Universal method for screening protein cancer biomarkers and application thereof in nasopharyngeal carcinoma

Abstract

The invention belongs to the technical field of biology, and particularly relates to a universal method for screening protein cancer biomarkers and application thereof. The universal method for screening the protein cancer biomarkers comprises the following steps: (1) preparing a specific enrichment material to carry out peptide fragment enrichment on an actual biological sample, and carrying out primary data processing through LC-MS (Liquid Chromatography-Mass Spectrometer); and (2) further retrieving the data obtained by the primary processing through a protein database, and screening target proteins with research potential for subsequent qualitative and quantitative research. The method disclosed by the invention can be used for simply, conveniently and quickly screening potential protein type biomarkers, and the research efficiency of the biomarkers is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a universal screening method for potential protein biomarkers, and in particular to its application in nasopharyngeal carcinoma and a potential nasopharyngeal carcinoma protein biomarker. Background Art

[0002] In proteomic analysis, direct protein analysis is extremely difficult due to the naturally low abundance of proteins in biological samples and the signal suppression effects of other interfering substances. Therefore, through bottom-up proteomics approaches, the use of functionalized materials to enrich peptides corresponding to post-translationally modified proteins can obtain protein information from complex biological samples, laying the foundation for the identification of potential biomarkers. However, the acquired data is complex, requiring efficient data processing methods. At the same time, with the establishment and improvement of public protein databases, data retrieval and screening of proteins in public protein databases can effectively reduce the difficulty of raw data processing and improve analysis efficiency. However, a large amount of data is required as a search basis to mine the valuable information contained in the raw data. Based on the complementary advantages of these two research methods, protein biomarker search methods based on material enrichment and database search have emerged.

[0003] Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors with a relatively high mortality rate. So far, the literature has reported that the main treatment for NPC is radiotherapy [1], and no protein molecules have been reported as diagnostic biomarkers for NPC. Due to the challenges of early diagnosis, the five-year survival rate of NPC remains poor. In this regard, the discovery of NPC biomarkers may be an effective method to provide a reliable way to promote and simplify the early diagnosis and treatment of NPC.

[0004] [1].Linglong Tang,Lin Chen,Guiqiong Xu,Ning Zhang,etc.Reduced-volumeradiotherapy versus conventional-volume radiotherapy after inductionchemotherapy in nasopharyngeal carcinoma:An open-label,noninferiority,multicenter,randomized phase 3trial[J].CA:A Cancer Journal for Clinicians.2025. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the present invention aims to provide a novel universal screening method for potential protein biomarkers and a nasopharyngeal carcinoma biomarker discovered using this method.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A general method for screening protein cancer biomarkers comprises the following steps:

[0008] (1) Prepare specific enrichment materials to enrich peptides from actual biological samples and perform preliminary data processing by LC-MS;

[0009] (2) The data obtained from the preliminary processing are further searched through the protein database to screen target proteins with research potential for subsequent qualitative and quantitative research.

[0010] Furthermore, the specific enrichment material can be designed specifically according to different enrichment targets. Preferably, the targeted design includes hydrophilic surface modification for glycosylated peptide enrichment and high-valent metal ion surface modification for phosphorylated peptide enrichment.

[0011] Furthermore, the specific enrichment material is a material with stable properties, specifically including silicon-based nanomaterials, metal organic framework materials, covalent organic framework materials, titanium dioxide materials, graphene materials, and other qualified materials not included in the list; preferably, the material morphology can be nanoparticles, two-dimensional nanosheets, mesoporous skeletons, microporous skeletons, and other qualified morphologies not included in the list.

[0012] Furthermore, various specific enrichment materials can be used in combination to complement each other to enhance the efficiency and accuracy of peptide enrichment.

[0013] Furthermore, the actual biological sample is derived from blood, tissue, urine, and saliva.

[0014] The present invention also provides a universal screening method for potential protein biomarkers for use in nasopharyngeal carcinoma biological samples; the biological samples are blood and tissue samples; and the biomarker is afamin (P43652, Afamin, AFM).

[0015] The present invention also provides a potential cancer protein biomarker, which is a known protein, afamin (P43652, Afamin, AFM). Afamin is the scientific name of this protein, which was published in Alessandra Altamirano, Andreas Naschberger, Barbara G. Fürnrohr, etc. Expression, Purification, and Biochemical Characterization of Human Afamin. Journal of Proteome Research. 2018, 17(3): 1269-1277.

[0016] Furthermore, the cancer is nasopharyngeal carcinoma, and the above method is applied for the first time to discover expression differences in nasopharyngeal carcinoma samples.

[0017] The present invention also provides a use of the cancer biomarker in preparing or screening cancer diagnostic products. Preferably, the use includes drug development and detection technology.

[0018] Furthermore, the nasopharyngeal carcinoma drug developed based on the invention is a drug that degrades afamin and / or blocks the synthesis of afamin and / or converts afamin.

[0019] Furthermore, the detection technology includes nasopharyngeal carcinoma test kits, test strips, chips, and high-throughput sequencing platforms. Detailed description of the invention:

[0021] The first aspect of the present invention provides a universal screening method for potential protein biomarkers. The method comprises:

[0022] Acquisition module: Enrich the peptides in the biological sample using specific enrichment materials; obtain the raw data by liquid chromatography-mass spectrometry detection of the enriched samples;

[0023] Analysis module: The raw data is input into the protein database for screening; potential research subjects are identified through the screening results, and subsequent qualitative and quantitative analysis is performed.

[0024] The term "enrichment" refers to the process of concentrating and extracting low-abundance post-translationally modified peptides from biological samples through materials.

[0025] Preferably, the biological sample can be derived from blood or tissue.

[0026] Preferably, the specific enrichment material used for enrichment can be designed and prepared according to different needs.

[0027] Preferably, different protein databases can be selected for data screening according to different research needs.

[0028] Compared with the prior art, the advantages of the universal screening method for potential protein biomarkers applied in the present invention are:

[0029] The screening method of the present application can be used to screen potential protein-type biomarkers simply and quickly, thereby improving the efficiency of biomarker research.

[0030] The second aspect of the present invention provides a protein biomarker for nasopharyngeal carcinoma discovered based on the screening method, wherein the tumor biomarker is afamin (P43652, Afamin, AFM).

[0031] The term "biomarker" refers to a biological molecule that is differentially expressed in the human body under different conditions. Biomarkers include, but are not limited to, nucleic acids, proteins, and other biological molecules. In a specific embodiment of the present invention, the biomarker described is afamin (P43652, Afamin, AFM).

[0032] Preferably, the expression of afamin is upregulated in nasopharyngeal carcinoma patients compared to healthy controls.

[0033] Preferably, the up-regulated expression value is at least about 1.5 times the normal value.

[0034] Preferably, the biomarker samples are derived from human nasopharyngeal carcinoma tissue and non-cancerous hyperplasia tissue of the nasopharynx.

[0035] The afamin is highly expressed in nasopharyngeal carcinoma tissue and is lowly expressed in non-cancerous hyperplastic tissue of the nasopharynx. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The present invention provides a design process for two specific enrichment materials according to an embodiment of the present invention. The two materials are used to study the screening method proposed in the present invention for the afamin.

[0037] Figure 2 This is the basic process of the screening method according to one embodiment of the present invention. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0040] The specific implementation process of the present invention for screening and verifying protein-based nasopharyngeal carcinoma biomarkers is as follows:

[0041] First, plasma from one nasopharyngeal carcinoma patient, two nasopharyngeal carcinoma tissues, and one nasopharyngeal non-cancerous hyperplasia tissue were selected as samples for this experiment.

[0042] 1. Experimental instruments and reagents

[0043] (1) Reagents

[0044] Ammonium bicarbonate (NH4HCO3), trypsin, urea, dithiothreitol (DTT), formic acid (FA), and iodoacetamide (IAA) were purchased from Sigma-Aldrich (USA). Trifluoroacetic acid (TFA) and acetonitrile (ACN) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. PNGase F was purchased from Annolon Biotechnology Co., Ltd. (USA). Protease inhibitor cocktail was purchased from Roche (Switzerland).

[0045] 2. Sample Collection and Preparation

[0046] The obtained 10mg to 100mg tissue sample was immersed in 100μL 8M Urea PBS lysis buffer (pH=7.4, containing 1% protease inhibitor cocktail) and pre-cooled in an ice bath for 3 minutes. After the lysate has cooled, it was ground at 60Hz for 10 seconds using a handheld grinder in an ice bath, and then cooled for 30 seconds. The above grinding operation was repeated three times. After the grinding was completed, the lysate was transferred to an ultrasonic machine, ultrasonically treated for 60 seconds in an ice bath, and then cooled for 30 seconds. The above ultrasonic operation was repeated three times. After the ultrasonication was completed, the lysate was centrifuged at 20000rpm for 20 minutes at 4°C, the supernatant was aspirated, and its protein concentration was determined by the BCA method.

[0047] Accurately measure the above tissue lysate containing 1 mg of protein and ultrasonically dissolve it in 100 μL Urea solution (8 M, dissolved in NH4HCO3 buffer). Subsequently, add 5 μL DTT solution (200 mM, dissolved in NH4HCO3 buffer) and incubate in a 56°C constant temperature shaker for 45 minutes. After the reaction is completed, add 20 μL IAA solution (200 mM, dissolved in NH4HCO3 buffer) and incubate in a 37°C constant temperature shaker in the dark for 30 minutes. Subsequently, add NH4HCO3 buffer until the final volume of the solution is 1 mL, and add 0.04 mg Trypsin, and incubate in a 37°C constant temperature shaker for 17 hours. Finally, add 5 μL FA to terminate the reaction to obtain a protein concentration of 1 mg ml -1 of tissue enzymatic hydrolysate.

[0048] The enzymatic hydrolysis process of NPC patient plasma was the same as described above, and the final protein concentration was 1 mg ml -1 of plasma enzymatic hydrolysate.

[0049] 3. Enrichment of target peptides

[0050] Using two self-prepared specific enrichment materials, the glycosylated peptides were enriched in the plasma hydrolysate and nasopharyngeal carcinoma tissue hydrolysate of nasopharyngeal carcinoma patients based on the complementary enrichment specificity generated by different pore structures. 2 μL of the above plasma / tissue hydrolysate was centrifuged and lyophilized and then redissolved in 400 μL of glycosylated peptide loading solution (V 乙腈 :V 水 :V 三氟乙酸 =90:8:2). After dissolution, 100 μL of glycosylated peptide loading solution (containing 200 μg of material) was added to the solution, and the mixture was vortexed at room temperature for 30 minutes. Subsequently, after thorough centrifugation at 10,000 rpm, the supernatant was discarded and 100 μL of glycosylated peptide eluent (V 乙腈 :V 水 :V 三氟乙酸 =90:9.9:0.1) dispersed material, the mixture was vortexed at room temperature for 5 minutes, centrifuged thoroughly at 10,000 rpm, and the supernatant discarded. The elution step was repeated three times. After the final centrifugation and supernatant discarded, 100 μL of glycosylated peptide eluent (3% TFA solution) was added, the mixture was vortexed at room temperature for 20 minutes, and centrifuged thoroughly at 10,000 rpm. The supernatant was collected and the elution step was repeated three times. The resulting supernatant was the enriched glycosylated peptide solution, which was used for subsequent LC-MS mass spectrometry analysis.

[0051] The enriched glycosylated peptide solution was deglycosylated. The collected eluate was lyophilized, and 17 μL of H₂O, 2 μL of 10X GlycoBuffer 2, and 1 μL of PNGase F were added to the solution. The lyophilized powder was reconstituted and incubated at 37°C with shaking for 17 hours. After incubation, the solution was centrifuged and lyophilized again for subsequent LC-MS analysis.

[0052] 4. Obtaining Raw Data Using LC-MS

[0053] (1) Sample preparation

[0054] The sample was desalted and redissolved in 20 μL of 1% FA solution, from which 5 μL was taken for subsequent LC-MS detection.

[0055] (2) Liquid chromatography conditions

[0056] Liquid chromatography conditions: Thermo Scientific TM Acclaim TM PepMap TM 100C18 (75μm×50cm, 2μm particle size, Pore ​​diameter); Pressure limit: 900 Bar; Mobile phase composition: Mobile phase A is a mixture of FA and H2O in a volume ratio of 0.1 / 99.9, and mobile phase B is a mixture of FA, ACN, and H2O in a volume ratio of 0.1 / 80 / 19.9. Elution gradient: 0-1 min: 2%-8% B, 1-90 min: 8%-25% B, 90-110 min: 25%-55% B, 110-111 min: 55%-95% B, 111-132 min: 95% B, 132-162 min: 100% A; Mobile phase flow rate: 0.2 mL min -1 .

[0057] (3) Mass spectrometry conditions:

[0058] Electrospray voltage: 2200 V; column temperature: 60°C; operating mode: positive ion mode; detection resolution: 6000; primary mass spectrometry detection range: 350–1600 m / z; maximum activation time: 45 ms; AGC: 300,000. In MS / MS, HCD energy: 28%; resolution: 15,000; maximum activation time: 22 ms, dynamic exclusion time: 15 s; AGC: 100,000.

[0059] (4) Preliminary data processing

[0060] Raw data (.raw) were uploaded to Proteome Discoverer (2.2.0.388) and matched against the UniProt-SwissProt database using the Sequest HT engine. Search parameters were as follows: trypsin digestion type; maximum mass tolerance: parent ion 10 ppm, fragment ion 0.05 Da; maximum allowed missed cleavage sites: 2; modification type: methionine oxidation and asparagine deamidation as variable modifications, cysteine ​​carbamidomethylation as fixed modification; false positive rate: less than 1%. N-glycosylation sites must meet the sequence pattern of N-!PS / T / C.

[0061] (5) Database screening and subsequent quantitative analysis

[0062] The data sets generated through preliminary LC-MS data processing were intersected and then screened using the UniProt protein database. Alternatively, they could be screened using Al-related big data models, and potential research subjects were selected based on protein-related diseases and physiological manifestations. After this screening, alfamyl was selected as the subject for further research.

[0063] Furthermore, enzymatic hydrolysates from two nasopharyngeal carcinoma tissues and one nasopharyngeal non-cancerous hyperplasia tissue were selected for quantitative analysis of afamin using a commercially available ELISA kit. The specific operation procedures were consistent with the kit instructions.

[0064] According to quantitative results, the content of afamin in cancer tissues accounts for about 1.5% of the total protein content, while in non-cancerous hyperplastic tissues it only accounts for about 1%.

[0065] This study shows that the content of afamin in nasopharyngeal carcinoma tissue is higher than that in non-cancerous hyperplasia tissue of the nasopharynx, and it is a potential protein biomarker for nasopharyngeal carcinoma.

[0066] (6) Application of afamin in detection method development and drug development

[0067] Based on the differences in the expression of alphamin in affected tissues of patients and corresponding tissues of healthy controls, various detection methods for alphamin can be designed to detect its expression in corresponding parts of the human body, such as fluorescence and electrochemical methods, thereby enabling early diagnosis of nasopharyngeal carcinoma.

[0068] Based on the differences in the expression of afamin in the affected tissues of patients and the corresponding tissues of healthy people, drugs can be designed that target afamin to degrade afamin and / or block afamin synthesis and / or convert afamin, thereby providing a certain degree of treatment for nasopharyngeal carcinoma.

[0069] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A universal method for screening protein cancer biomarkers, characterized in that: The steps include: (1) Prepare specific enrichment materials to enrich peptides from actual biological samples and perform preliminary data processing by LC-MS; (2) The data obtained from the preliminary processing are further searched through the protein database to screen target proteins with research potential for subsequent qualitative and quantitative research.

2. The universal method for screening protein cancer biomarkers according to claim 1, characterized in that: The specific enrichment material can be designed specifically according to different enrichment targets; preferably, the targeted design includes hydrophilic surface modification for glycosylated peptide enrichment and high-valent metal ion surface modification for phosphorylated peptide enrichment.

3. The universal method for screening protein cancer biomarkers according to claim 2, characterized in that: The specific enrichment material is a material with stable properties, specifically including silicon-based nanomaterials, metal organic framework materials, covalent organic framework materials, titanium dioxide materials, graphene materials, and other qualified materials not listed; preferably, the material morphology can be nanoparticles, two-dimensional nanosheets, mesoporous skeletons, microporous skeletons, and other qualified morphologies not listed.

4. The universal method for screening protein cancer biomarkers according to claim 2, characterized in that: Various specific enrichment materials can be used in combination to enhance the efficiency and accuracy of peptide enrichment.

5. The universal method for screening protein cancer biomarkers according to claim 1, characterized in that: The actual biological samples are derived from blood, tissue, urine and saliva.

6. Application of a universal screening method for potential protein biomarkers in nasopharyngeal carcinoma biological samples; the biological samples are blood and tissue samples; the biomarker is afamin (P43652, Afamin, AFM).

7. A potential cancer protein biomarker, characterized in that: The biomarker is the known protein Afamin P43652, Afamin, AFM.

8. The potential cancer protein biomarker according to claim 7, characterized in that: The cancer is nasopharyngeal carcinoma, and the method described in claim 1 is applied to nasopharyngeal carcinoma samples for the first time to discover expression differences.

9. Use of the cancer biomarker according to claim 7 in the preparation or screening of cancer diagnostic products, preferably, the use includes drug development and detection technology.

10. Use of the cancer biomarker according to claim 9 in preparing or screening cancer diagnostic products, characterized in that: The nasopharyngeal carcinoma drug developed based on the invention is a drug that degrades afamin and / or blocks afamin synthesis and / or converts afamin; the detection technology includes a nasopharyngeal carcinoma kit, test paper, chip, and high-throughput sequencing platform.

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