TW200817682A - Method of gastric juice protein analysis for stomach cancer diagnosis - Google Patents

Abstract

A method and biomarkers for detection of a gastroduodenal disease are provided. The method includes the use of mass spectrometry, two-dimensional gel electrophoresis analysis, and/or neural network for analyzing gastric juice samples and detecting elevated and/or suppressed levels of stomach disease-associated proteins and peptides. The biomarkers, peptide fragments, and proteins identified herein can be used in assays and diagnosis kits for a gastroduodenal disease.

Description

200817682 IX. Description of the invention: [Technical field to which the invention pertains] A biomarker and method for detecting gastric and duodenal diseases. [Prior Art]

After the completion of the phenotype of the tongue and the ft, the proteosome study yields a high-yield two-two white body analysis to provide an effective study of disease occurrence and the total protein type and quantity of the disease. Use this Qing; the theory is called the sensitivity and resolution of the detection is not limited, can be protein corpus callosum: quality ί if this „ two protein composition, and produce the test sample 单 single-test sample ^ from =;ΐί 白^ It takes a lot of time and money, not to mention that only a few are the steps to determine the sequence of the acid. The protein identification and/or amine use of serum samples for mass spectrometry is a serum towel. A large number of eggs are self-contained in the mass spectrometer. It is not easy to accurately measure. It is compared with normal 丄 and = suffering: = 罕5 200817682 and screening can be applied to the diagnosis of ovarian cancer and prostate cancer, although Qiu Shi =, its accurate analysis is still difficult. Use the serum sample = 3 · the natural protein content of the specific protein content in the serum, the same nutritional, environmental and psychological state.;: 乂; = = produced Change, a specific protein In patients with serum-like dimorphism, the expectation of early cancer diagnosis is not a problem: Μ成i---the test sample obtained from the affected area instead of the blood is highly invasive and can obtain a higher content of tumor-associated protein. Most of the diseased fields and the wide-ranging cases are not willing to give such sampling. On the contrary, it is relatively simple to obtain the protein f/polypeptide for analysis by mass spectrometer from the moon ^. cancer cancer or gastric cancer Gc), which is the world's second syndrome. It is difficult to diagnose in the early phase. Gastric cancer can exist in the stomach-segment, and grows into a large size. In the early stage of gastric cancer, ί^ίΐΐΐΐϊ is bad, stomach discomfort, After meals, the feeling of the building is slight, and the nausea and sputum may be in the advanced stage of gastric cancer. There may be bloody stools, vomiting, or heavy weight. So far, there is no good stomach cancer so far. There is no reliable, non-invasive screening resistance; the general test for monitoring cancer and measuring the cancer is related to the biomarker. This type of test measures the significant release or distribution of tumor cells. $4 of protein or For example, only a few of the two white blood cells release the cancer embryo antigen (Carcin〇embryonic antigel1, tA) 'carbohydrate antigen (CA19_9) or other currently known biological standard 6 200817682 into I:: two The amount of multiple cancer-specific or cancer-associated proteins is generally associated with the disease; can be diazepam, antigen (detec_^ [invention content] Shengshiguan supply of stomach _ method and degree. Biological information points; ^, year The increase and/or inhibition of the compound peptide is defined in the test and diagnostic kits for intestinal diseases. 1 μ in the stomach | 4 — - square; One or two Shi 2 = one: set to: ===, Ben, == line mass mass spectrometry, and identify a spectrum of 3 or more molecular systems for the stomach or duodenal disease of the organism to listen to "^ one in another-specific In the embodiment, it is provided that - sieve 2 1 = package contains = personal, the quality of the mass spectrum and the quality of the base line is in the - part of the invention, and the base = line ^ = disease_1. The polypeptide is identified. The one or more polypeptides; the sub-packages 200817682 The amino acid sequence of identification numbers 1-3. After the present invention, the one or more polypeptides are increased. 'The amount added to the baseline amount is identified by H or more t protein protease precursors. Examples of greedy white shells contain alpha-anti-pancreas in another specific example, one or more Tianyiyi signs The system is identified. For example, the biomarker of gastric cancer and gastric ulcer in the organism of duodenal diseases = ^^=, identified as - the new ones are included as containing the amino acid phase, 7. The target is identified by a biomarker number 1- The amino acid sequence of 5, , and the like, are similar to the selected sequence. [Embodiment] The specific embodiment of the present invention provides a higher sensitivity of the stomach finger f_ Twelve $ compare the test samples of patients and healthy people and determine the amount of protein expression and the amount of inhibition of the mass spectrometry. They may or may not have one or more polymorphisms and /, disease detection / diagnostic test silk two For example, after the normalization analysis of the stomach and the twelve fingers ^ for the cancer, the stomach ulcer, the busy finger ulcer and the disease, the 200817682 of the stomach and the twelve fingers is adjusted to adjust the identification of one or more biomarkers. One or more of the corresponding strains of the stomach and duodenal diseases may be developed for treatment and/or heal detection. Comparison of Gastric Juice Samples Mass Spectrometry + One or More Specific Examples , providing a method for identifying biomarkers associated with disease and stomach disease. In another embodiment, the composition spectrum analysis = or = fixed stomach and ten m-like biological view, in another In addition, provide - use the use of == w to the stomach and duodenal diseases of human patients in the court. Sleeve sample collection method to collect gastric juice samples. - Method is to use the method. Endoscope to absorb gastric juice, can also use other Non-endoscopic side I and ten In the case of duodenal diseases, 'getting a sample of gastric juice relative to two = ί ' Γ millions of patients and the general subject are undergoing annual brother examination. Non-invasive methods without endoscopy are also available to collect the stomach , this / for example / when collecting gastric juice samples - non-invasive wire rope test

C (T^ng/eH non-invasive cord test was applied to Helicobacter pylori in the stomach to help epidemiological studies to assess asymptomatic 2, dioxin, bacillus. Another - get gastric juice samples Examples include - 40 capsules' which contain - a plastic line covering the perforated plastic along the absorbent sheet and a nylon thread that rests in the length of the field to the inner capsule, swallowing the capsule and allowing the capsule to saturate the absorbent sheet. The gastric juice is absorbed, and then the pull-month I line is removed from the stomach cavity and the gastric juice is obtained from the absorbent sheet. The monthly liquid system contains several compounds 'containing hydrochloric acid factors, polypeptides, acid suppression and electrolytes, etc. ί i in the liquid component 'gastric and duodenal reflux contains bile, and; immunoregulatory factor of the moon and moon, and blood and cancer from the stomach cancer 9 200817682. The gastric juice sample is intended to be used in the gastric juice sample protein or peptide The spectrum analysis of the number, the non-ΪΧΞ3 environment = the = prime, the wall of the ΐ ΐ white matter can not exist, we have a large mouth in the knife protein, the molecular weight of the ancient time, the typical mass spectrometer resolution is very limited, two It is quite difficult to use high-quality protein. However, the example of distinguishing samples within the circumference is shorter: the baibai shell is decomposed by flattening, such as gastric juice: relatively high-resolution 夕 polypeptide can be obtained. The search biometrics produced by the polymorph (4) tid〇mic) eggs are used as a diagnosis of the disease. The body of the knife is analyzed as a distribution pattern to the blessing of the brake::u, the hunting is made by the method of the present invention, Zhong Ding, and the content of certain polypeptides is not known. However, 'multiple polymorphisms are compared to the pairing: two, most peptides: combined with stomach 4 and with different stomachs and twelve phases = hair = * and specific stomach and stomach, special mass spectrometry twelve fingers win knee and other Colon phase age 丨 200817682 method analysis. It can be found that the gastric juice mountain (less acid) of the gastric cancer patient and the higher degree of Huayu 1 have a PH value or a plurality of specific examples, fs according to the present invention - here, two and two fingers The detection and diagnosis of intestinal diseases and the successful identification of peptide fragments and proteins and the successful identification of gastric cancer are more sensitive and can also be attributed to the analysis of gastric juice than the serum analysis according to it. For example, when: = protein purification to extract the protein and / or peptide of the gastric juice sample ^ ί : ί you, 'centrifugation can be used to remove contaminants. This, magnetic 2 i grain: is - simple; A quick and easy way to use the protein in the gastric juice sample is also used, including diamond nanoparticle early, HPLC, ion exchange column chromatography, two-dimensional gel, Western: decapillary, etc. The protein extraction method is not limited to the above. The method and the consistent application system utilize the matrix-assisted laser desorption ionization: ^1X_aSS1Sted laSer deqingtion/ion h (time_of_fllght mass spectrometer; TOF-MS) to obtain the prosthetic limb Mass spectrometry by massi-tof-ms, a mass spectrometer G can be obtained in a few seconds =. In general, only one or two charges can be obtained via MALDI, and the resulting map is significantly easier than other types of mass spectrometry. However, other two types of mass spectrometers are also utilized, However, it is not limited to the above mass spectrometer, electrospray mass spectrometry, desorption electrospray mass spectrometry, laser induced acoustic desorption mass spectrometry, and the like. Various mass spectrometers are also used. Electrospray is a mild ionization method and is easy to generate a large charge in large proteins, making it suitable for the analysis of proteins and peptides with high molecular weight. Although EFI mass spectrometers are usually very Complex, however, the mass spectrometry of the peptide is not as good as the protein state. 200817682 f 'Therefore, the electrospray mass spectrometer can use this advantage to divide & the peptide has a charge of less than 3. According to the mass selection, the magnetic part And the Fourier transform (F0urier transf〇rm), the quality η change. Because the Fourier transform quality instrument has a very solution ^ > 2_00), Mass difference is the measured one; Chang, mass spectrometry-based aspect of polypeptides and proteins can be used to resolve gastric Wu itch-like sample measured. The mass spectrometry can also be used to distinguish gastric ulcers from ulcer-free human subjects. #胃料本和一

C, the sample comparison mass spectrum can also be finer than (10) or n-f cancer, which should be directly compared with the cancer cells collected from the cancer (four) package. The process of obtaining the sample is invasive, and is collected from the disease location, serum samples. The blood sample is difficult to be revealed = 扪 ^ The sample of the sample of the cell is also smeared in this 'quality= and other more quarantines two; two types of r / 曰: two materials and other raw (four) recorded body% ^ road Program-derived data 'Compared in mass spectrometry' ===: A large number of sample-analyzed samples that can be applied to normal and patient samples/two-dimensional electrophoresis of a sample of gastric juice in a different embodiment , performing two-dimensional electrophoresis to divide P I I ΐ to improve. In addition to the molecular weight, the two-dimensional electrophoresis system uses only the t-point oblique red-shot to hide, and the fine-mass spectrometry divides the gastric juice samples of patients with different stomach and duodenal diseases into two different bands, including three different strip states, including the basic strip. Band state 様, special 12 817682 = band state 様 杂 。 。 。 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐 罐By using a two-dimensional gel to read the abnormal protein protein, the pattern is used to reduce the degree of bifurcation. For example, a two-transfer test sample. Two-dimensional transfer from the sample and the general tester The test and the normal non-stomach ulcer health are affected by ^ ^. = 辫 stomach ulcer cancer f-like enteropathy. The sample of the patient and the sample of healthy people., 7 outside 'also compare the stomach and fingers and ^ Wei coagulation With the appearance of the sample, one or more protein bands ^ is ^I, #, the intensity of the band implies one of them. By the two-dimensional silicone gut good biomarker or multiple protein bands special = It can be found from polyacids that the frequency from healthy people and + two Hu La, and sputum is relatively low 'both are about 6;,: to 5: state f; f suffering from the county The frequency of the special strip state 様 is: f2 7 i * as a special protein organism of gastric ulcer and gastric cancer H. It can be analyzed. We also Wei, stomach and stomach hang, stomach is obviously increased (about 1 · 〇 6 mg / ml and about 2, / rut health mg / ml, individually), and twelve _ ulcer patients with:: = 48 Qualitative concentration compared with healthy people (about 26.26 mg / mlv low ^液^蛋白: Buqi aging Λ is the second son of the gastric acid influencing gastric juice. This = 2'^ η^32·9 (95^(4) is the biomarker of Zhongding from gastric juice and its application is identified from the biomarker of the method of the present invention. The system can be applied to a diagnostic test or screening kit for gastric and duodenal diseases. In addition, the biomarker identified from the method of the present invention can be applied to the treatment of gastric and duodenal diseases or the stomach and twelve. The standard of treatment of intestinal diseases. A special group of polypeptides can be used as a reliable disease. A valuable biomarker is provided. For example, the present invention provides specific polypeptides and proteins in gastric juice as biological markers for diagnosis of gastric cancer, including polypeptides. Gig? m/z, peptide 2387 m/z, polypeptide 3573 m/z, peptide 2573 m/z, peptide 4132 m/z, α-antitrypsin precursor and others. The amino acid sequence series of these peptides is given by the serial identification number ship S ··.

1 of 2187, polypeptide 2387 m/z and polypeptide 3573 m/z (sequence recognition leg: 1-3) was found to be lower in patients with gastric cancer than in general health. The amount of the second polypeptide 2573 111/2 and the polypeptide 4132111/2 (sequence identification number 3:4_5) was found to be increased in comparison with the general health. Polymorphism 2187 111/2 (sequence identification number: 1111101 «^1^^) and multiple ^ 387 m / z (order 2, leg _p mineral EA) is a part of the original protein S #. Peptide 3573 m/z (sequence ^^_νί^®Ε〇α(_Ρδνι^Ό#, a polypeptide fragment of the leucme ziPper protein. The importance of this inhibition is still under investigation. Xifu Naiqi and peptide 2573 m/z (sequence identification number.4 DAHKSEVA_LGEE_VL) is a polypeptide fragment of albumin. It is generally believed that 'the possibility of higher bleeding in cancer patients makes the white egg free blood f into the protein. In the stomach of the acidic stomach, (4) the sputum into a small peptide and the interaction between the polypeptide and the cancer cells is high and is detected by the method of the present invention and observed in gastric juice. The polypeptide 4132 m The importance of /z (sequence identification number · 5 = ρ^_^_Ε_^ΡΙ^Μ(}κννΝρτ(^κ)) is still under study. The m-specific special band state of gastric cancer samples from two-dimensional electrophoresis The protein shellfish and the amine-grade oil of the band show that the α-anti-pancreatic egg (four) precursor system is 14 200817682; ί; = the main protein of the special band of cancer t, as shown in Figure 18, B1 = egg biomarker of gastric cancer i. protein feeding system can be used as early and advanced by I2, human suffering from duodenal diseases Ben, from the collection of his biological disclosure - 4. Collecting gastric juice test samples. The peptides and protein lines of the woody H' sample can be partially purely by various detection methods, with the amount of stomach cramps The system can be screened. For example, the relevant biomarker features are used to screen the stomach eleven fingers = shellfish, the moon liquid, and the sample system can be used for screening. The polypeptide can be used for screening. The invention of the prescription ir stomach and/or gastric cancer and other applications. The diagnosis and diagnosis of gastric and duodenal diseases are provided - the diagnosis of simple gastric cancer ^ The enzymes and protein of this kind of oyster performance are white Biometric identification, identification, and other possible treatments or detection of many biological diseases. When used as a disease diagnosis;: Yes! Second, the quantitative detection of enzymes and peptides can be compared with the sample of the subjects. The basic line phase energy can be used for the stomach and the twelve test sample. If the amount of the biomarker 15 of the test sample is the same as the amount of the baseline, the subject is negative. EXAMPLES The present invention was implemented as follows Examples are further clarified, but the scope of the invention is not This eye

囷l^nsteruetrap In general, subjects, such as human subjects, are banned for nighttime accuracy and avoid complexity in sample analysis. Other methods of non-mirror are also utilized. For example, the '-a highly absorbent strand is wrapped around the inside of a heavy and heavy capsule. P female swallowed the scale and took it out of the stomach after 15 minutes. The distal end of the line segment is clipped for use as a sample of gastric fluid. Various methods of collecting gastric fluids are followed by periodic examination of the upper gastroduodenal duct, and biopsies obtained from the larger curved antrum are subjected to rapid urease analysis to determine the ugly/oW infection. Subjects who consented to a topographical histopathological study were examined with a tissue for the stomach. Part of the small and large curved part of the midantmm (the pyloric gland and the middle b〇dy). The living body shouting sample is fixed with 1G% cushioning formalin. The stone was embedded and sectioned. The section was stained with H&E. The section was subjected to intestinal metaplasia). In order to adjust the clinical characteristics, the following data are included: age, gender, family history of gastric cancer, and situation. Diagnostic review. Each of the tested subjects was evaluated by Ascending _ Sydney pure evaluation of gastrient infiltration (rophilic inflltration), monocyte infiltration (m〇n〇nudear cell infiltmtion), glandular atr〇phy, and small intestine degeneration. Smoking history and drinking list 2: Collecting gastric juice 檨 too 16 200817682 After measuring instrument (glass_electrodepHmeter) collected 4 °c, centrifuge ^ 11 value. The collected gastric fluid sample was then passed at 10,0 (8) xg, and stored in a clock to remove cell fragments and contaminants. The upper cobalt hydroxide was evenly distributed from ^70 to further experimental analysis. The gastric juice sample supernatant after centrifugation at a desired pH of about 1150 mM / about 1 〇 μ1 is adjusted to one, one or more specific examples, when the gastric juice sample is collected and removed, the protein is deficient Protein or polypeptide purified to extract the gastric juice sample

Oh. For example, protein extraction by nanoparticle beads. 'Changdi is a sample chart for collecting gastric juice samples for mass spectrometry analysis. The stomach secret is combined with the copper magnetically unbound portion. The bound part can be separated by a slow buffer or a meso = μ1 elution buffer, and the precipitation buffer used after the magnetic force disappears is 5 〇〇 / 0 acetonitrile (acet〇 nitrile) ). Various surface modified magnetic beads, such as C8, C18, magnetic nanoparticles, diamond nanoparticles or other metal nanoparticles, can be utilized. Figure 4 shows that the gastric sample is collected in 50% acetonitrile precipitation buffer and ready for mass spectrometry. Partial protein purification, HPLC, ion exchange, two-dimensional gel point and capillary electrophoresis systems make mass spectrometry between cancer and non-cancer patients easier.

U0 consecutive healthy subjects (HSs), 39 gastric ulcer patients (GU), 38 = duodenal ulcer patients (DU), and 31 gastric cancer (GC) patients were involved in this 2D gel electrophoresis study. The healthy subject was recruited at a health check-up clinic and did not have a history of gastroduodenal disease. Endoscopic examinations of healthy subjects were normal or showed only mild gastritis. The patient exclusion principle was included in the prosthetic pump to inhibit the use of back-J, H2 receptor antagonists (3⁄4 receptor antagonists) or non-lipid alcohol anti-inflammatory drugs 17 200817682 within the first 4 weeks of the study, two months 11 fingers The coexistence of intestinal lesions, the presence of upper gastric duodenal blood, and the common systemic disease.曰 诊断 诊断 藉 藉 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃 胃#由开前前—The living group with the width known = the pliers measure the size of the ulcer. Qiaojing is determined by histology and is based on Lauren's classification method. Eight from the intestinal type of gastric cancer (n=19), diffuse gastric cancer (n=9) and mixed gastric cancer (n=3) The degree of tumor invasion in patients with gastric cancer (n=27) is based on the criteria proposed by the Japanese Society for Gastrointestinology 1 (the Japanese Society for the Study of Gastric Cancer), and is divided into f-phase* progressive-gastric cancer samples. The study was approved by the Second Research Committee of Kaohsiung Rongmin General Hospital. All patients and control groups were informed. Recent studies indicate that the occurrence of intestinal type gastric cancer is a multi-step event, 1 ^(superficial gastritis) > f ^ (atr〇ph; 'gastritis), small intestine degeneration (intestinal (10) postal (four), hair dysplasia evolved into a malignant tumor, through cumulative multiple genetic changes x ^ = quality rich, structural or functional changes. Disseminated gastric cancer, 1 adhesion molecules ^ ^ i ^ li ^ l ^ (E-cadherb) > loss of catenin and annexin_7 are also involved in its canceration. w, (1) k Because the gastric epithelial cells in the stomach will cause changes in gastric juice composition during the process of gastric cancer, the gastric juice protein described here finds a new type of tumor special marker to screen for gastric malignant tumors. The gastric autologous concentration of the gastric and duodenal patients' gastric juices and the biomarkers for the discovery of new diseases. m The total protein production of gastric juice is determined by the Bradford method (21). 5Gpg per Tablet gel) is used in 2D electrophoresis $2 = protein dot by mass spectrometry analysis. - contains about 5 samples with acetone (1 to 2Wv) at about _2Gt: sinking the temple for about 2 hours, 18 200817682 The solution was centrifuged at 10,000 xg for 10 minutes at 4 ° C. The precipitate was rehydrated (9 M urea, 35 mM Tris, 42 mM DTT, 2% CHAPS, 0.66% SDS, 2% IPG buffer, trace bromophenol blue) Mix and add to a fixed pH gradient IPG strip, 11 cm, non-linear, pH 4-7 (Amersham Phamacia Biotech, Immobiline Dry-Strip, Uppsala, Sweden) for overnight overwatering at 30V. Two-dimensional electrophoresis was performed on the second-dimensional gel electrophoresis. The first-dimensional gel separation was performed using a commercially available, specialized apparatus, for example, IPGphor (Amersham Phamacia Biotech). The protein was continuously concentrated at about 200 V, about 1 Hours, about 500V, about 1 hour, about 1000V, about 1 hour. Then carry out a gradient of about 1000V to 8000V for about 30 minutes. Then continue to concentrate on 8000V for about 8 and a half hours to get a total amount of IPGphor. 70kVh. In the second dimension, the PG is balanced 50 mM tris-CL, pH 8.8, 6 M urea, 30% glycerol, 1% DDT, and bromophenol blue for about 12 minutes, followed by 50 mM tris-CL, pH 8.8, 6 M urea, 30% glycerol, 1.2% SDS , 2% iodoacetamide, and bromophenol blue for about 12 minutes. The strip was placed on a 15% gradient poly-amine gel plate (190 X 140 X 1 mm) and the gel was electrophoresed to the next day. The gel was dyed with silver nitrate. The protein in the gel was recorded as a digital image by a GS-710 Calibrated Imaging Densitometer (Bio-Rad). Gel image alignment was performed using PDQuest software (Bio-Rad). Further, prior to the extraction of the protein from the two-dimensional gel, digestion of the gel fragment by porcine trypsin (Promega, Madison, USA) is ideally performed to digest the gelatin membrane protein S#. The resulting digested and decomposed polypeptide fragment was extracted with trifluoroacetic acid and acetonitrile (ACN) and subjected to mass spectrometry to determine its mass and amino acid sequence. The uranium at mass spectrometry was purified by ZipTip C18 (Millip〇re, Bedford, USA) according to the manufacturer's instructions. 19 200817682 Example 4: Mass spectrometry is divided into a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (1^^]〇1 TOF-MS) analysis, generally, with a 2,5-dihydroxy group Mixing about 1 μl of 2,5-dihydroxybenzoic acid (5〇ηηη〇ι/μΐ in 5〇% acetonitrile) and phosphonic acid (final concentration about 〇·4%) ! A sample of gastric juice precipitated from μΐ. Other substrates known to be suitable for the preparation of protein ions are also tested in the same wire. Next, about 丨μ1 of the resulting mixture was made into a MALDI unstained steel sample pan and allowed to air dry at room temperature. Modified by ABI Voyager DE_STR MALDI-T0F mass spectrometer

A MALDI-T0F mass spectrometer (Applied Biosystems, Foster City, CA, USA) produces mass spectrometric profiles of the polypeptide and protein of the precipitated sample. The spectrometer operates on a positive ion junction with an accelerating voltage of -25 kV. The miscellaneous mass valve is set at 500 m/z. Protein library search tool "pr〇f〇und,, (http://www.i29.85J92/profoundbinIWebp^^ is used to compare the single isotope m/z value of the trypsin fragment with its known protein in the database The knowledge of the micr〇 channel plate and the ability to measure laser energy. The external laser system is used to obtain the tender (10) spectm. With a mass and charge ratio (M/Z) ranging from 1 〇〇〇 to 1 〇, the 〇〇〇Yang yang sub-mode obtains most of the spectrum. From about 2G mj/em2 to about 3〇〇=j/:m > The laser fluence and the accumulation of 〇〇 雷 laser emissions yield a typical texture map. Samples of normal and cancer patients are performed under the same laser fluence. MALDI-TOT New Zealand provides clear The sample of protein properties was further studied by electrospray MS/MS. The pancreatic egg was purified and concentrated by C18 HPLC from the enzymatic digestion. The concentrated protein/peptide sample was then used. The spray (nanoflow elect ray) is sprayed from gold-coated glass capillaries. The MS/MS map is obtained by A FT-ICR mass spectrometer (Therm〇, B dirty 20 200817682 was used in the database search, identified by amino acid by MASC0T amino acid phase search (httP://www.matriXscience.com) Confirmed with the library. The typical mass spectra of normal P and cancer patients are presented separately in Figure 5A. By comparing the abnormal values of gastric juice samples & Yu & non-cancer, this towel, a small part of the mass spectrometer __ different systems can be by the pro, '', other additional complex analysis is required. Therefore 'about this mass peak ° (pea) 夕 peptide is It is identified as a good student for the diagnosis of gastric cancer. It has 5 mass peaks associated with gastric cancer, with mass and charge in two cases (m/z) at 2187, 2387, 3572, 2573 and 4132. , peptide 2 broad 1m peptide 2387 _ and polypeptide 3572 _, the amount of peak ^ in cancer

However, the polypeptide 2573 m/z and the polypeptide 4 mm/z J showed an increase in expression (see Fig. 6). Close; ^ Γϋ Detailed content is presented and compared in Figure 6Α6Β. Obviously, 丄/peptide 2387111/2 and polymorphic 3572 m/z 3 masses. 2 表现 performance but disappeared in cancer samples or lower

In the V sample, the disappearance of the heptapeptide 2i73m/z * polymorphism of 4132m/z is shown in the cancer sample in the normal harvesting white a曰 non-hanging weak. Analysis. Samples of normal subjects and cancer patients were also statistically sampled. Two from my ^, the main, and ^^ were 6 samples from healthy normal subjects with ulcers* from the monthly disease. 37 samples from duodenal disease * diazepam and 34 patients from gastric cancer. There are important differences between cancer and non-cancer ^ more than 15 peptide mass peaks. The results of the eight-pregnancy 乂Beibei are presented in the table. 21 200817682 The first table: inhibition of gastric cancer biomarkers, signal 2187 m / z sample positive negative normal 84 --- 22 gastric ulcer 27 —— 11 duodenal ulcer 35 2 gastric cancer 5 29 Second table: enhanced gastric cancer biomarker signal 2573 m / z 4132 m/z — positive sample ^ negative positive negative _ normal 8 98 6 100 _ gastric ulcer 7 31 9 28 duodenal ulcer 0 37 0 37 ___ gastric cancer 23 11 —— 16 18 ~---- ~ 2387 m/ z 3572 m/z Sexual negative positive negative ~80 26 84 22 24 — 14 23 15 ^37 0 35 2 4 ^--- 30 8 26 If we calculate the sensitivity and specificity of gastric cancer for each peptide Specificity, polypeptide 2187 m/z, polypeptide 2387 heart, polypeptide 3572 m/z and polypeptide 4132 m/z, the mass spectrometry sensitivity was 85%, 88%, 76% and 53%. ' Polypeptide 2187 m/z, polypeptide 2387 111/2, polypeptide 3572 m/z and polypeptide 4132 m/z, and its shell specificity was 80%, 80%, 79% and 92%. The results obtained from this comparison with other sera currently available for detection of gastric cancer are significantly more sensitive than other peptide biomarkers. For example, on the L bed, the cancer embryo antigen antigen (cea) and the carbohydrate antigen (CA19-9) are serum biomarkers commonly used in gastric cancer. The sensitivity of serum CEA and CA19-9 in gastric cancer detection was only 18% and 29%. A recent study showed that the sensitivity of CEA and CA19-9 increased in the monthly fluid at the time of gastric cancer detection was 27% and 17%, respectively = in addition to 'other recently identified organisms labeled 'for example, serum p53 antibodies and Tissue inhibition of matrix metall〇 proteinase-1 (TIMP-1) 22 200817682 The sensitivity of the drug used in the detection of gastric cancer is very low (15% and 17%, individually). Therefore, the utility of gastric juice CEA and CA19-9 in the diagnosis of gastric cancer is very limited. However, the polypeptide biomarkers identified here have a relatively high sensitivity and are very useful for gastric cancer detection. The advantages of peptide and protein biomarkers here are their high sensitivity and specificity. For example, in the detection of gastric cancer, the sensitivity and specificity of the gastric juice α-antitrypsin precursor were 93% and 83%, respectively. As far as we know, α-antitrypsin is a novel biomarker in gastric juice, and among other currently available biomarkers, it has the highest sensitivity for gastric cancer detection.

Example 5: Statistics of the public Douli ^ 〃 SPSS program (the first ιο ί version, Q ^ gojuinois, USA) statistic scores two ίο map showing healthy subjects, gastric ulcer (GU}, duodenal ulcer (Du) and stomach cancer (GC) The demographic characteristics of patients. Patients with gastric cancer were significantly older than those who were healthy (67.51^4^, ρ <0·001). In addition, the gastric cancer patients and healthy subjects were compared. There is a higher ratio of male to female (p=〇〇54). There is no significant difference in family history of gastric cancer and drinking history among the identified ethnic groups. However, the proportion of smoking in patients with monthly/beat ulcer and duodenal ulcer is obvious. More healthy *嶋, individually). Furthermore, the sensory rate of patients with duodenal ulcer is also significantly higher than that of healthy subjects ((ρ=〇〇34). Generation. The first t-test of gastric juice samples of patients with stomach and duodenal diseases In the second, the difference between gastric acid pH and total protein concentration in the gastric ulcer, duodenal ulcer (Du) and gastric cancer (gc) group is determined by the single factor variable ^ (one_wayANOVAtest). The polypeptide content in the moon liquid is also - Important indicators, in the disease = significantly higher than the healthy subjects (44% and 55% vs, 27%; p < 〇 = Bay = t. (4), in the twelfth of the series turned over the tester, and Significantly different. The pH value of gastric juice is higher in health than in gastric ulcer and gastric cancer. (P, 01% 23 200817682 The duodenal silk has a low gastric gastric concentration. V:8, ΐί 〇= refers to the total protein concentration of gastric cancer patients in all the protein groups of 2 intestinal ulcers and gastric cancer patients. It is clearly 'in all the patients with egg-gut ulcers in 4 groups of white matter concentration days. Low total protein compared with 12 standard deviation plus 2, quality concentration; and 22%, 36%, 8% and ten-Μ of gastric cancer patients Patients with ulcer disease at m, with or without Yates correction contnnnty) and Fisher's exact test (Fi^ _ (i lost t used in the analysis of human stomach affects all protein estimates total = 2 Ϊ ^ kg regression (1〇 The giStic regression analysis is based on the independent factor of ί whiteness (higher than 〇·5 tons of all eggs J ^) or low (less than 〇.5mg/ml of total protein concentration). The factor system has included at least 11 clinical factors, bacterial factors, gastric juice factors, and others. For example, the factor of the study includes age (below or higher than 6 years old), gender (male or female), and gastric cancer. History (with or without), smoking history (less or more than 1 pack/week), drinking history (less than (one) or more (+) 8〇g/day), bacterial infection (ugly sand/〇rz• infection) The performance of the situation does not show), gastric fluid characteristics (less than or higher than 5 ml of gastric juice; acidity of gastric juice is lower than or about pH 3.5), gastric ulcer (with or without), duodenal ulcer (with or without), gastric cancer ( Performance with or without performance. On the one hand, multiple factors affecting protein concentration in gastric juice are Time assessment. For example, '11 clinical factors, bacterial factors and gastric juice factor are compared and found independent parameters. Figure 12 shows the results of single variable analysis affecting the total protein concentration factor in human stomach. At />< 0.05 The degree of dissimilarity is considered to be meaningful. The results show that the factors up to 24 200817682 ΙΙΐίΛ are clearly associated with high protein concentrations (eg, above about ο. 5 + two' including aging (P = aG() 1), gastric cancer Family history (P = G.054), protein hiding _ relying on the line - money age analysis, in search of all the eggs of any '° figure 13 shows human stomach effects. 1-26-8.2 Τ);,, 95% ^ fBl(CI)): - Amateurs also Γί, TG is an active protein decomposing pH in a gastric juice. The effect of 6 liters is not readable. It is not activated but stable at pH 4-6. (4). Γ t egg homemade group (4) is affected by acid gastric acid trypsin elimination f low acidity gastric juice high protein concentration is understandable. Less, prostaglandin ϋίί, gastric ^ physiological changes (for example, mucosal blood flow is reduced by ί ί ί gastric ulcer and the main risk factors for the occurrence of gastric cancer, ^ white matter iiiitt) high incidence. But there is a high egg in the gastric juice: research comes from the type according to its neural network, the new type of neural network - the neural network surface is in a single network node (10) =:==) 25 200817682 clustering, classification, Functional approximation (functi〇n appr〇ximati〇n) and easy evaluation, Reference: 1 Chen, Υ· Υ · (2005) · Neural networks, fuzzy logic and statistical inference. Neural Networks ((2006) to appear); 2 Chen? YY and Chen, JJ (2004). Neural networks and belieflogic, Proceedings of the Fourth International Conference on Hybrid Intelligent Systems (HIS904)} IEEE, 460-461; 3. Chen, YY (2002). Plausible neural networks. Advance in Neural Networks World. Ed

Grmela, A· and Mastorakis, Ν· E· WSEAS Press, 180_185·). our

The results indicate that the difference between normal and ulcerated patients is very clear and the PNN procedure for mass spectrometry combined with gastric fluid samples can distinguish between cancer samples and normal samples. Example 7: Applying a Protein Purification Layer; j: Primary color layer analysis to identify a candidate molecule Because a mass spectrometer with many proteins in the human body is manufactured with a relatively low mass spectrometer mass spectrometer is not accurate enough to determine these mass peaks A corresponding protein or polypeptide. Many proteins have molecular weights that can be combined into one mass peak. A two-step method was applied to identify these biological targets. First, HPLC was utilized to previously separate these precipitated samples. A typical knot is presented in Figure 7, which introduces an HpLe map of a identified peptide biomarker from each peak of HPLC. We further perform a mass map = analysis, which identifies which HPLC interval contains the interest. The mass of the peak material. The results are presented in Fig. 8A_8C, which shows that the mass spectrum of the three fractions of the moon is in the mass range of 500 m/z to 5000 m/z after the purification of the layer of the column layer. The spectrometer purifies the candidate biomarker iron molecules and identifies the target molecules of the amino acid hydrazone I by protein chromatography analysis, for example, C18 'pure 1 匕 and concentration to prepare for further amino acid sequence identification. . This 9 map was then obtained on an ABI 4700 mass spectrometer. A typical map is shown in Figure </ RTI> which shows the results of MS/MS analysis of an experimental polypeptide (2187 m/z) amino acid and /. The sequence of the polypeptide sub-protein can be identified by a search of the mscot amino acid sequencing search engine. In addition to the polypeptide 2187m/Z, the polypeptide 2387m/Z, 26 200817682 polypeptide 3572m/z, the polypeptide 2573m/z, and the paste of the iron-like body *Z according to the present invention were also identified. Obtaining gastric juice and continuing to analyze the proteomic analysis of some patients with gastric cancer and existing patients, 'can be used in ^ yt5 CEA ^ CA ^ test, describe the diagnosis / screening method and field value of blood / month higher Sensitivity and specificity to go with "biomarkers, for gastric cancer detection

C 3 gastric electrophoresis 3 gel electrophoresis 3 suffering from '33 duodenal ulcer patients and 29th month / shell disease: the results of the mass spectrometry image classification into three kinds of: the basin stone 4 strips, special Striped and non-special strips. ,- Department w Tuben two Jiankang ΐ ΐ ΐ stomach and duodenal ulcer patients with gastric juice sample band can ^ field: Η = basic strips, special strips and non-special health test c The calculated frequency of the patient sample is individual; the 1i map introduces the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Tian No. 16 is a two-dimensional electrophoresis pattern in a gastric juice sample, showing a fire K=degree __. Small body gland (four) contraction and stomach low acid = special = 幽 crocodile crocodile new height phase _ all are ~ view): base f take band, inch "and non-special band frequency in low acidity gastric juice two-dimensional electropherogram The individual was 27 200817682 0〇/〇, 93% and 7%. The health of one of the 9 patients who had undergone gastric mucosal examination did not have atrophy of the stomach, resulting in two-dimensional coagulation of gastric juice and Kang, and the gel electrophoresis study showed a The non-special strip pattern. The text of the text is in the two-dimensional condensation. Figure 17 shows an example of two-dimensional gel electrophoresis. The strip is in acid Ρΐ (4.ϋ·5·α) can be observed: ^ The molecular weight of the strip 42 kDa is named as, and =

-TOF and MS/MS mass spectrometry analysis of the dream or sleep Ια A 7 stalks by MALDI regress = TOF * in the analysis of the two apparent polypeptide peaks at mz 538. The trypsin A precursor from the _ two peptide peaks was identified as the ^ column (QJFTVFDR). U Same Domain Acid Sequence Figure 18 is an illustration of a two-dimensional gel electrophoresis image showing the same. The final interpretation of the basic stripe pattern is notable and can be detected significantly. The band from the bad sample, therefore, corresponds to the observation of the trypsin A precursor and the ίίίΐΪΪΐ strip. In the non-specific two-dimensional gel electrophoresis strips, the distribution of the enzymes is very diverse. Partial lymphoid elastin inhibitorXM, 43kDa; pi, 5.90) . I"^ gldn_1 division (10)·) (攸114kDa; P1, 4.90), bridge 叩 test) (money 332kDa; pI, 6.44) and 1g K The fragment of the chain C (the mitochondrial (8) fragment is composed of two layers of the gel. The basic strip pattern of the electrophoresis image contains two main lines composed of trypsin II. This basic strip pattern is in the residual drive (and duodenal ulcer disease _. Phase = the electrophoresis image is mainly the special coffee strip pattern (93%). The special stripe pattern of the image and the non-special stripe pattern are highly variable; the electrophoresis 3 ΐ; she points out that at least _ of the gastric cancer patients in the gastric juice analysis and 42 methods for detecting gastric cancer are very sensitive The system is - in the patient's sputum, in the main liquid, "the anti-trypsin anterior contraction and the low acidity of the stomach are highly correlated. It may be, - the excess corpus callosal inflow / part 3,,, Can stand in low acidity gastric juice to avoid being wounded by ίί white water. The stomach of the tester does not have glandular atrophy. - Healthy subjects have obvious treasure compartment density =: septic degeneration. Potential conditions for gastric cancer, such as atrophy and small use of the aforementioned process, determine the gastric cancer with a permanent polypeptide 2187 m / z, 1 and have a relative 'contains A polypeptide biomarker, FLKKHNLNPARKYFpqw, Μ ^ proprotein propeptide #日# m/Z, also has a corresponding secretion from the pancreatic flkkhnlnparkyfp^^a and ^2 amino acid sequence (chiefcell). The production of sputum sputum/protease is reduced by the number of primary cells in the stomach, i trypsin, stomach wilting, the process, which leads to the inhibition of the pro-protein proteasome fragment of the main cell. The resulting gastric cancer is inhibited for many months in Taishan for many months. It contains a leucine zipper protein domain with a leucine zipper protein domain. The leucine zipper protein region is expressed in the leucine zipper protein domain. Tumor suppressor protein (tum〇r suppress〇rping said coffee). The expression of certain tumor suppressor proteins was found to be inhibited in human gastric cancer cell lines. The inhibition of acid zipper proteins was also observed in pancreatic cancer and mouth. Squamous cell carcinoma ((10)^ squamous cell carcinoma) and bladder cancer (Wadder cancer) have been reported. The expression of certain leucine zipper proteins induces apoptosis (叩叩t〇sis) and decreases in some human fines. The cell fraction of Lin. The leuco-chain egg from the f-peptide # segment, polymorphous 3 Μ Γ == should be - potential, tumor suppressor protein f, its surface in the stomach and pancreatic cancer cell line = oyster The identification of gastric juice in gastric cancer patients by the ''Hayalan zipper polypeptide fragment (3572m/z) implies that the corresponding spleen scorpion protein is inhibited in gastric cancer cells or atrophic gastric mucosa. The amino acid identification results from mass spectrometry also identified polypeptides whose gastric cancer was up-regulated. A polypeptide, peptide 2573 m / z, from the egg self-recorded. Because white (Lui bl / and self-formed ulcers in the stomach cancer _ tumor hidden in the bleeding of the corpse two cockroaches see the phenomenon, it may be in the gastric cancer and tumor bleeding 'liquid comes into the human gastric juice. So, the Albumin peptide ί ^1 1 ^^ t:zhi. Furthermore, the presence of albumin in gastric juice can be used as a warning, and the possibility of the presence of lunar cancer is more carefully observed. Mass spectrometric analysis of the amino acid sequence results in 11 up-regulated proteins. One protein, peptide 4132m/z, A white enzyme precursor, shore, % 疋 mouth is α1 - antitrypsin is reported in the blood, and Paniculitis, ^ trypsin can be used in the process of gastric cancer, tumor blood hemorrhage, a few because of the identification of the polypeptide and protein, in the w into the moon. The specificity is very high, higher than any ^ ^ ^ gastric cancer Sensitivity and the biomarker of gastric cancer in the stomach, in the gastric juice 30 200817682 The emergence of protein and peptide fragments in the scrapie • The early strain resistance system can be developed for screening or diagnosis of gastric cancer. , the application, the identification of the polypeptide on the shore γ The digestive decomposition of the prime is observed by the disease, and the result is a good biomarker. *Shuyi peptide tablets &amp; (not the full-length protein gastric cancer. Therefore, the difference between the different sources of cancer pointed out but not little ^ M /, from the Ministry of the Ministry. Gan also #匕出+广自Θ质和; Fragments 'Other possible tests ^,, and two are less likely to be from other organs. - Can exist in gastritis and stomach. 'Inhibition of f-albumin's upward regulation and growth are both pre-cancerous: § § shape,: atrophic stomach qier with the detection of f occurs to mention the same performance of the phfield 1 special polypeptide peptide between the inflammatory and gastric cancers. 'It is still - to determine gastric cancer or cancer: ίίίίΞ stomach: this The protein concentration of gastric juice in patients with 仏巧TM is healthier than the test 31 200817682 In terms of -, 彳目^^^^1度_立因子. Can be used as a new type of H secret gastric juice and continued protein body analysis The method and the duodenal disease 2 patients ^=ϊί'3ί ° in another aspect, the various gastric stomach and duodenum di: liquid to find the main protein components are clearly provided in the first f disease of each embodiment of the present invention The concentration of protein in the gastric juice is very different. In the gastric juice /, the stomach / field digestive biomarkers are identified as special strip states. In the gastric juice, at least $ of the new gastric juice protein, ! Becky analysis. 4 found highly pointed out, measuring the inflammatory ice ~, 'Chen Ya,,, mouth ^ protein body analysis to analyze the gastric juice in the protein Check out the new type of stomach and duodenal diseases, and ^: ^ according to the tree 0 shape specific compensation, this batch of other or further 1 /,, yoke example can apply for patent without deviating from the invention = / The scope of the invention is defined by the illustration of a simple method according to one or more of the first embodiment of the present invention. Lai Ming, one of the silk age corrections or Fig. 3 is an illustration of the collection of gastric juice samples t by mass spectrometry using different magnetic beads, such as C8 or C18, according to a specific example of the present invention. 4 is a view showing a sample of gastric juice collected by a microcentrifuge tube and preparing for mass spectrometry according to a specific embodiment of the present invention. The image of the gastric juice collected from a normal, unaffected subject is a gastric juice. Sample of 32 200817682 cases ^1_ bead, According to a specific embodiment of the present invention, according to an exemplary mass spectrum of a gastric juice sample of a patient, the root is 6B: an example of a mass spectrum of a gastric juice sample collected from a cancer subject, and the figure is an identified polypeptide biomarker. Η χ χ χ χ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Mass spectrometry of m/z, a region of gastric juice sample = 曰,,, according to a specific embodiment of the present invention. rpf: The figure is a sample of gastric juice purified by HPLC layer chromatography, after mass purification It is in accordance with a particular embodiment of the invention. r円第图图 is a mass spectrum of a gastric juice sample purified by HPLC layer chromatography, and then subjected to a mass spectrum of ϋ〇0 to 5〇〇〇m/z, and another fraction of the gastric juice sample, It is in accordance with a particular embodiment of the invention. 9/Fig. Illustrative illustration of amino acid sequence determination, which presents the results of a polypeptide flute 2 q/MS analysis, in accordance with a particular embodiment of the present invention. Wada W., a healthy tester and a population of stomach ulcers (duodenal, duodenal ulcer (DU) and lunar cancer (GC). 1 chart for healthy subjects and various gastroduodenal diseases, including gastric ulcers Gastric fluid characteristics of sputum 1, duodenal ulcer (DU) and gastric cancer (GC). Section 12® presents a single variable analysis of u clinical, bacterial and lunar fluid factors associated with high protein concentrations in gastric juice. Figure 13 shows the analysis of single variables. After that, the results of the logistic regression analysis further showed that it did not affect the independent factors of gastric protein concentration. Figure 14 shows various gastroduodenal diseases, including gastric ulcer, duodenum, TB, and gastric cancer ( GC), Statistical analysis of two-dimensional electrophoretic patterns of gastric juice. 33 200817682 Figure 15 shows the results of a cancer evolution analysis of α-antitrypsin precursors, which confirmed α-antitrypsin precursors specifically and advanced cancers ( 90%) is associated with early cancer (100%). Figure 16 shows the association between atrophic gastritis and low acidity in the stomach in a two-dimensional gel electrophoresis pattern of gastric juice. Figure 17 shows a two-dimensional gel electrophoresis pattern. Exemplary base strip This is a specific embodiment of the present invention. Figure 18 is a diagram showing an exemplary special strip pattern of a two-dimensional gel electrophoresis image, which is based on a specific embodiment of the present invention. An exemplary non-special strip pattern of a gel electrophoresis image is according to a specific embodiment of the present invention. [Description of main component symbols] (none) 34

Claims (1)

200817682 X. Patent Application Range: 1. A method for identifying biomarkers of gastric and duodenal diseases, comprising: collecting one or more gastric juice samples from a human subject; performing a mass spectrometry analysis on the one or more gastric fluid samples Generating one or more mass spectra; comparing the mass spectrum of the one or more mass spectra and a baseline quality value; and identifying an abnormal mass value of one or more molecules that are biomarkers of the stomach or duodenal disease . 2. The method of claim 1, wherein the stomach and duodenal disease are selected from the group consisting of gastric cancer, gastric ulcer, duodenal ulcer, and combinations thereof. 3. The method of claim 1, wherein the quality of the baseline quality is obtained from a sample of gastric juice of a normal, unaffected subject. 4. The method of claim 1, wherein the one or more gastric fluid samples are collected from a test selected from the group consisting of endoscopic analysis, a capsule having an absorbent cord, and combinations thereof. 5. The method of claim 1, further comprising separating the protein from the one or more gastric juice samples by a protein purification assay selected from a magnetic nanoparticle-containing protein purification test, diamond nanoparticle Groups of protein purification assays, HPLC protein purification chromatography assays, ion exchange protein purification chromatography assays, two-dimensional gels, Western blots, capillary electrophoresis, and combinations thereof. 6. The method of claim 1, wherein the mass spectrometry is selected from the group consisting of MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry, desorption electrospray 35 200817682 two-spectrum analysis, and thunder (four) acoustic separation (four) spectrometer Analyze the method of the family, the stomach, and the human subjects to collect one or more gastric juice samples; 2 the one or more gastric juice samples to perform a two-dimensional gel electricity = or more a special strip of gastroduodenal diseases; one or more strips of two different patterns of gastric juice samples that are not affected by the disease; and more than one abnormal strip of one or more molecules The one is a biomarker of duodenal diseases. Knife Department 8 · The method of claim 7 is selected from the group consisting of a stomach itch, Tian Sheng /, μ month . Guangshan monthly ulceration, duodenal ulcer and its group 9. If the scope of the patent application is 7th, the sample is collected from the test; the group consisting of one or more gastric juices and the combination of the capsules and their combinations. If there is a suction, 10. If one of the patent applications, item 7 of the 田 田 哎 / / 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂 乂Nai Lizi egg ^, diamond nanoparticle protein f purification test, muscle j test gel, Western ink point purification chromatographic analysis test, two η. For example, please include the scope of the patent =. Gel electrophoresis extraction - one or more molecules 36 200817682 Multiple anomalous bands were subjected to mass spectrometry selected from a MALDI-TOF mass spectrometry, electrospray mass spectrometry, desorption electrospray mass spectrometry and laser induced acoustic sound The population consisting of the desorption mass spectrometer analysis. 12. A method of screening a stomach and duodenal disease, comprising: collecting one or more gastric fluid samples from a human subject; screening the stomach and duodenal disease for the one or more gastric fluid samples via a qualitative analysis Or a plurality of biologically-labeled values to produce one or more mass spectra; and identifying the abnormal mass value of the one or more biomarkers as positive for the stomach or duodenal disease. 13. The method of claim 12, wherein the stomach and duodenal disease is selected from the group consisting of gastric cancer, gastric ulcer, duodenal ulcer, and combinations thereof. 14. The method of claim 12, wherein the mass spectrum of the baseline quality value is obtained from a gastric juice of a normal, unaffected subject. 15. The method of claim 12, wherein the abnormal mass value is identified as a inhibition of one or more polypeptides as compared to a baseline value. 16. The method of claim 15, wherein the one or more polypeptides comprise an amino acid sequence selected from the group consisting of sequence numbers Nos 1-3. 17. The method of claim 12, wherein the quality value of the abnormality is identified as an upwardly regulated value of one or more polypeptides as compared to a baseline value. The method of claim 17, wherein the one or more polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 4-5. 19. If the method of claim 12 is applied, the quality value of the anomaly is identified as an upward adjustment of one or more proteins compared to the baseline value. 20. The method of claim 19, wherein the one or more polypeptides comprise an alpha-antitrypsin precursor. 21. Biomarkers for stomach and duodenal diseases are identified by the method of claim 1 of the scope of the patent application. 22. The biomarker of claim 21, which comprises an amino acid sequence selected from the sequence identification numbers Nos 1-5. 23. The biomarkers of the stomach and duodenal diseases are identified by the method of claim 7 of the scope of the patent application. 24. The biomarker of claim 23, which contains an alpha-antitrypsin precursor. 25. The gastric cancer biomarker comprises an amino acid sequence which is at least 90% similar to an amino acid sequence selected from the sequence number Nos 1-5. 38