CN112881694A - Renal cancer serum polypeptide marker and application thereof - Google Patents

Abstract

The invention discloses a renal cancer serum polypeptide marker and application thereof. The amino acid sequence is shown in SEQ.ID.NO. 1. The molecule is named ATP1B4-A, is a fragment of human protein ATP1B4, has the accurate molecular weight of 4214 daltons ATP1B4-A, shows obvious high expression in the serum detection of a renal cancer patient, and can be used as a detection method of renal cancer serum by detecting ATP1B4-A by a matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or detecting the expression level of ATP1B4 by an ELISA method.

Description

Renal cancer serum polypeptide marker and application thereof

Technical Field

The invention belongs to the technical field of kidney cancer detection, and relates to a kidney cancer serum polypeptide marker ATP1B4-A and application thereof.

Background

The kidney cancer is one of the most common malignant tumors of the urinary system, the incidence rate of the kidney cancer accounts for 2% -3% of adult malignant tumors, about 209000 new-onset kidney cancers worldwide every year and about 102000 deaths are caused, and the incidence rate of the kidney cancer is in a steadily rising trend. Early renal cancer may have no symptoms, and only can be found by means of imaging examination such as ultrasound, CT, MRI and the like, and when patients have classic renal cancer triple symptoms such as hematuria, lumbago, abdominal mass and the like, the diagnosis is often in an advanced stage. About 50% of patients have advanced at the time of first visit, about 40% of patients have postoperative metastasis or relapse, metastatic renal cancer has a poor prognosis, the average survival time is less than 1 year, and the 3-year survival rate is less than 5%. In addition, the clinical problems that the differential diagnosis of certain benign and malignant tumors of the kidney is difficult, and the postoperative micrometastasis cannot be evaluated are also often encountered. Therefore, the clinical significance of early diagnosis and early treatment of the kidney cancer is very important. The accurate and sensitive kidney cancer marker is searched, and the early screening and early diagnosis of the kidney cancer are facilitated, so that the threat of the kidney cancer to the human health is reduced.

Serum diagnosis is considered to be the most recent and effective method for early diagnosis of cancer. The method judges the occurrence and development of the tumor by searching tumor markers in blood, particularly protein markers in the blood, thereby realizing early diagnosis of the tumor. A large amount of proteins and polypeptides exist in human serum, and the existence, deletion and expression of partial proteins and polypeptides are closely related to the health degree of human beings, so that the human serum becomes a biomarker for disease diagnosis.

Therefore, if the biomarker capable of being applied to screening the serum protein of the kidney cancer can be researched, the early screening and diagnosis of the serum protein of the kidney cancer are facilitated, and the research on the pathogenesis of the kidney cancer, the establishment of the diagnosis standard and the development of therapeutic drugs are also of great significance.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a renal cancer serum polypeptide marker ATP1B4-A and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a kidney cancer serum polypeptide marker ATP1B4-A, the amino acid sequence of which is shown in SEQ.ID.NO. 1.

Preferably, the serum polypeptide marker ATP1B4-A is a fragment of human protein ATP1B4, and has a molecular weight of 4214 daltons.

Preferably, the detection parameter in the serum is 9.63-17.89 ng/mL.

The invention also discloses application of the kidney cancer serum polypeptide marker ATP1B4-A as a kidney cancer serum diagnostic drug target.

The invention also discloses application of the kidney cancer serum polypeptide marker ATP1B4-A in preparation of a kidney cancer serum diagnosis medicament.

Preferably, the renal cancer serum diagnosis drug is a drug for detecting a renal cancer serum polypeptide marker by ELISA.

The invention also discloses application of the molecule combined with the kidney cancer serum polypeptide marker ATP1B4-A in preparing a kidney cancer serum diagnosis medicine.

The invention also discloses application of the ATP1B4 protein as a renal cancer serum diagnosis drug target.

The invention also discloses application of the ATP1B4 protein in preparation of a renal cancer serum diagnosis medicament.

The invention also discloses application of the molecule combined with the ATP1B4 protein in preparing a renal cancer serum diagnosis medicament.

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

the invention discloses a serum marker ATP1B4-A of a renal cancer patient, and the amino acid sequence of the serum marker is shown in SEQ.ID.NO. 1. This molecule is a fragment of human protein (ATP1B4), with an exact molecular weight of 4214 daltons, expressed in serum in the normal control population in the range: 5.19-10.21 ng/mL; the expression range in the serum of renal cancer patients is: 9.63-17.89 ng/mL, it can be seen that it exhibits significantly high expression in the serum of renal cancer patients, and has very significant difference between groups (p < 0.001).

In view of the fact that the serum marker ATP1B4-A is remarkably and highly expressed in the serum of the kidney cancer, ATP1B4-A can be used as a serum diagnostic marker of the kidney cancer; and the maternal protein ATP1B4 shows specific high expression in the serum of the renal cancer patient, therefore, ATP1B4 can be applied to the serum diagnosis of the renal cancer patient: the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is used for detecting ATP1B4-A or an ELISA method is used for detecting the expression level of ATP1B4, and the method can be used as a method for detecting renal cancer patients. For ELISA detection of renal cancer serum diagnosis, ATP1B4 can be used as a new target point for ELISA detection of drugs.

Drawings

FIG. 1 is a protein polypeptide map of three replicates (1 kDa-10 kDa) of a serum sample of the same renal cancer patient;

FIG. 2 shows the difference in protein polypeptide expression of protein polypeptide peaks m/z 4214 in renal cancer patients and normal control groups;

FIG. 3 is a MS/MS mass spectrometric identification profile of ATP1B 4-A;

FIG. 4 shows the expression level of ATP1B4 protein in serum of renal cancer patients and normal control groups.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the renal cancer serum polypeptide molecule provided by the invention is a newly screened renal cancer serum diagnostic marker, has specificity in expression, and can be applied to renal cancer diagnosis. .

Specifically, the screening of the renal cancer serum diagnostic marker comprises the following steps:

the method comprises the steps of firstly, separating and extracting serum protein polypeptides of a kidney cancer patient and a normal control group by using a liquid protein chip technology, capturing protein polypeptide spectrograms of the kidney cancer patient and the normal control group by using MALDI-TOF-MS, comparing and analyzing the difference of the serum protein polypeptide spectrograms of the kidney cancer patient and the normal control group by using ClinProTools2.1 software, finding out the protein polypeptide score which is obviously differentially expressed among groups, and screening a kidney cancer serum tumor marker from the protein polypeptide peak which is obviously highly expressed in the serum of the kidney cancer patient.

The verification of the screened renal cancer serum diagnostic markers is as follows:

the protein polypeptide mixture separated from the serum of the kidney cancer patient is divided into 20-30 components by HPLC, secondary mass spectrum identification is carried out on the protein polypeptide mixture, the identified protein polypeptide is subjected to serum regression analysis by an enzyme-linked immunosorbent assay, and the result of serum regression verification proves that the protein polypeptide mixture is remarkably high in expression and specificity in the serum of the kidney cancer patient and can be used as a biomarker for screening the serum of the kidney cancer patient.

1. Collecting and processing a sample:

100 (50 men; 50 women; mean age 58 years) clinically confirmed renal cancer patients and 100 normal healthy control groups (50 men; 50 women; mean age 56 years) collected from the first subsidiary hospital of the university of transport of Xian (1 month-2020-1 to 12 months 2019). The sample considers factors such as age, sex, collection time, whether the storage conditions are consistent, whether basic diseases exist and the like. Collecting blood of the collected person with fasting state, collecting 5mL whole blood with vacuum blood collection tube (yellow cap, with isolation gel), and standing at room temperature for 30 min; centrifuging at room temperature for 5min (3000g), subpackaging the upper layer serum into 100 μ L/tube, immediately storing at-80 deg.C, and avoiding repeated freeze thawing.

Reagents and instrumentation: the serum proteins were extracted using the magnetic bead kit "weak cationic" (MB-WCX) from Bruker, Germany, and spectrally pure (HPLC grade) acetonitrile, trifluoroacetic acid (Merck, Germany), alpha-cyano-4-hydroxycinnamic acid (HCCA) (Sigma, USA).

Magnetic bead separator, 384AnchorChip target plate and AutoFlex III matrix assisted laser desorption ionization time of flight mass spectrometry MALDI-TOF-MS (Bruker Daltonics, Germany).

2. Preparation of serum protein samples

Capturing serum protein polypeptide by weak cation (MB-WCX) magnetic beads, which comprises the following specific operation steps:

completely mixing the magnetic bead suspension for 1min by using a mixer;

adding 10 mu L of MB-WCX binding solution and 10 mu L of MB-WCX magnetic beads into a PCR tube, uniformly mixing, adding 5 mu L of serum, uniformly mixing for at least 5 times, and standing for 5 min;

thirdly, placing the PCR tube into a magnetic column separator, making the magnetic beads adhere to the wall for 1min, and removing the supernatant after the liquid is clear;

adding 100 mul MB-WCX flushing fluid, moving the PCR tube on a magnetic column separator back and forth for 10 times, and removing supernatant after the magnetic beads are attached to the wall;

repeating the third step and the fourth step twice;

fifthly, adding 5 mu L of MB-WCX eluent to wash the adherent magnetic beads, repeatedly blowing and beating for 10 times, allowing the magnetic beads to adhere for 2min, and transferring the supernatant into a clean centrifugal tube;

sixthly, adding 5 mu L of MB-WCX stable solution into a centrifuge tube and mixing uniformly, wherein the extracted protein polypeptide can be used for direct MALDI-TOF-MS detection or frozen in a refrigerator at the temperature of-20 ℃ for mass spectrometry within 24 h.

Mass spectrometry analysis: mixing 1 μ L of the separated and collected protein sample with 10 μ L of matrix alpha-cyano-4-hydroxycinnamic acid, and spotting 1 μ L on an Anchorchip target plate, wherein each sample is spotted with three target spots for three times. And (3) after drying at room temperature, putting the target plate into a mass spectrometer for analysis, correcting the standard substance by adopting FlexControl 2.0 software, and then starting sample detection, wherein each sample generates a mass spectrogram after being subjected to laser targeting for 300 times (5 times of point targeting and 2 times of 30 times of targeting each time), so as to obtain protein polypeptide spectrograms consisting of different mass-to-nuclear ratios (m/z). The protein polypeptide maps of the two groups of serum samples are analyzed by using ClinProTools2.1 software, a genetic algorithm and other biological statistics and bioinformatics methods. Carrying out normalization smoothing treatment on the total ion flow diagram, and eliminating chemical and electro-physical noises; analyzing the difference protein among groups, calculating the difference size, and arranging the difference size from large to small to find out the peak value (P <0.001) of the protein polypeptide with obvious difference expressed among groups.

After the blood serum samples of the kidney cancer patient group and the normal control group are processed by a magnetic bead separation system, protein polypeptide maps of each sample of the kidney cancer patient group and the normal control group are drawn after MALDI-TOF-MS analysis, 79 protein polypeptide peak maps are detected in the molecular weight range of 1000Da to 10000Da, and the three-time repeated stability of each sample is high, as shown in figure 1.

The method comprises the steps of adopting ClinProTools2.1 software to analyze serum protein polypeptide spectrums of a kidney cancer patient and a normal control group captured by mass spectrometry, comparing and analyzing the serum polypeptide spectrums of the kidney cancer patient with normal people, and detecting that a protein polypeptide peak with the molecular weight of 4214 daltons is remarkably and highly expressed in the serum of the kidney cancer patient (a kidney cancer group vs healthy control group: 13.55 +/-2.37 vs7.82 +/-1.74, and p is less than 0.001). As shown in FIG. 2, when the expression of M/Z:4214 in renal cancer patients (red, curve with upper peak) and normal control (green, curve with lower peak) are compared, the protein polypeptide peak pattern of M/Z:4214 is remarkably high expressed in the serum of renal cancer patients, so that the protein polypeptide peak pattern is subjected to sequence identification and is used as the first choice for further identification of a marker.

3. Sequence identification of potential markers of kidney cancer serum

Specifically, a technology of combining liquid chromatography separation and mass spectrometry is adopted to identify a kidney cancer serum polypeptide marker M/Z:4214, two-dimensional gel chromatography separation is carried out on the serum protein polypeptide remaining after the magnetic bead separation and collection of the mass spectrometry sample loading by using Nano acquisition UPLC of Waters company, and 15-30 parts of peptide fragment fractions are collected: detecting the protein of interest in the collection; and then, the sequence identification is carried out on the protein polypeptide M/Z:4214 with the expression up-regulated in the serum of a renal cancer patient by using an LTQ Orbitrap XL mass spectrum system of Thermo Fisher company.

The specific operation steps are as follows:

3.1 sample Pre-treatment

Mixing the extracted protein samples, rotating at 1300 deg.C for 10min, collecting supernatant, freeze drying to obtain liquid A with a final volume of 50ul, extracting with Agilent Ziptip column, and concentrating to obtain liquid A. The treatment method comprises the following steps: blowing and beating a ziptip column for 5 times by using 100% acetonitrile, and activating the column; secondly, repeatedly blowing and sucking the activated ziptip in the liquid 1 for 10 times to avoid bubbles as much as possible; ③ 50% ACN 0.1% TFA aqueous solution, washing the ziptip column for 3 times; fourthly, repeatedly blowing and sucking the ziptip column in 0.1% TFA to elute the sample to obtain eluent 2; fifthly, repeating the steps from the first step to the fourth step for 30 times; sixthly, combining 30 times of eluates 2, freezing and drying to 10ul, and using for mass spectrum identification.

3.2, chromatographic separation:

the original sample was added with 10ul of mobile phase A and transferred to a sample vial for a total of 20 ul.

One-dimensional ultra-high performance liquid phase system: nano Aquity UPLC (Waters Corporation, Milford, USA). A chromatographic column:

a trapping column:

5μm,180μm×20mm,nanoAcquity^TMColumn

and (3) analyzing the column:

3.5μm,75μm×150mm,nanoAcquity^TMColumn

mobile phase A: 5% acetonitrile, 0.1% formic acid in water

The mobile phase B is 95 percent of acetonitrile and 0.1 percent of aqueous solution of formic acid; all solutions were HPLC grade.

The trapping flow rate is 15 mul/min, the trapping time is 3min, and the analysis flow rate is 400 nl/min; the analysis time is 60min, and the temperature of a chromatographic column is 35 ℃; the Partial Loop mode was injected in a volume of 18. mu.l.

The gradient elution procedure is shown in table 1 below:

TABLE 1

Time	Flow rate of flow	Mobile phase A%	Mobile phase B%
				40.0	0.400	95.0	5.0
41.0	0.400	55.0	45.0
				45.0	0.400	20.0	80.0
45.50	0.400	95.0	5.0
				60.00	0.400	95.0	5.0

The results of gel chromatography are shown in FIG. 3. In the chromatogram, the abscissa represents the sample outflow time, the ordinate represents the relative abundance of the polypeptide, the set time of the chromatogram is 60min, fractions are collected and collected from 10min, the polypeptide components are mainly separated after 15min, a gradient elution mode is adopted, the elution efficiency is improved, and the capture time is set to collect the fractions: collecting 15-30 parts of peptide fragment fraction.

3.3LTQ-Orbitrap XL Mass Spectrometry:

the Thermo Fisher LTQ orbitrap XL mass spectrometry system was used. Nano ion source (Michrom biosources, Auburn, USA), spray voltage 1.8 kV; scanning time of the mass spectrum is 60 min; the experimental modes are Data dependence (Data dependency) and Dynamic Exclusion (Dynamic Exclusion), and parent ions are added into an Exclusion list for 90 seconds after being cascaded for 2 times within 10 seconds; the scanning range is 400-2000 m/z; the first-order scanning (MS) uses Obitrap, and the resolution is set to be 100000; CID and secondary scanning use LTQ; selecting single isotope of 10 ions with the strongest intensity as parent ion to carry out MS/MS (single charge exclusion without being used as parent ion) in MS spectrogram. The results of the detection are shown in FIG. 3.

And (3) data analysis: sequest was performed using the data analysis software Bioworks Browser 3.3.1SP1^TMAnd (5) searching. The parent ion error is set to 100ppm, the fragment ion error is set to 1Da, the enzyme digestion mode is non-enzyme digestion, and the variable modification is M (Methionine) methionine oxidation. Search result parameter is set to deltacn>0.10. The retrieval result is as follows: 4214.08 for m/z; IPI is IPI 00013724.1; gene Symbol is ATP1B4 Isoform Aof X/potassium-transporting ATPase subbnit-m; the sequence is as follows:

IMSEYLWDPERRMFLARTGQSWSLILLIYFFFY。

4214 isolated M/Z:4214, designated ATP1B4-A, is a fragment of human protein (ATP1B4) with an exact molecular weight of 4214 daltons, its amino acid sequence:

IMSEYLWDPERRMFLARTGQSWSLILLIYFFFY (shown in SEQ. ID. NO. 1).

Therefore, the matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) is used for detecting ATP1B4-A or the ELISA method is used for detecting the expression level of ATP1B4, and the method can be used for detecting the renal cancer patients.

ATP1B4-A as a fragment of ATP1B4 suggested that ATP1B4 is a protein specifically associated with kidney cancer, and was further verified by ELISA detection.

4. ELISA serum validation analysis of kidney cancer serum ATP1B4 expression:

1) serum samples: serum of 80 patients with renal cancer (40 cases in men, 40 cases in women; mean age 56 years) and 80 cases in normal control group (40 cases in men, 40 cases in women; mean age 55 years) were collected and subjected to serum-validation analysis by ELISA. All serum samples were obtained from the subsidiary Children hospital of the university of Xian traffic, from 2019, 1 month to 2020, 12 months.

2) The detection method comprises the following steps: the expression levels of serum ATP1B4 of renal cancer patients and normal control groups are detected by adopting an enzyme-linked immunosorbent assay (ELISA), and the kit is purchased from Jiangsu enzyme-labeled biotechnology limited company. The kit adopts a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA): to the coated microwells previously coated with anti-human ATP1B4 protein (ATP1B4) antibody, the specimen, the standard, and the HRP-labeled detection antibody were added in this order, incubated, and washed thoroughly. The color is developed with the substrate TMB, which is converted to blue by the catalysis of peroxidase and to the final yellow color by the action of an acid. The shade of the color was positively correlated with the ATP1B4 protein in the sample. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the sample concentration was calculated. The specific experimental steps refer to the kit specification, and the positive judgment standard is defined according to the kit specification.

3) The statistical method comprises the following steps: one-way analysis of variance (ANOVA) and T-test of independent samples were performed using graphpad. prism. v5.01 software.

4) And (4) analyzing results: the results of enzyme-linked immunosorbent assay analysis show that the expression level of ATP1B4 in the kidney cancer and normal control detection group is 13.55 + -2.37 (9.63-17.89) vs7.82 + -1.74 (5.19-10.21) of the kidney cancer vs normal control group, p is less than 0.001, and the specific results are shown in FIG. 4.

The ELISA detection is carried out on ATP1B4 in the serum of a normal control population and a renal cancer patient, and the result shows that the ATP1B4 in the serum of the renal cancer patient is obviously highly expressed (renal cancer vs normal control group: 13.55 +/-2.37 vs7.82 +/-1.74): the expression range in the normal control group serum is: 5.19-10.21 ng/mL; the expression range in the serum of renal cancer patients is: 9.63-17.89 ng/mL, and has very significant difference (p <0.001) among groups. This indicates that: ATP1B4 is a protein closely related to the occurrence of kidney cancer, and can be used as a primary kidney cancer detection index.

Therefore, whether the cancer of the kidney exists can be preliminarily judged through the expression of ATP1B4 of a serum sample to be detected by an ELISA experiment: patients with renal cancer (9.63-17.89 ng/mL); normal population (5.19-10.21 ng/mL).

In conclusion, the invention discloses a novel renal cancer serum polypeptide molecule ATP1B4-A and a detection method and application thereof. The amino acid sequence of ATP1B4-A is shown in SEQ ID No. 1. This molecule is a fragment of the human protein ATP1B4, with an exact molecular weight of 4214 daltons. ATP1B4-A shows remarkably high expression in the detection of the serum of a kidney cancer patient, ATP1B4-A is detected by a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or the expression level of ATP1B4 is detected by an ELISA method, and the method can be used as a detection method of the serum of the kidney cancer.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence listing

<110> first subsidiary Hospital of medical college of Western-Ann transportation university

<120> renal cancer serum polypeptide marker and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 33

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ile Met Ser Glu Tyr Leu Trp Asp Pro Glu Arg Arg Met Phe Leu Ala

1 5 10 15

Arg Thr Gly Gln Ser Trp Ser Leu Ile Leu Leu Ile Tyr Phe Phe Phe

20 25 30

Tyr

Claims (10)

1. A kidney cancer serum polypeptide marker ATP1B4-A is characterized in that the amino acid sequence is shown in SEQ ID No. 1.

2. The renal cancer serum polypeptide marker ATP1B4-A as claimed in claim 1, wherein the serum polypeptide marker ATP1B4-A is a fragment of human protein ATP1B4, and has a molecular weight of 4214 daltons.

3. The renal cancer serum polypeptide marker ATP1B4-A according to claim 1, wherein the detection parameter in serum is 9.63-17.89 ng/mL.

4. The use of the renal cancer serum polypeptide marker ATP1B4-A of claim 1 as a renal cancer serum diagnostic drug target.

5. The use of the renal cancer serum polypeptide marker ATP1B4-A of claim 1 in the preparation of a renal cancer serum diagnostic drug.

6. The use according to claim 5, wherein the renal cancer serum diagnostic drug is a drug for detecting a renal cancer serum polypeptide marker by ELISA.

7. Use of a molecule that binds to the renal cancer serum polypeptide marker ATP1B4-a of claim 1 in the preparation of a renal cancer serum diagnostic drug.

The application of the ATP1B4 protein as a renal cancer serum diagnosis drug target.

Application of ATP1B4 protein in preparing renal cancer serum diagnosis medicine.

10. The application of the molecule combined with ATP1B4 protein in preparing renal cancer serum diagnosis medicine.

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