CN110669104B - A group of markers derived from human peripheral blood mononuclear cells and their applications - Google Patents

Abstract

Translated fromChinese

本发明公开了一组来源于人外周血单核细胞的标志物及其应用，涉及质谱蛋白质组学与临床医学及生物信息学领域，所述标志物为一组具有如序列表SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13所示的氨基酸序列的肽段。本发明提供的肽段标志物具有来源易获取、无侵入性或微侵入性、提取成本低的优势，且可通过质谱分析等方法实现对良恶性肺结节的特异性、高敏感性和准确性的诊断，还可以结合影像学等医学检查实现对肺结节恶性程度的判别，相较于现有技术具有显著的进步。

The invention discloses a group of markers derived from human peripheral blood mononuclear cells and their application, and relates to the fields of mass spectrometry proteomics, clinical medicine and bioinformatics. 1. SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, Peptide fragments of amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. The peptide marker provided by the present invention has the advantages of easy source, non-invasive or minimally invasive, low extraction cost, and can be specific, highly sensitive and accurate for benign and malignant pulmonary nodules by mass spectrometry and other methods The diagnosis of pulmonary nodules can also be combined with medical examinations such as imaging to determine the degree of malignancy of pulmonary nodules, which is a significant improvement compared to the existing technology.

Description

Group of markers derived from human peripheral blood mononuclear cells and application thereof

Technical Field

The invention relates to the fields of mass spectrometry proteomics, clinical medicine and bioinformatics, in particular to a group of markers derived from human peripheral blood mononuclear cells and application thereof.

Background

Lung cancer is the leading cause of cancer death in humans. In order to detect and monitor lung cancer at an early stage, the search for protein biomarkers of lung cancer becomes a research hotspot in recent years. Proteomics, and in particular quantitative proteomics, can identify and quantify proteins/polypeptides in complex biological samples for the study of tumor-associated proteins. Blood samples are easy to collect, can be repeatedly reserved in the treatment process, and are increasingly used for research of finding potential markers of lung cancer.

Peripheral Blood Mononuclear Cells (PBMC) are a class of blood cells with an intact round nuclear structure, including T cells (70%), B cells (15%), natural killer cells (10%) in blood, and mononuclear cells (5%) and dendritic cells (1%), which further differentiate into dendritic cells and macrophages. PBMC are important components of the immune system of the body and play an important role in the innate and adaptive immune functions of the body. PBMCs, like scouts of organisms, constantly monitor and maintain the homeostasis of the organism, and they respond rapidly in the event of infection or disturbance of the organism. PBMCs are more and more valued and utilized by researchers because they are easier to obtain and lower in extraction cost, and more importantly, they contain many key inflammatory-related factors such as cytokines and interferons. Some genomics studies show that PBMC plays an important role in revealing autoimmune diseases and chronic inflammation mechanisms, such as systemic lupus erythematosus, psoriasis, rheumatoid arthritis, inflammatory bowel disease and the like. However, no studies have been reported so far for predicting benign and malignant pulmonary nodules by using high resolution mass spectrometry to characterize the proteome of PBMCs.

Mass spectrometry has become the most common method for clinical proteomics research due to its characteristics of high efficiency, simplicity and high throughput. Mass spectrometry is the most complex and critical component of proteomic analysis. Unlike shotgun protein analysis, which is commonly used in exploratory studies, targeted analysis is a candidate protein-based proteomics technology that is capable of detecting specific peptides representing a target protein in a complex background based on the quality and/or cleavage characteristics of the peptides.

Therefore, those skilled in the art are devoted to develop a marker derived from human peripheral blood mononuclear cells, and can perform targeted mass spectrometry on the marker to realize noninvasive or micro-invasive, specific and accurate diagnosis of benign and malignant pulmonary nodules.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is to provide a specific marker from human peripheral blood mononuclear cells, so that noninvasive or micro-invasive, specific and accurate diagnosis of benign and malignant pulmonary nodules can be achieved by quantitative analysis of the marker.

In order to achieve the above objects, the present invention provides a set of markers derived from human peripheral blood mononuclear cells, wherein the markers are a set of markers having the sequences shown in SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13, or a peptide fragment of the amino acid sequence shown in figure 13.

Further, the peptide fragment is a specific peptide fragment on a lymphocyte membrane protein in the human peripheral blood mononuclear cell.

Further, the marker can be selected from one or a combination of several of the peptide fragments.

Further, the expression profile of the peptide fragment in patients with malignant lung nodules compared to the differential profile in patients with benign lung nodules comprises up-regulation and down-regulation of expression.

The invention also provides application of the marker from the human peripheral blood mononuclear cells in diagnosis of benign and malignant lung nodules.

Further, the detection and analysis are carried out by using a proteomics quantitative mass spectrometry analysis method, and the method specifically comprises the following steps:

step 1, collecting a blood sample of a patient, and extracting lymphocytes in the blood sample;

step 2, preparing the lymphocytes extracted in thestep 1 into a polypeptide sample suitable for proteomics analysis;

and 3, respectively carrying out separation operation and detection analysis on the target peptide fragments of the markers contained in the polypeptide sample prepared in thestep 2 by using a quantitative mass spectrometry method.

Further, the blood sample instep 1 is selected from early morning fasting venous blood.

Further, the separation operation instep 3 can be performed according to the quality, cleavage characteristics or relative retention time of the target peptide fragment.

Further, the application may also be used for diagnosis in connection with medical examinations, wherein the medical examinations comprise imaging.

Further, the use may also be diagnostic in conjunction with flow cytometry.

Compared with the prior art, the invention at least has the following beneficial technical effects:

(1) the benign and malignant lung nodule diagnostic marker provided by the invention is derived from human peripheral blood mononuclear cells, so that a sample is easier to obtain and can be left for multiple times, the invasiveness is lower, and the extraction cost is lower;

(2) the polypeptide fragment combination detection model provided by the invention has extremely high sensitivity and specificity for diagnosing benign and malignant lung nodules, and the accuracy can reach more than 90%;

(3) the polypeptide segment combination detection model provided by the invention can be combined with medical examination technologies such as imaging and the like to judge the malignancy degree of lung nodules;

(4) the polypeptide fragment combination detection model provided by the invention can be combined with flow cytometry to further promote the technology of predicting cancer by blood detection.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a ROC curve of a polypeptide fragment combination model according to a preferred embodiment of the present invention;

FIG. 2 is a schematic illustration of the principal component analysis of lung nodules for benign and malignant purposes in accordance with a preferred embodiment of the present invention;

FIG. 3 is a schematic diagram of the expression difference of peptide segment markers in patients with benign and malignant lung nodules according to a preferred embodiment of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

The invention takes peripheral blood mononuclear cells of a pulmonary nodule patient as a research object, and performs full spectrum analysis on PBMC of the pulmonary nodule patient by applying proteomics technology based on high resolution mass spectrum to obtain 13 specific peptide segments which respectively have the sequences shown in sequence tables SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof. The PBMC protein expression profiles of the patients were compared to determine the likelihood of malignant lung nodules. The method comprises the following specific steps:

first, collect the blood sample of the patient, and extract the peripheral lymphocytes of the blood sample

1. In all cases, 4-5mL of venous blood is extracted in an EDTA-Na anticoagulation tube on an empty stomach in the early morning before the operation;

2. taking a human lymphocyte separation tube, pouring blood into the separation tube in an ultra-clean workbench, and centrifuging for 15 min;

3. the PBMC cells in the middle layer of the separation tube were aspirated. Adding 1.5mL Phosphate Buffer Solution (PBS), transferring into a 1.5mL centrifuge tube, centrifuging at 1300rpm for 10min, pouring off waste liquid, absorbing the waste liquid from the tube opening by absorbent paper, and retaining PBMC cells at the tube bottom;

4. cells were cryopreserved in a-80 ℃ freezer for use.

Secondly, preparing the extracted lymphocytes into polypeptide samples suitable for proteomics analysis

1. Adding a lysis solution to the collected peripheral blood mononuclear cells;

2. cracking on ice for 25min by a horizontal shaking table;

3. pre-cooling the centrifuge to 4 ℃, 1300rpm, centrifuging for 20min, and preserving the protein solution at-80 ℃;

4. taking 30 mu g of protein, adding 12M urea into a protein sample for protein denaturation (the final concentration of urea is 6M), and uniformly mixing at 37 ℃ for 30 min;

5. adding the prepared TCEP to the final concentration of 5mM, carrying out protein reduction reaction at 55 ℃ for 30 min;

6. adding IAA to make the final concentration be 6.25mM, and carrying out protein alkylation reaction in the dark for 1 hour at room temperature;

7. urea concentration was made below 1M with 6 sample volumes of 50mM ammonium bicarbonate;

8. adding prepared CaCl₂Promoting the digestive activity of pancreatin, diluting 1000 times to make the final concentration be 1mM, and checking that the pH value at this moment is larger than 7 and close to 8;

9. adding pancreatin for enzymolysis, wherein the mass ratio of the added pancreatin to the protein sample is 1: reacting at 50-1:100 and 37 ℃ overnight;

10. after enzymatic hydrolysis, the pH was adjusted to 3 with phosphoric acid, the sample was applied to a C18 column and 0.1% trifluoroacetic acid (TFA/H)₂O) rinse thecolumn 3 times. The sample was eluted 3 times with 0.1% TFA/80% ACN;

11. the samples were concentrated and dried by a centrifugal concentrator and stored at-80 ℃ for loading.

Thirdly, detecting and analyzing the target peptide segment in the polypeptide sample by using a quantitative mass spectrometry method

Data acquisition was performed using a Data Dependent (DDA) data acquisition mode, and mass spectrometry data acquisition was performed using an EASY nLC-1000UPLC system in combination with an Orbitrap mass spectrometer (QE plus, Thermo Scientific) and electrospray ion source. The analytical column is a laboratory self-made analytical column with 75 mu m (i.d. inner diameter) multiplied by 15cm, and the filler is C18 filler with the grain diameter of 3 mu m x 100 i. The pre-column is 2cm and is composed of 5 μm

The column is made by filling. Separating liquid: phase a was 0.1% TFA and phase B was 0.1% TFA, 80% acetonitrile. The separation gradient was: 0-5min, 3-7% of B; 5-55min, 7-22% of B; 55-65min, 22-35% B; 65-68min, 35-80% B; 68-75min, 80% B (as shown in Table 1)Shown).

TABLE 1 Low PH liquid phase separation gradient

Time/min	Comparative example B
		0	3
5	7
		55	22
65	35
		68	80
75	80

The experiment adopts a tMS2 mode of orbitrap fusion, an orbit trap (orbitrap) is used as a mass analyzer of a secondary mass spectrum, a target peptide segment and relative retention time thereof are used as target list for separation and analysis, the primary mass spectrum resolution is 60,000, the secondary mass spectrum resolution is 30,000, and the target peptide segment is analyzed in specific retention time. The collected data are subjected to extraction of mass spectrum peaks and calculation of peak areas by using skyline. The calculated peak area is used as a quantitative result of the target protein to assist medical examinations such as imaging and the like to judge the malignancy degree of the lung cancer.

In this example, the expression difference of the peptide segment markers provided by the present invention in malignant lung nodules and benign lung nodules is shown in fig. 3, and there are significant differences, including peptide segments with up-regulated expression and peptide segments with down-regulated expression.

In addition, the sensitivity and accuracy of the peptide fragment combination marker model provided by the invention for diagnosing benign and malignant lung nodules are evaluated. As shown in fig. 1, the Area integration is performed on the fitted receiver operating characteristic Curve (ROC Curve for short), so as to obtain an AUC (Area over rock Curve) value which is as high as 0.98, which is significantly higher than the prior art in the field, and shows excellent sensitivity and accuracy. And in the graph (as shown in fig. 2) of the benign and malignant main component analysis of the lung nodule, it can be further seen that the peptide fragment combination marker model of the present invention achieves good distinction (PC1 orientation) between benign (benign) and malignant (malignant) of the solid nodule.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

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Claims (1)

Translated fromChinese

1.一种一组来源于人外周血单核细胞的标志物在制备预测肺结节良恶性试剂中的应用；所述标志物为一组具有如序列表SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13所示的氨基酸序列的肽段。1. The application of a group of markers derived from human peripheral blood mononuclear cells in the preparation of a reagent for predicting benign and malignant pulmonary nodules; the markers are a group of markers with the following sequences: SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. The peptide segments of the amino acid sequences shown.

Images