Biomarkers for lung cancer detection and their applications

Abstract

The present invention belongs to the technical field of cancer diagnosis, and specifically relates to biomarkers for lung cancer detection and their applications. The present invention has found, through research on non-small cell lung cancer, amino acid modifications with diagnostic and therapeutic significance in hemoglobin α subunit and β subunit and pulmonary surfactant-associated protein A1 of patients with non-small cell lung cancer, thereby providing a new non-small cell lung cancer detection marker. The difference in amino acids reveals from another level the impact of the diversity of biological macromolecules on the homeostasis of the body, and the abnormal modification or variation of macromolecules is the original inducing factor of many diseases, especially chronic diseases and cancer. Therefore, compared with conventional abnormal protein quantification, the present invention has earlier and more effective diagnostic value, as well as higher therapeutic reference value, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer diagnosis, and in particular relates to biomarkers for lung cancer detection and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Lung cancer is divided into small cell lung cancer and non-small cell lung cancer, of which non-small cell lung cancer accounts for about 80% to 85%. It is estimated that 80% of malignant tumors are related to environmental influences, and the cancer caused by environmental factors and genetic carcinogenic factors comes from the damage of cell DNA structure.

[0004] More than 95% of hemoglobin in a normal adult is a tetramer composed of two α subunits and two β subunits (α2β2). Hemoglobin is composed of about 96% globulin and 4% heme, and each subunit contains one molecule of globulin and one molecule of ferrous heme. Each globulin is connected by hydrogen bonds, and its polypeptide chain is mostly folded in the form of α helix, and the helices are connected by non-helical short segments, thus forming a pocket-like structure that firmly binds heme.

[0005] As one of the most important respiratory proteins, hemoglobin is the most widely distributed and can reversibly bind to oxygen molecules (oxygenated hemoglobin). With the in-depth study of hemoglobin, its energy storage, phenoloxidase-like activity, antibacterial and antiviral functions have also been reported one after another. It has now become one of the ideal model molecules for studying the multiple biological functions of proteins. Recent studies have found that hemoglobin is also expressed in non-erythroid cells, and plays an important role in resisting pathogen invasion and regulating the body's natural immunity. Abnormal hemoglobin is a group of hereditary hemoglobin diseases caused by defects in the globin gene, which changes the primary structure of globin, thereby causing changes in the molecular structure of hemoglobin. At present, more than 700 abnormal hemoglobins have been discovered, most of which are mutations of the α chain and β chain, and are mainly single base substitutions, and most of them are clinically asymptomatic. About 30% of abnormal hemoglobins can cause abnormal physical and chemical properties and physiological functions of hemoglobin, resulting in various degrees of clinical symptoms.

[0006] Existing studies have confirmed that tumor lesions often have hypoxia symptoms. Hemoglobin is the main oxygen carrier of the human body, and its concentration can directly affect the blood oxygen content of the human body and the local oxygen supply of the tumor. Literature reports that hemoglobin concentration has a significant effect on the short-term efficacy and local control rate of tumor radiotherapy, and the possibility of hemoglobin as a tumor marker remains to be explored.

[0007] At the same time, pulmonary surfactant-associated protein A1 (SP-A or PSP-A or SP-A1) is the most abundant protein component in pulmonary surfactant, mainly distributed on the bronchial surface and alveolar air-liquid interface. Studies have found that pulmonary surfactant is a lipid and protein complex synthesized and secreted by alveolar type II epithelial cells and bronchiolar cells, of which 90% is a phospholipid part mainly composed of dipalmitoyl phospholipids (DP-PC) and phosphatidylglycerol (PG), and 8% to 10% is a protein that specifically binds to lipids, namely pulmonary surfactant-associated protein (SP). Currently, four types of SP have been discovered, namely SP-A, SP-B, SP-C, and SP-D. Among them, SP-A is the most abundant, accounting for about 50% of the total SP. Studies have also found that changes in the quality and quantity of SP-A are closely related to lung diseases in mammals. It participates in the metabolism of PS and reduces surface tension, plays an important role in the host defense of the lung, connects innate and acquired immunity, and has the effects of inhibiting inflammation and enhancing antigen clearance. It plays an important role in maintaining the normal physiological function of PS and regulating local immune and inflammatory responses. However, it has not been reported in lung cancer detection. Summary of the invention

[0008] Based on the above-mentioned deficiencies of the prior art, the present invention provides biomarkers for lung cancer detection and their applications. The present invention has discovered amino acid modifications with diagnostic and therapeutic significance in hemoglobin α subunit and β subunit and pulmonary surfactant-associated protein A1 through research on lung cancer, especially non-small cell lung cancer, thereby providing new non-small cell lung cancer detection markers. Based on the above-mentioned research results, the present invention is completed.

[0009] The specific technical solutions of the present invention are as follows:

[0010] In a first aspect of the present invention, a biomarker for lung cancer detection is provided, wherein the biomarker comprises amino acid site modifications of related proteins and / or protein subunits;

[0011] Wherein, the proteins and / or protein subunits include mitochondrial single-stranded DNA binding protein, hemoglobin α subunit and β subunit, and pulmonary surfactant-associated protein A1;

[0012] The amino acid site modifications of the related proteins and / or protein subunits include:

[0013] a) Tyrosine modification at position 43 of hemoglobin alpha subunit 1 (HBA1);

[0014] b) modification of tryptophan 15, tryptophan 37, lysine 66 and threonine 84 of the hemoglobin β subunit;

[0015] c) Single oxidation modification of proline 97 in pulmonary surfactant-associated protein A1.

[0016] The lung cancer is preferably non-small cell lung cancer;

[0017] In the mass spectrometry detection of non-small cell lung cancer and normal lung tissue, it was found that whether the molecular weight of the 43rd tyrosine in the hemoglobin α subunit 1 protein molecule shifted to -44.03Da also had a significant statistical difference, which can be used to determine the occurrence of non-small cell lung cancer.

[0018] At the same time, according to the present invention, the molecular weight of lysine at position 66 of hemoglobin β subunit in cancer patient lesions is shifted by 42.01Da, which is lower than that in normal tissues; it has been verified that the above mass shift is derived from the acetylation of amino acids, and the content of acetylated lysine in lung cancer tissues is greatly reduced. The cancer tissue of lung cancer patients is lower than the hemoglobin β subunit with a molecular weight shift of 79.966Da at position 84 of threonine in normal tissues, and the above mass shift is derived from phosphorylation. The data of the present invention show that the phosphorylated threonine at position 84 of hemoglobin β subunit in lung cancer tissues is greatly reduced.

[0019] Existing studies have shown that tryptophan residues have a large hydrophobic surface, and the indole ring structure of tryptophan is highly reactive and particularly susceptible to attack by reactive oxygen species, resulting in corresponding modifications, playing a unique role in protein folding and function; the tryptophan side chain of hemoglobin can be catalyzed by its own heme to undergo oxidation reactions, resulting in a series of modifications such as single oxidation, double oxidation, trioxidation, and kynurenine. The two tryptophan residues of the β subunit of hemoglobin are located at positions 15 and 37, respectively. The present invention has verified that a series of oxidative modifications of the 15th and 37th tryptophans of the β subunit of hemoglobin were detected in non-small cell lung cancer tissues, and these oxidative modifications were significantly downregulated in cancer tissues, suggesting that the oxidative modification levels of the 15th and 37th tryptophans of the β subunit of hemoglobin can be used to characterize the hypoxia of cancer tissues.

[0020] In addition, through the analysis of mass spectrometry detection results, lung surfactant-associated protein A1 was obtained, in which the 97th monooxidized proline accounted for 81.6% of the total modification of the protein; the P value of the 97th monooxidized proline was 8.6e-4, which is less than 0.001, that is, the 97th monooxidized proline modification of lung surfactant-associated protein A1 had a very significant statistical difference between non-small cell lung cancer and normal tissues, and the data showed that the modification was downregulated 11.6 times in non-small cell lung cancer tissues than in normal tissues.

[0021] Therefore, the above biomarkers can be used for the diagnosis of lung cancer, especially non-small cell lung cancer.

[0022] It should be noted that the non-small cell lung cancer referred to in the present invention includes but is not limited to squamous cell carcinoma, large cell carcinoma or adenocarcinoma.

[0023] The second aspect of the present invention provides the use of a substance for detecting the above-mentioned biomarker in the preparation of a lung cancer diagnosis product.

[0024] The product can be a lung cancer diagnosis kit.

[0025] Therefore, the third aspect of the present invention provides a lung cancer diagnosis kit, which includes substances for detecting the above-mentioned biomarkers;

[0026] Specifically, it may include cell / tissue sample processing reagents, protein extraction reagents, peptide enzymatic hydrolysis reagents, mass spectrometry detection auxiliary reagents, etc. Those skilled in the art may select appropriate reagents or products based on actual conditions and use corresponding devices for detection, which will not be described in detail here.

[0027] In a preferred embodiment, the lung cancer is non-small cell lung cancer, including but not limited to squamous cell carcinoma, large cell carcinoma or adenocarcinoma.

[0028] At the same time, the above biomarkers can also be used as corresponding targets for lung cancer treatment. Therefore, the fourth aspect of the present invention provides the use of the above biomarkers as drug targets in the preparation of anti-lung cancer drugs.

[0029] In a preferred embodiment, the lung cancer is non-small cell lung cancer, including but not limited to squamous cell carcinoma, large cell carcinoma or adenocarcinoma.

[0030] The medicament may also include at least one pharmaceutically inactive ingredient.

[0031] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy. Moreover, according to the usual methods, the drug can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0032] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0033] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0034] In another specific embodiment of the present invention, the medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be optionally administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention can vary greatly depending on various factors, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc. It is not specifically limited here.

[0035] Beneficial technical effects of the above technical solution:

[0036] The above technical solution provides a series of amino acid modification sites on proteins and protein subunits that can be used as diagnostic markers for lung cancer, especially non-small cell lung cancer. The difference in amino acids reveals the influence of the diversity of biological macromolecules on the homeostasis of the body from another level, and the abnormal modification or mutation of macromolecules is the original inducing factor of many diseases, especially chronic diseases and cancer. Therefore, compared with conventional abnormal protein quantification, the present invention has earlier and more effective diagnostic value, as well as higher therapeutic reference value, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 is the secondary spectrum of the target peptide in Example 1;

[0039] Figure 2 The detection information of tyrosine 43 (-44.03Da) of HBA1 in the sample of Example 1;

[0040] Figure 3is the ROC curve in Example 1. Note: the AUC of the ROC curve is 0.87, and the critical value is 3.5;

[0041] Figure 4 It is an indicator map of acetylated lysine sites in intact hemoglobin;

[0042] Figure 5 This is a simulation diagram of the interaction between lysine 66 and the heme porphyrin ring through water;

[0043] Figure 6 is the secondary spectrum of the target peptide described in Example 2;

[0044] Figure 7 is the distribution of acetylated lysine in the sample described in Example 2;

[0045] Figure 8 is the ROC curve of non-small cell lung cancer and adjacent tissues described in Example 2;

[0046] Fig. 9 This is the secondary spectrum of the peptide segment containing the 84th phosphorylated threonine in Example 3;

[0047] Fig.10 The phosphorylated threonine detection information in the sample described in Example 3;

[0048] Fig.11 is the ROC curve of phosphorylated threonine at position 84 of hemoglobin in cancer tissue and adjacent tissue in Example 3;

[0049] Fig.12 Schematic diagram of the positional relationship between the 15th and 37th tryptophan residues of the hemoglobin β subunit and ferrous protoporphyrin in Example 4;

[0050] Fig.13 The secondary spectrum of the target peptide (hemoglobin 15W (+15.99)) described in Example 4;

[0051] Fig.14 is the secondary spectrum of the target peptide (hemoglobin 15W (+31.99)) described in Example 4;

[0052] Fig.15 is the secondary spectrum of the target peptide (hemoglobin 37W (+15.99)) described in Example 4;

[0053] Fig.16 is the secondary spectrum of the target peptide (hemoglobin 37W (+31.99)) described in Example 4;

[0054] Fig.17 The detection information of oxidized tryptophan at positions 15 and 37 of hemoglobin in Example 4;

[0055] in, Fig.17 A is the expression of hemoglobin 15W (+15.99) in non-small cell lung cancer tissues and adjacent tissues;

[0056] Fig.17 B is the expression of hemoglobin 37W (+15.99) in non-small cell lung cancer tissues and adjacent tissues;

[0057] Fig.17 C is the expression of hemoglobin 15W (+31.99) in non-small cell lung cancer tissues and adjacent tissues;

[0058] Fig.17 D is the expression of hemoglobin 37W (+31.99) in non-small cell lung cancer tissues and adjacent tissues;

[0059] Fig.18 is the ROC curve of the oxidized tryptophan at positions 15 and 37 of hemoglobin in Example 5;

[0060] in, Fig.18 A is the ROC curve of hemoglobin 37W (+15.99);

[0061] Fig.18 B is the ROC curve of hemoglobin 37W (+15.99);

[0062] Fig.18 C is the ROC curve of hemoglobin 15W (+31.99);

[0063] Fig.18 D is the ROC curve of hemoglobin 37W (+31.99);

[0064] Fig.19 is the secondary spectrum of the target peptide in Example 6;

[0065] Fig. 20 The detection information of monooxidized proline in the sample of Example 6;

[0066] Fig.21 This is the ROC curve in Example 6. Note: the AUC of the ROC curve is 0.85, and the critical value is 4.5. DETAILED DESCRIPTION

[0067] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0068] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0069] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0070] Example 1: Data analysis method for the molecular weight deviation of tyrosine at position 43 of hemoglobin alpha subunit 1 (HBA1) by 44.03 Da

[0071] A total of 17 pairs of samples were used in this experiment, 17 of which were cancer tissues and 17 were normal lung tissues.

[0072] The sample tissue was processed into pieces smaller than 1 mm 2 The tissue block was added with RIPA lysis buffer (tissue: lysis buffer = 1:100 (1mg / 100μl)), oscillated and mixed, and treated on ice for 30min; then the grinding beads after ice bath were added to the sample tube. The sample tube was ground 3 times, each treatment time was 30 seconds; the grinding beads were taken out and subjected to ice bath ultrasonic treatment, and the specific treatment conditions were: 5 seconds each time, ultrasonic 3 times, vortex treatment for 30 seconds; then treated at 4℃, 10000g for 10min, and centrifuged. The supernatant was taken into a new EP tube, and 5 times the volume of -40℃ pre-cooled acetone was added to the EP tube, and vortexed for 10 seconds. Then placed at -40℃ to precipitate protein for 1 hour. Centrifuged at 4℃, 10000g for 10 minutes, and then the supernatant was discarded. The same volume of 80% acetone (water) was added to the precipitate, and the protein precipitate was washed by shaking again. 100mM ammonium bicarbonate solution was added to the precipitate to dissolve the precipitate, and solubilized under low temperature ultrasonic conditions. The protein concentration of the samples was determined using the BCA method.

[0073] Trypsin [KR | P] (abbreviated as Trypsin) was added to the sample tube at a ratio of total sample protein: Trypsin = 50:1 (μg:μg), and incubated at 37°C and 700 rpm for 12 hours under a nitrogen atmosphere. Then, 1 / 2 of the first amount of Trypsin was added to the sample, and the incubation was continued at 37°C and 700 rpm for 2 hours under a nitrogen atmosphere.

[0074] The digested peptides were desalted using a desalting column (Waters Sep-Pak Vac 1cc (50mg) C18 Cartridges), and the concentration of the desalted peptides was determined using a BCA peptide concentration determination kit.

[0075] Specifically, 100 μg of peptide sample was taken and fractionated using Waterse2695 (Separations Module) liquid phase. Each sample was divided into three levels. The following fractionation process was followed (conventional peptide high pH reverse phase fractionation): ① Buffer A: 10 ml 100× stock solution, 970 ml water, 20 ml acetonitrile; Buffer B: 10 ml 100× stock solution, 10 ml water, 980 ml acetonitrile; 100× stock solution (100 ml): 13.4 ml concentrated ammonia water, 86.6 ml water. ② Run the chromatograph and flush the chromatograph pipeline. ③ Take 100 μl of sample into a sample bottle without residue, start sample loading, set the liquid phase flow rate to 0.1 ml / min, the temperature of the chromatographic column thermostat was controlled at 50°C, wavelength (Wavelength): 214 nm; bandwidth (bandwidth): 12 nm. ④ Prepare 2 ml flat-bottom EP tubes, label them in sequence and place them in the sample collector to collect the components of the sample. ⑤ Divide the collected components into three groups in time sequence, thereby combining the sample components of each group, and then vacuum centrifuge to dry the sample.

[0076] The samples were dissolved in 0.1% formic acid (water) and centrifuged at 20,000 g at 4°C for 10 min.

[0077] The separation was performed using an EASY-nLC 1200 liquid phase system, and the detection in DDA mode was performed using an online Thermo QE-HFX mass spectrometer. The liquid phase separation system parameters included: precolumn: 150 μm*3 cm C18 1.9 μm Reprosil-Pur120; chromatographic column: 150 μm*25 cm C18 1.9 μm Reprosil-Pur 120; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); flow rate: 600 nl / min; specific gradient elution program: 4%-7% B for 1 min, 7%-25% B for 94 min, 25%-40% B for 16 min, 40%-100% B for 5 min, 100% for 4 min; the sample load was controlled to 5 μl.

[0078] Mass spectrometry detection parameters include: scanning range: 150-2000 m / z; data acquisition mode adopts DDA acquisition mode, top 20 fragmentation spectrum; MS1 resolution (200 m / z): 60,000; MS1 AGC target: 3e 6 ,maxIT:50ms;MS2 resolution (200m / z):15,000;MS2 AGC target:5e 4 ,maxIT:45ms; Normalized Collision Energy (NCE): 28%, isolation window specifically 1.6m / z.

[0079] The mass spectrometry results were analyzed for hemoglobin alpha subunit 1 (HBA1), in which the molecular weight of tyrosine at position 43 was shifted by 44.03 Da, accounting for 4.3% of the total modification of the protein.

[0080] The secondary spectrum of the peptide TY(-44.03Da)FPHFDLSHGSAQVK containing the 43rd tyrosine with a molecular weight offset of 44.03Da ( Figure 1 ), the secondary spectrum contains more than 82.1% of the ion fragments (except for the daughter ions of b1 and y1), and the presence of y14 and b3 and 82.1% of the b ions indicates the credibility of the modification and related information.

[0081] The invention point of the present invention is: the molecular weight of tyrosine at position 43 is shifted by 44.03Da. Its P=3.2e-4<0.001, that is, the molecular weight of tyrosine at position 43 of hemoglobin alpha subunit 1 (HBA1) is shifted by 44.03Da, which has a very significant statistical difference between non-small cell lung cancer and non-small cell lung cancer adjacent tissues, and the data show that the modification is downregulated 18.9 times in non-small cell lung cancer tissues than in normal lung tissues. ( Figure 2 )

[0082] The relevant information of Deltamass is as follows: the protein to which Deltamass belongs: P69905 (hemoglobin α subunit 1, HBA1); the position of Deltamass in HBA1: 43; the peptide to which Deltamass belongs: TYFPHFDLSHGSAQVK; the molecular weight of Deltamass: 44.03; Deltamass score: 0.745 (Deltamass score means: in mass spectrometry detection, due to various extremely small effects of Deltamass, the result of a single Deltamass measured by mass spectrometry is normally distributed within the range of accuracy and error, and the score is the degree of fit to the normal curve, with a score of 0 to 1. The higher the score, the more reliable the test result. A secondary spectrum that can reach 0.2 will be recognized).

[0083] The ROC curve of the molecular weight deviation of 44.03Da of tyrosine at position 43 of HBA1 in non-small cell lung cancer and normal tissue is as follows ( Figure 3 ).

[0084] From the above information, it can be seen that the tyrosine with a molecular weight offset of 44.03 Da at position 43 of the HBA1 protein has important guiding and suggestive significance for the diagnosis and treatment of patients with non-small cell lung cancer.

[0085] In the above content of this example, the target peptide segment was subjected to subsequent experiments after the sample protein was hydrolyzed using Trypsin [KR|P]. In mass spectrometry, a variety of enzymatic sample digestion schemes can also be used, such as Trypsin (semi) [KR | P]; Trypsin / p [KR | -]; Trypsin K [K | P]; Trypsin R [R | P]; ChymoTrypsin [FWYL | P]; Arg C [R | P]; Asp N [- | D] n-term; Clostripain [R | -]; CNBr [M | P]; Elastase [GVLIA | P]; Formic Acid [D | P]; Glu C [DE | P]; Glu C bicarb [E | P]; Iodosobenzoate [W | -]; Lys C [K | P]; Lys C / P [K | -]; Lys N [- | K] n-term; Lys N promisc [- | KASR] n-term; Pepsin A [FL | P]; Protein endopeptidase[P|-];Staph protease[E|-];Trypsin-CNBr[KRM|P];Trypsin-GluC[DEKR|P], etc. The molecular weight shift of 44.03Da of tyrosine at position 43 of HBA1 can also be detected in samples treated by these enzymatic hydrolysis or physical schemes such as ultrasound. In addition, the modification described in the present invention can also be enriched and detected by immunology and protein interaction schemes.

[0086] Application of the molecular weight shift of 44.03Da for tyrosine 43 in HBA1

[0087] A rapid mass spectrometry method for detecting the molecular weight shift of 44.03 Da at tyrosine 43 in HBA1.

[0088] The mass spectrometry data measured in Example 1 were analyzed using skyline analysis to obtain the chromatographic retention time of the target peptide TY (-44.03Da) FPHFDLSHGSAQVK. The retention time was combined with the target peptide 2-valent and 3-valent parent ions and their corresponding daughter ions to construct an ion list of PRMs collected during mass spectrometry detection. (See Table 1. Ion list. Each parent ion can correspond to multiple 1-valent, 2-valent, and 3-valent daughter ions. For ease of detection, the present invention only lists the daughter ion fragments with relatively high relative abundance)

[0089] Table 1. Divalent and trivalent precursor ions of target peptides and their corresponding product ions

[0090]

[0091]

[0092] The collected samples were processed according to Example 1.

[0093] The separation was performed using an EASY-nLC 1200 liquid phase system and detected using an online Thermo QE-HFX mass spectrometer. The liquid phase separation system parameters were as follows: precolumn: 75μm*2cm PepMap C18 3μm Thermo; chromatographic column: 75μm*15cm PepMapC18 2μm Thermo; flow rate: 500nl / min; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); elution gradient: 6%-25% B for 21min, 25%-40% B for 4min, 40%-100% B for 3min, 100% B for 2min; sample loading volume: 3μl. Mass spectrometry detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragmentation spectrum; MS2 resolution (200m / z): 30,000; MS2 AGC target: 2e 5 ,maxIT:70ms; Normalized Collision Energy (NCE):28%; Isolation Window:1.6m / z.

[0094] Adjust the mass spectrometer parameters to quickly acquire and detect samples based on the PRM ion list.

[0095] Skyline is used to analyze the data measured by the mass spectrometer, and the target ion fragments are relatively or absolutely quantified using internal or external standards. Then, the samples are qualitatively or quantitatively analyzed based on the quantitative results of the ion fragments.

[0096] Determine whether a patient has non-small cell lung cancer based on the qualitative or quantitative precursor of the sample, or use it as an indicator to assist its diagnosis.

[0097] Conventional immunoassay schemes or protein interaction schemes can also detect target modifications. The present invention only describes the PRM scheme in detail.

[0098] The tyrosine molecular weight offset of 44.03 Da at position 43 of hemoglobin alpha subunit 1 (HBA1, Uniprot ID: P69905) described in this example can also be used as a therapeutic target for non-small cell lung cancer. Different forms of protein or small molecule drugs can be prepared for this target.

[0099] Detection validation set: 3 healthy samples and 3 clinically diagnosed non-small cell lung cancer patient samples (not belonging to the samples in the analysis set of Example 1) were used as research objects for validation, and the detection results were consistent with the known results.

[0100] Example 2: Screening and diagnostic method for acetylation modification of lysine 66 at hemoglobin as a marker

[0101] In this example, 17 pairs of lung cancer and distal cancer samples were used to screen for non-small cell lung cancer markers. The screening step includes processing tissue samples in non-small cell lung cancer and confirming the acetylated amino acid sites according to the mass spectrometry detection results. According to the analysis results of this example, the acetylation modification of lysine 66 at the hemoglobin β subunit has diagnostic significance for non-small cell lung cancer.

[0102] 1. Screening of acetylation modification of lysine 66 at hemoglobin as a marker

[0103] 1. Mass spectrometry method for non-small cell lung cancer specimens: The samples, sample processing and mass spectrometry detection method are the same as in Example 1.

[0104] 2. Data analysis method for acetylated lysine 66 modification of hemoglobin β subunit (hemoglobin)

[0105] The mass spectrometry results were analyzed to obtain the modification of hemoglobin β subunit (hemoglobin), in which the acetylated lysine at position 66 accounted for 89.4% of the acetylated modification of the protein. Among them, the peptide segment with acetylated modification of lysine at position 66 K (+42.01)VLGAFSDGLAHLDNLK's secondary spectrum ( Figure 6 ), the secondary spectrum contains more than 87% of the ion fragments (excluding daughter ions of b1 and y1), and the existence of 80% (excluding b1) of b ions including b2~b4 fully proves the credibility of the modification.

[0106] Delta mass belongs to protein: P68871 (hemoglobin); Delta mass position: 66 (K); Delta mass belongs to peptide: KVLGAFSDGLAHLDNLK; Delta mass molecular weight: 42.01Da; Delta mass score: 0.912. (Delta mass score means: In mass spectrometry detection, due to various extremely small effects, the single Delta mass result measured by mass spectrometry is normally distributed within the accuracy and error tolerance range, and the score is the fit to the normal curve, with a score of 0 to 1. The higher the score, the more reliable the test result. A secondary spectrum that can reach 0.2 will be recognized.

[0107] According to the statistical analysis results, the P value of acetylation of lysine at position 66 of hemoglobin β subunit was 0.015 (P<0.05), which means that this modification has a significant statistical difference between lung cancer tissue and adjacent tissue. The acetylated lysine at position 66 of hemoglobin β subunit in cancer tissue is downregulated by 2.02 times compared with adjacent tissue (see Figure 7 ). At the same time, the ROC curves of non-small cell lung cancer and adjacent tissues are as follows Figure 8 From the above information, we can know that the acetylation of lysine 66 in hemoglobin β subunit (hemoglobin, Uniprot ID: P68871) is of great significance and value for the diagnosis of non-small cell lung cancer, and can also be used as a therapeutic target for non-small cell lung cancer. Different forms of protein or small molecule drugs can be prepared for this target.

[0108] 2. Method for diagnosing non-small cell lung cancer based on acetylation modification of lysine 66 of hemoglobin

[0109] In this embodiment, a method for diagnosing non-small cell lung cancer based on acetylation modification of lysine 66 at position of hemoglobin β subunit is also provided.

[0110] The diagnostic method comprises:

[0111] 1. Collect samples: Process the collected samples according to the sample pretreatment method in Example 1.

[0112] 2. Rapid mass spectrometry detection of acetylated lysine modification at position 66 of hemoglobin in the sample: The mass spectrometry data measured by skyline analysis was used to obtain the chromatographic retention time of the target peptide K (+42.01) VLGAFSDGLAHLDNLK. The retention time was combined with the target peptide 2-valent and 3-valent parent ions and their corresponding daughter ions to construct an ion list of PRMs collected for mass spectrometry detection. (See Table 2. Ion list. Each parent ion can correspond to multiple 1-valent, 2-valent, and 3-valent daughter ions. For ease of detection, this example only lists the daughter ion fragments with relatively high relative abundance)

[0113] Table 3. Divalent and trivalent precursor ions of target peptides and their corresponding product ions

[0114]

[0115] The separation was performed using an EASY-nLC 1200 liquid phase system and detected using an online Thermo QE-HFX mass spectrometer. The liquid phase separation system parameters were as follows: precolumn: 75μm*2cm PepMap C18 3μm Thermo; chromatographic column: 75μm*15cm PepMapC18 2μm Thermo; flow rate: 500nl / min; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); elution gradient: 6%-25% B for 21min, 25%-40% B for 4min, 40%-100% B for 3min, 100% B for 2min; sample loading volume: 3μl. Mass spectrometry detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragmentation spectrum; MS2 resolution (200m / z): 30,000; MS2 AGC target: 2e 5 ,maxIT:70ms; Normalized Collision Energy (NCE):28%; Isolation Window:1.6m / z.

[0116] Adjust the mass spectrometry parameters to quickly collect and detect samples according to the PRM ion list. Use skyline to analyze the data measured by the mass spectrometer, use internal or external standards to perform relative or absolute quantification of the target ion fragments, and then perform qualitative or quantitative analysis of the samples based on the quantitative results of the ion fragments. Determine whether the patient has non-small cell lung cancer under the precursor of sample qualitative or quantitative analysis, or use it as a criterion for its diagnosis.

[0117] In other feasible embodiments, conventional immunoassays or protein interactions for the detection of acetylated lysine modification at position 66 of hemoglobin can also be used as a basis for the diagnosis of non-small cell lung cancer. In the above embodiments, only the PRM scheme is described in detail.

[0118] Example 3: Screening and diagnostic method of phosphorylated threonine at position 84 of hemoglobin as a marker

[0119] 1. Screening of phosphorylated threonine at position 84 of hemoglobin as a marker

[0120] Based on the mass spectrometry method of non-small cell lung cancer specimens in Example 1, mass spectrometry data of protein fragments in non-small cell lung cancer specimens and adjacent tissues were obtained. Analysis of the mass spectrometry detection results also revealed that phosphorylation at position 84 of the hemoglobin β subunit accounted for 84% of the total phosphorylation of the protein. T The secondary spectrum of FATLSELHCDKLHVDPENFR (such as Fig. 9 ), the secondary spectrum contains more than 67.5% of the ion fragments (except for the daughter ions of b1 and y1), and the presence of y20, y21 ions and a large number of b ions fully proves the credibility of the modification.

[0121] According to the statistical analysis results, the P value of phosphorylated threonine at position 84 in hemoglobin was 0.002 (P<0.01), indicating that this modification had a significant statistical difference between non-small cell lung cancer tissues and adjacent tissues, and the cancer tissues were downregulated by 6 times compared with adjacent tissues (see Fig.10 ).

[0122] Delta mass information, Delta mass belongs to the protein: P68871 (hemoglobin); Delta mass position: 84 (T); Delta mass belongs to the peptide: GTFATLSELHCDKLHVDPENFR; Delta mass molecular weight: 79.96Da; Deltamauss score: 0.886.

[0123] 2. Diagnostic method based on phosphorylated threonine at position 84 of hemoglobin

[0124] The diagnostic method comprises the following steps:

[0125] (1) Processing the collected samples according to the sample pretreatment method in Example 1;

[0126] (2) The mass spectrometry data measured by the data analysis method in Example 1 were analyzed using the skyline to obtain the chromatographic retention time of the target peptide GT (+79.96) FATLSELHCDKLHVDPENFR. The retention time was combined with the target peptide 2-valent and 3-valent parent ions and their corresponding daughter ions to construct an ion list of PRMs collected during mass spectrometry detection. (See Table 3. Ion list. Each parent ion can correspond to multiple 1-valent, 2-valent, and 3-valent daughter ions. For ease of detection, this example only lists the daughter ion fragments with relatively high relative abundance)

[0127] Table 3. Divalent and trivalent precursor ions of target peptides and their corresponding product ions

[0128]

[0129] The separation was performed using an EASY-nLC 1200 liquid phase system and detected using an online Thermo QE-HFX mass spectrometer. The liquid phase separation system parameters were as follows: precolumn: 75μm*2cm PepMap C18 3μm Thermo; chromatographic column: 75μm*15cm PepMapC18 2μm Thermo; flow rate: 500nl / min; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); elution gradient: 6%-25% B for 21min, 25%-40% B for 4min, 40%-100% B for 3min, 100% B for 2min; sample loading volume: 3μl. Mass spectrometry detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragmentation spectrum; MS2 resolution (200m / z): 30,000; MS2 AGC target: 2e 5 ,maxIT:70ms; Normalized Collision Energy (NCE):28%; Isolation Window:1.6m / z.

[0130] Adjust the mass spectrometry parameters to quickly collect and detect samples according to the PRM ion list. Use skyline to analyze the data measured by the mass spectrometer, use internal or external standards to perform relative or absolute quantification of the target ion fragments, and then perform qualitative or quantitative analysis of the samples based on the quantitative results of the ion fragments. Determine whether the patient has non-small cell lung cancer under the precursor of sample qualitative or quantitative analysis, or use it as a criterion for its diagnosis.

[0131] Example 4

[0132] 1. Screening of oxidized tryptophan at positions 15 and 37 of hemoglobin as markers

[0133] Based on the mass spectrometry method of non-small cell lung cancer specimens in Example 1, mass spectrometry data of protein fragments in non-small cell lung cancer samples and adjacent normal tissues were obtained, and the mass spectrometry detection results were analyzed to obtain information about the hemoglobin β subunit, including oxidized tryptophan at positions 15 and 37, which accounted for 14% of the total modification of the protein, and there were significant differences in both single oxidation and double oxidation at these two positions.

[0134] Secondary spectra of peptides LLVVYPWTQR and SAVTALWGK containing oxidized tryptophan at positions 15 and 37 ( Figure 13-16). The secondary spectra all contain oxidatively modified tryptophan and nearly 86-100% of the peptide-related daughter ions, indicating the credibility of the modification and related information.

[0135] Statistical analysis of the above test results showed that the oxidized tryptophan at positions 15 and 37 of hemoglobin had significant statistical differences between non-small cell lung cancer tissues and adjacent tissues, and the data showed that the amounts of modification at positions W15 and W37 in non-small cell lung cancer tissues were downregulated by 19 times, 16.8 times, 13.8 times, and 12 times, respectively, compared with adjacent tissues. Fig.17 The relevant information of Deltamass is as follows: the protein to which Delta mass belongs: P69905 (hemoglobin β subunit, hemoglobin); the position of Deltamass in hemoglobin: 15 and 37; the peptides to which Delta mass belongs: LLVVYPWTQR and SAVTALWGK; the molecular weight of Deltamass: 15.99 and 31.99; the Delta mass score: 15.99 is 0.919; 31.99 is 0.937.

[0136] In addition, the ROC curves of oxidized tryptophan at positions 15 and 37 of non-small cell lung cancer and adjacent hemoglobin are shown in Figure 2. Fig.18 As shown. The above analysis results show that the oxidized tryptophan at positions 15 and 37 of hemoglobin has important guiding and suggestive significance for the diagnosis and treatment of patients with non-small cell lung cancer. They can be analyzed separately, while simultaneous analysis can improve accuracy.

[0137] 2. Diagnostic method based on oxidation of tryptophan at positions 15 and 37 of hemoglobin

[0138] The diagnostic method comprises the following steps:

[0139] (1) treating the sample to be tested according to the sample pretreatment method in Example 1;

[0140] (2) Use the skyline to analyze the mass spectrometry data and obtain the chromatographic retention time of the target peptide LLVVYPW(+15.99)TQR and SAVTALW(+15.99)GK. Combine the retention time with the target peptide 2-valent and 3-valent parent ions and their corresponding daughter ions to construct an ion list of PRMs collected during mass spectrometry detection. (See Table 4-7, each parent ion can correspond to multiple 1-valent, 2-valent, and 3-valent daughter ions. For ease of detection, this example only lists the daughter ion fragments with relatively high abundance)

[0141] Table 4. Divalent and trivalent parent ions of LLVVYPW(+15.99)TQR and their corresponding daughter ions

[0142]

[0143] Table 5. Divalent and trivalent parent ions of SAVTALW(+15.99)GK and their corresponding daughter ions

[0144]

[0145]

[0146] Table 6. Divalent and trivalent parent ions of SAVTALW(+31.99)GK and their corresponding daughter ions

[0147]

[0148] Table 7. Divalent and trivalent parent ions of LVVYPW(+31.99)TQR and their corresponding daughter ions

[0149]

[0150] The separation was performed using an EASY-nLC 1200 liquid phase system and detected using an online Thermo QE-HFX mass spectrometer. The liquid phase separation system parameters were as follows: precolumn: 75μm*2cm PepMap C18 3μm Thermo; chromatographic column: 75μm*15cm PepMapC18 2μm Thermo; flow rate: 500nl / min; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); elution gradient: 6%-25% B for 21min, 25%-40% B for 4min, 40%-100% B for 3min, 100% B for 2min; sample loading volume: 3μl. Mass spectrometry detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragmentation spectrum; MS2 resolution (200m / z): 30,000; MS2 AGC target: 2e 5 ,maxIT:70ms; Normalized Collision Energy (NCE):28%; Isolation Window:1.6m / z.

[0151] Adjust the mass spectrometry parameters so that the sample can be quickly collected and detected according to the PRM ion list. Use skyline to analyze the data measured by the mass spectrometer, use internal or external standards to perform relative or absolute quantification of the target ion fragments, and then qualitatively or quantitatively analyze the sample based on the quantitative results of the ion fragments. Determine whether the patient has non-small cell lung cancer under the premise of qualitative or quantitative analysis of the sample, or use it as an indicator to assist in its diagnosis. Conventional immunoassay schemes or protein interaction schemes can also detect target modifications. This embodiment only describes the PRM scheme in detail.

[0152] Example 5: Data Analysis Method for Modification of Monooxygenated Proline 97 of Pulmonary Surfactant-Associated Protein A1

[0153] The mass spectrometry results were analyzed to obtain the pulmonary surfactant-associated protein A1, in which the 97th monooxidized proline accounted for 81.6% of the total modification of the protein.

[0154] The secondary spectrum of the peptide GPP(+15.99)GLPAHLDEELQATLHDFR containing the 97th monooxygenated proline is shown in Fig.19 As shown, the secondary spectrum contains more than 89.5% of the ion fragments (except for daughter ions b1 and y1), y18 and y19 in the spectrum are both high-peak daughter ions, and the existence of 78.9% b daughter ion type (all b ions are related to modification) fully proves the credibility of the modification and related information.

[0155] The core content of this example is: in non-small cell lung cancer, the 97th monooxygenated proline of SP-A protein in cancer tissue is significantly downregulated by 11.6 times compared with that in normal tissue (p=8.6e-4). Fig. 20 shown.

[0156] Delta mass related information is as follows: Delta mass belongs to the protein: Q8IWL2 (pulmonary surfactant-associated protein A1, SP-A); Delta mass position in SP-A protein: 97; Delta mass belongs to the peptide: GPPGLPAHLDEELQATLHDFR; Delta mass molecular weight: 15.99; Delta mass score: 0.919 (Delta mass score means: in mass spectrometry detection, due to the influence of instrument accuracy and environmental influences, the result of a single Delta mass measured by mass spectrometry is normally distributed within the accuracy and error allowable range, and the score is the fit to the normal curve, with a score of 0 to 1. The higher the score, the more reliable the test result. A secondary spectrum that can reach 0.2 will be recognized).

[0157] The ROC curve of the 97th monooxygenated proline in non-small cell lung cancer and adjacent SP-A protein is shown in Figure 2. Fig.21 shown.

[0158] From the above information, we can know that the monooxygenated proline at position 97 of SP-A protein has important guiding and suggestive significance for the diagnosis and treatment of patients with non-small cell lung cancer.

[0159] In the above content of this example, the target peptide segment was subjected to subsequent experiments after the sample protein was hydrolyzed using Trypsin [KR|P]. In mass spectrometry, a variety of enzymatic sample digestion schemes can also be used, such as Trypsin (semi) [KR | P]; Trypsin / p [KR | -]; Trypsin K [K | P]; Trypsin R [R | P]; ChymoTrypsin [FWYL | P]; Arg C [R | P]; Asp N [- | D] n-term; Clostripain [R | -]; CNBr [M | P]; Elastase [GVLIA | P]; Formic Acid [D | P]; Glu C [DE | P]; Glu C bicarb [E | P]; Iodosobenzoate [W | -]; Lys C [K | P]; Lys C / P [K | -]; Lys N [- | K] n-term; Lys N promisc [- | KASR] n-term; Pepsin A [FL | P]; Protein endopeptidase[P|-];Staph protease[E|-];Trypsin-CNBr[KRM|P];Trypsin-GluC[DEKR|P], etc. The 97th monooxygenated proline of SP-A protein can also be detected in the samples treated by these enzymatic hydrolysis or physical schemes such as ultrasound. In addition, the modification described in the present invention can also be enriched and detected by immunology and protein interaction schemes.

[0160] Application of monooxygenated proline 97 in SP-A protein

[0161] A rapid mass spectrometry detection method for the 97th mono-oxidized proline modification of SP-A protein. The mass spectrometry data measured by skyline analysis were used to obtain the chromatographic retention time of the target peptide GPP(+15.99)GLPAHLDEELQATLHDFR. The retention time was combined with the target peptide 2-valent and 3-valent parent ions and their corresponding daughter ions to construct an ion list of PRMs collected during mass spectrometry detection. (See Table 8. Ion list. Each parent ion can correspond to multiple 1-valent, 2-valent, and 3-valent daughter ions. For ease of detection, the present invention only lists the daughter ion fragments with higher relative abundance)

[0162] Table 8. Divalent and trivalent precursor ions of target peptides and their corresponding product ions

[0163]

[0164] The collected samples were processed according to "Example 1".

[0165] The separation was performed using an EASY-nLC 1200 liquid phase system and detected using an online Thermo QE-HFX mass spectrometer. The liquid phase separation system parameters were as follows: precolumn: 75μm*2cm PepMap C18 3μm Thermo; chromatographic column: 75μm*15cm PepMapC18 2μm Thermo; flow rate: 500nl / min; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); elution gradient: 6%-25% B for 21min, 25%-40% B for 4min, 40%-100% B for 3min, 100% B for 2min; sample loading volume: 3μl. Mass spectrometry detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragmentation spectrum; MS2 resolution (200m / z): 30,000; MS2 AGC target: 2e 5 ,maxIT:70ms; Normalized Collision Energy (NCE):28%; Isolation Window:1.6m / z.

[0166] Adjust the mass spectrometer parameters to quickly acquire and detect samples based on the PRM ion list.

[0167] Skyline is used to analyze the data measured by the mass spectrometer, and the target ion fragments are relatively or absolutely quantified using internal or external standards. Then, the samples are qualitatively or quantitatively analyzed based on the quantitative results of the ion fragments.

[0168] Determine whether a patient has non-small cell lung cancer based on the qualitative or quantitative precursor of the sample, or use it as an indicator to assist its diagnosis.

[0169] Detection verification: 4 pairs (cancer tissue and normal tissue) of clinically diagnosed non-small cell lung cancer patient samples (not included in the analysis set of Example 1) were used as research objects for verification, and the single oxidation modification of proline 97 of the pulmonary surfactant-associated protein A1 of the above samples was detected. Results: When the single oxidation modification of proline 97 of SP-A protein was used for separate diagnosis, the cancer tissue and normal tissue of the 4 pairs of samples could be correctly distinguished.

[0170] Conventional immunoassays or protein interaction schemes can also detect target modifications. The present invention only describes the PRM scheme in detail. The monooxygenated proline at position 97 of the pulmonary surfactant-associated protein A1 (Uniprot ID: Q8IWL2) described in this embodiment can also be used as a therapeutic target for non-small cell lung cancer; different forms of protein or small molecule drugs can be prepared for this target.

[0171] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. Use of a biomarker detection reagent in the preparation of a lung cancer diagnosis product, characterized in that: The biomarker is a modification of the amino acid site of a related protein; the protein is pulmonary surfactant-associated protein A1; the modification of the amino acid site of the related protein is a single oxidation modification of the 97th proline of pulmonary surfactant-associated protein A1; The pulmonary surfactant-associated protein A1 Uniprot ID: Q8IWL2; The lung cancer is non-small cell lung cancer.

2. The use according to claim 1, characterized in that: The product is a lung cancer diagnosis kit; the lung cancer diagnosis kit includes cell / tissue sample processing reagents, protein extraction reagents, peptide enzymatic hydrolysis reagents and mass spectrometry detection auxiliary reagents.

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