected protein is normalized with the fluorescence intensity of the Cy3 internal standard sample from the Cy5 fluorescence image derived from each sample in these gel images. Output as a list of

<Statistical analysis of detection spots>
In order to obtain highly reliable results, spots with a high ratio (appearance) that were not detected between gels were first excluded from the analysis target from the list of standardized fluorescence intensities. The exclusion criteria here are set so that the appearance does not meet 70%. For those spots that have cleared this standard, a total of 1,075 spots with missing values are supplemented with the values generated statistically by the k-nearest-neighbor method (k = 10), and random fores According to the statistical analysis of the Tsu method, the top 30 spots were output that have the ability to significantly distinguish the prognosis good and bad groups that are the clinical background of the original sample. For this extracted spot, the 2D-DIGE gel image is again verified in detail, and the fluorescence intensity is calculated by overlapping with other spots due to insufficient separation. 16 spots that finally identify proteins were narrowed down, excluding spots where reliability was not sufficiently secured, such as those with a large influence of fluorescence intensity.

  Tables 1 and 2 summarize the increase and decrease of each expression level and the fluctuation ratio between these 16 spots in the lung adenocarcinoma prognosis good group and the bad group. The spots extracted here were found to show a difference of 1.2 times or more in the comparison between the groups as the expression level.

<Preparation of protein identification sample>
In the analysis of lung adenocarcinoma tissue by 2D-DIGE method, protein was identified by a method using a mass spectrometer for 16 spots in which expression differences were recognized due to differences in prognosis. Samples for this analysis were prepared by the following in-gel digestion method.

  First, as a sample for protein identification, a lung adenocarcinoma tissue extract containing 500 μg of protein was obtained by referring to the method described in the Ettan DIGE simple manual (DeCyder version 6.0 compatible version, manufactured by GE Healthcare Biosciences). Cy3 fluorescent labeling was performed in the same manner as the saturated fluorescent labeling method. This sample was subjected to two-dimensional electrophoresis in the same manner as described above, and then a Cy3 fluorescence image was obtained by a fluorescence scanner. This two-dimensional electrophoresis image was transferred from the Image Quant TL software to DeCyder, and the position of the spot to be identified on the gel was confirmed with this software, and the position data of each XY coordinate was acquired. Next, the gel is set on an Ettan spot picker (manufactured by GE Healthcare Bioscience), and the gel corresponding to the position of the XY coordinate data is sampled with a diameter of 2.0 mm, and a cylindrical gel with a thickness of 1 mm. Collected as a piece. The gel pieces were washed four times for 10 minutes while shaking vigorously with 100 mmol / L ammonium bicarbonate solution containing 100 μL of 60% acetonitrile, and then further shaken in 100 μL of acetonitrile for 5 minutes. The solution was removed and completely dried with a centrifugal evaporator, and then swollen with a 50 mmol / L ammonium bicarbonate solution containing 20 μL of 0.1% RapiGest SF (manufactured by Waters) at 37 ° C. for 10 minutes. After removing the excess solution and drying again with a centrifugal evaporator, 1 mmol / L Tris-HCl buffer pH 8 containing 200 ng lysyl endopeptidase (Acromobacter protease I (Lys-C), Wako Pure Chemical Industries, Ltd.). 5, 20 μL was added and left on an ice bath for 45 minutes. After removing the solution that did not soak in the gel, 20 μL of 1 mmol / L Tris-HCl buffer, pH 8.5 was further added, and incubated at 37 ° C. for 16 hours or longer. After the enzyme digestion reaction, transfer this solution to a new tube, add 50 μL of 60% acetonitrile solution to the remaining gel piece, add the solution shaken for 15 minutes, and combine them to obtain the protein digested peptide recovery solution. did. After completely drying this solution with a centrifugal evaporator, using a ProteoMass Guanigination Kit (Sigma-Aldrich), all lysine residues contained in the peptide were converted to homoarginine and dried with a centrifugal evaporator. Digested peptide samples were obtained.

  Proteins were identified using the MALDI (Matrix Assisted Laser Desorption / Ionization Method) Mass Spectrometer and ESI (Electro Spray Ionization Method) Mass Spectrometer using the samples obtained here as materials. Details are described below.

<Identification of protein (MALDI mass spectrometer)>
Protein identification using a MALDI mass spectrometer is based on the peptide mass fingerprinting (PMF) method, which identifies proteins from the masses of multiple generated peptide fragments, and the internal amino acid sequences of several detected peptides. Two methods were used, MS / MS ion search method (MS / MS analysis) to identify proteins.

  In the analysis with the MALDI type mass spectrometer, the above-mentioned in-gel digested sample was first redissolved in 4 μL of 0.2% trifluoroacetic acid, and this was dissolved in the literature (Johan G. et al., Anal. Chem. 73, 434-438 ( 2001)) using the matrix reagent α-Cyano-4-hydroxycinnamic Acid (CHCA), the target plate for MALDI mass spectrometry, MTP AnchorChip 600/384 TF (Bluker-Daltonics) ), And the plate was set on a MALDI-TOF / TOF type mass spectrometer Ultraflex II (manufactured by Bluker-Daltonics) and then measured. In the PMF method, data obtained by MS measurement in reflectron mode is used, and in MS / MS analysis, measurement is performed in LIFT mode for several peaks with relatively high ionic strength as a result of MS measurement. The performed data was used. The MS and MS / MS measurement data obtained here were subjected to database search using MASCOT software (Matrix Science) to determine the protein.

<Protein identification (ESI mass spectrometer)>
Protein identification using an ESI type mass spectrometer was performed by performing MS / MS analysis of a plurality of peptides using a measuring instrument connected to liquid chromatography (LC) and a mass spectrometer.

  First, after re-dissolving the above-mentioned in-gel digested sample in 20 μL of 0.1% formic acid and 0.01% TFA solution, HiQ sil C18W (inner diameter 0.2 mm x length 100 mm, manufactured by KA Technologies) was used. It was applied to the connected Nanoflow LC system, DiNA (manufactured by KE Technologies). After flowing with 0.1% formic acid and 0.01% TFA at a flow rate of 200 nL / min, fractionation was performed with a linear gradient of acetonitrile solution with 0.1% formic acid and 0.01% TFA added, and sequentially eluted peptides were ligated. MS / MS measurement was performed using an ESI type mass spectrometer, Q-tof ultima (Waters). The MS / MS measurement data obtained here was subjected to database search by MS / MS ion search of MASCOT software in the same manner as the measurement of the MALDI type mass spectrometer, and the protein was determined.

<Lung adenocarcinoma prognostic marker>
The 16 proteins identified by the above method are shown in Tables 1 and 2. Of the 16 proteins determined by MALDI mass spectrometer and ESI mass spectrometer, 5 were aldehyde dehydrogenases and 3 were different isotypes of tropomyosin. The molecular weight and isoelectric point assumed from the amino acid sequences on the NCBI and SWISS Prot databases of the protein found here, and the prognostic marker for lung adenocarcinoma calculated from the migration position of two-dimensional electrophoresis (FIG. 1) As a result of comparing these proteins with those of 12 proteins, 12 of 16 proteins were found (Table 1), and these proteins can be used as prognostic markers for lung adenocarcinoma in the present invention. It was. Such proteins are known to have undergone post-translational modifications in vivo, and some of the proteins are subject to post-translational modifications, such as phosphorylation in intracellular signal transduction. It is clear that it is greatly related to the function of. The 12 proteins identified as dissociated here are those that have undergone post-translational modifications, and their molecular weights or isoelectric points have shifted, and have been migrated to locations different from those assumed in two-dimensional electrophoresis. It was strongly suggested that there was. For example, the delta 3,5-delta 2,4-dienoyl-CoA isomerase (Delta 3,5-delta 2,4-dienoyl-CoA isomerase) shown by spot number 1B1023 in FIG. It is known to receive. The isoelectric point assumed from the amino acid sequence is pI 8.5, but the modified product observed from the two-dimensional electrophoresis image of this time had an isoelectric point of about pI 6.4 and was shifted to the acidic side. . This suggests that this protein has undergone modifications such as phosphorylation. In addition, Annexin A2 (Annexin A2) indicated by spot number 1B0816 is known to be modified by phosphorylation or acetylation. The isoelectric point assumed from the amino acid sequence is pI 7.6, but the modified substance observed from this two-dimensional electrophoresis image had an isoelectric point of about pI 6.6 and was shifted to the acidic side. . This suggests that this protein has undergone modifications such as phosphorylation.

  As representative examples found in the present invention, the expression distributions of poor prognosis cases (4 cases) and good prognosis cases (12 cases) of lung adenocarcinoma patients on 2D-DIGE gel in two markers are shown. This is shown in FIG. It can be seen that the expression level in the lung cancer tissue at each marker varies greatly depending on whether the prognosis is good or bad. Therefore, by comparing the expression levels of these markers, it is possible to discriminate between two groups with good prognosis and poor prognosis.

  In the analysis by two-dimensional electrophoresis, it is possible to separate with high resolution by the molecular weight and isoelectric point which are two physical properties in protein. This comparison of electrophoretic image profiles will directly compare the expression levels in a specific post-translational modified form. Furthermore, the 2D-DIGE method combining fluorescent labeling and two-dimensional electrophoresis can produce a protein profile with high sensitivity even when a sufficient amount of sample cannot be secured. In the present invention, a marker for predicting the prognosis of lung adenocarcinoma that is clinically required was found by taking advantage of these characteristics. The marker here is also a target of marker search for the increase or decrease in the expression level of a specific post-translational modified form, which is related to, for example, lymph node metastasis of lung adenocarcinoma reported in Patent Document 1 This is not possible with the marker search method. The prognostic markers for lung adenocarcinoma found here are those reported in the literature (Guoan C. et al., Proc. Natl. Acad. Sci. USA 98, 13537-13542, 2003). It is a novel protein that does not overlap with proteins searched for by conventional two-dimensional electrophoresis (detected by silver staining) for proteins whose expression varies from the survival rate of cancer patients.

  By the method of the present invention, the sample provider can determine the level of risk of recurrence of lung adenocarcinoma in the future. Based on this risk, sample providers can take preventive measures for recurrence of lung adenocarcinoma, and sample providers suspected of having recurrence of lung adenocarcinoma are more likely to be diagnosed with recurrence of lung adenocarcinoma, This is useful because treatment can be started early.

FIG. 1 is a diagram showing a two-dimensional electrophoresis image obtained by developing a lung adenocarcinoma tissue extract protein and detecting the protein by two-dimensional electrophoresis in the 2D-DIGE method for the proteins shown in Tables 1 and 2. is there. The protein spot numbers shown in the gel correspond to the protein spot numbers shown in Tables 1 and 2. FIG. 2 is a diagram showing an outline of the experimental design of the present invention. FIG. 3 is a diagram showing an example of a gel obtained by processing a fluorescent gel image acquired by two-dimensional electrophoresis for analysis. In addition, when using for an analysis, the acidic low molecular area | region and the basic area | region were cut out. FIG. 4 shows an example of comparison of expression distributions of poor-prognosis cases (4 cases) and good-prognosis cases (12 cases) of lung adenocarcinoma patients on 2D-DIGE gel on the markers found in the present invention. It is a figure, (A) is a figure regarding Tropomyosin alpha-3 chain, (B) is a figure regarding Aldehyde dehydrogenase. Each plot shows the average value of n = 3 measurements in each patient sample.

Claims (7)

  In biological samples derived from subjects, Delta 3,5-delta 2,4-dienoyl-CoA isomerase (Annexin A2), aldehyde dehydrogenase (Aldehyde dehydrogenase) ), Peptidyl-prolyl cis-trans isomerase A, Inorganic pyrophosphatase 2, Trotropyosin beta chain, Proteasome subunit beta type 2 a step of measuring the expression level of a modified form of at least one protein selected from the group consisting of beta type 2) and elongation factor 2 (step A), the protein in the biological sample derived from the subject A step of comparing the expression level of the expression level with a reference value (step B), and Subject when admit variation by compare is comprises determining that there is a high risk of recurrence lung adenocarcinoma (step C), recurrence prediction method of lung adenocarcinoma.   The prediction method according to claim 1, wherein the modified product is a modified protein whose molecular weight and / or isoelectric point deviates from a theoretical value.   The prediction method according to claim 1, wherein the measurement method is a two-dimensional electrophoresis method.   The prediction method according to any one of claims 1 to 3, wherein the biological sample is at least one selected from the group consisting of lung tissue, cells, body fluids, urine, and other biological protein extracts.   The prediction method according to any one of claims 1 to 4, wherein the protein is Delta 3,5-delta 2,4-dienoyl-CoA isomerase (Delta 3,5-delta 2,4-dienoyl-CoA isomerase).   The prediction method in any one of Claims 1-4 whose protein is Annexin A2 (Annexin A2).   Delta 3,5-delta 2,4-dienoyl-CoA isomerase (Annexin A2), aldehyde dehydrogenase, peptidyl-prolyl cis-trans isomerase A (Peptidyl-prolyl cis-trans isomerase A), Inorganic pyrophosphatase 2, Trotropyosin beta chain, Proteasome subunit beta type 2, and elongation factor A kit for predicting recurrence of lung adenocarcinoma, comprising an antibody against a modified form of at least one protein selected from the group consisting of 2 (Elongation factor 2).