CN110514838B

CN110514838B - Application of 184-bit N +22.968 of AKAP4 protein in preparation of diagnostic reagent for severe oligospermia

Info

Publication number: CN110514838B
Application number: CN201810489923.6A
Authority: CN
Inventors: 杨静华; 吕鑫; 王风芹; 陈子江
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-05-21
Filing date: 2018-05-21
Publication date: 2020-10-23
Anticipated expiration: 2038-05-21
Also published as: CN110514838A

Abstract

The invention discloses application of asparagine with mass shift of +22.968 +/-0.005 at 184 th site of AKAP4 protein as a biomarker in preparation of a diagnostic reagent for severe oligozoospermia. The invention discovers that: the detection frequency of asparagine with the mass shift of +22.968 +/-0.005 at the 184 th site of the AKAP4 protein can be used for diagnosing severe asthenospermia, and a new diagnosis and treatment target point is provided for severe asthenospermia.

Description

Application of 184-bit N +22.968 of AKAP4 protein in preparation of diagnostic reagent for severe oligospermia

Technical Field

The invention relates to the technical field of medicine and molecular diagnosis, in particular to application of asparagine (non-coding amino acid AKAP4@184N +22.968) with +22.968 +/-0.005 mass shift at 184 th position of AKAP4 protein as a marker in preparation of a diagnostic reagent for severe oligozoospermia.

Background

According to the world health organization survey, 15% of fertile couple have sterility problems, and infertility has become a global medical and social problem affecting human health and social development (Turner, T.T. and J.J.Lysiak, oxidative: a common factor in microbial stress. J. android, 2008.29(5): p.488-98). Asthenospermia can be diagnosed if the number of a-grade sperm is < 25%, the number of (a + b) -grade sperm is < 50%, and the sperm motility rate is less than 60%. Oligospermia refers to a condition in which the number of sperm in the semen is lower than that of a normal male with fertility, and is when the number of sperm per ml is lower than 2 million. Although many studies are now made on the pathogenesis of oligospermia, the exact mechanism is not known, which has hindered the development of new therapeutic approaches. Because the transcription and translation of the mature sperms are in a state of stagnation, the method provides convenience for researchers to research the physiological and pathological mechanisms of oligoasthenospermia on the level of proteome and posttranslational modification thereof.

There are many studies on sperm proteomes today, and a total of about 6238 non-redundant proteins have been identified (Semenprotomics and male fertility, Meritxell Journal, Ada Soler-Venturia, Rafael Oliva, Molecular Biology of Reproduction and Development research group, Journal of Proteomics 162(2017) 125-. The most recent human sperm proteome, Amaral et al, has now completed a total of 6198 proteins (Amaral A, Castillo J, Ramalho-Santos J, Olivar. the combined human sperm protein: cellular pathways and identities for basic and clinical science. human reproduction update,20(1),40-62 (2014)). Mayank et al used differential proteomics to quantify 667 proteins in the sperm cells of 5 groups of healthy and 8 groups of patients with oligospermia, 447 proteins in the seminal plasma, and 8 significantly down-regulated proteins were identified and subjected to pathway analysis (Human Spermatozoa Quantitative diagnostic Signature classes Normo-andAsdamenozopermia, Mayank Sayakat, Sakari Joenvarara, Tushar Jain, oil KumarTomar, Ashima Sinha, Sarman Singh, Savita Yadav, and Risto Renkon, Mol Cell Proteics.201Jan; 16(1): 57-72). To study the molecular mechanisms of azoospermia, Mehdi et al found 520 significant variant proteins including several key transcription factors in human obstructive and non-obstructive azoospermia testis tissues using a non-labeled Quantitative Proteomics method, which also laid the foundation for studying the molecular regulation mechanisms of spermatogenesis and human reproduction (Quantitative genetic analysis of human biological systems and cellular pathways with non-reciprocal cellular apoptosis, Mehdi Alikhani, Mehdi Mirzaei, Marnhagja, Pourisasa Paramat, Raziehkaramzadeh, SamaneAdib, Niloofa Sodeifi, Mohammad Alighighii, Massadzbed, Hayazam, Hayayu, Guikayama, Hayayu, Guikayu, Hayayu, Japan. Silencing of translational transcriptional activity in mature sperm also makes it an ideal cellular model for the study of post-translational modifications, but there has been little large-scale study of post-translational modifications in sperm based on mass spectrometry. The studies on modification have focused mainly on phosphorylation, glycosylation, acetylation and ubiquitination (The Challenge of HumanSpermatozoa proteins: A Systematic Review, Kambiz Gilany, Arash Minai-Tehrani, Mehdi Amini, Niloofar Agrarezaee, Babak Arjmand, J Reprod Infertil.2017 Jul-Sep; 18(3): 267-279.). The phosphorylation of tyrosine plays an important role in the processes of sperm movement, capacitation, super-excitation movement and the like. Chying-Chyuan Chan et al found that 12 proteins including TUBGCP2 were over-phosphorylated by proteomic analysis of sperm from 20 groups of normal and asthenospermia patients. Non-coding amino acids, including post-translational modifications and amino acid mutations, are important ways to regulate protein function and structure, and therefore, it is of great significance to use non-coding amino acids that are abnormal or have greatly changed amounts in disease states as biomarkers for disease and then to diagnose the course of disease.

The acrosin is the major protein of the acrosin matrix protein, a serine protease, and exists as an inactive pro-protease. Acrosin plays an important role in physiological processes such as acrosomal matrix protein disintegration, sperm binding to zona pellucida, acrosomal reaction, etc. (models of acrosin functionalizing and desiccating transduction). Studies have shown that Sperm fertilization rates are closely related to the activity of acrosin activity and fluorescence microscopic assessment of proacrosin/acrosin in ejaculates of fertility and fertility men.

Disclosure of Invention

At present, the pathogenesis of severe oligospermia is not clear to medical researchers, and the inventors of the present invention select sperm protein as a research object, analyze the mutation condition of non-coding amino acid in the sperm protein, and help to analyze the pathogenesis of severe oligospermia from a gene level. The treatment medicine for severe oligospermia is mainly prepared from deficiency-tonifying Chinese patent medicines and hormone medicines, and has low cure rate. The research on the non-coding amino acid mutation site is beneficial to providing a target for the treatment drugs of severe asthenospermia and providing more basis for the research and development of the drugs.

The present inventors have obtained certain results in conventional studies on biomarkers of severe asthenospermia and have published patent nos. CN106872630A, CN106932597A, CN106990177A, CN106996981A, CN106996979A, CN106996980A, CN107015005A, CN107024553A and CN 107037172A. As is well known, the inventors of the present invention have selected and studied the relationship between these mutation sites and the onset of severe oligoasthenospermia, but in fact, the mutation at these sites may be associated with the occurrence of various diseases in the human body, and screening as many non-coding amino acid sites with mutations as possible is of great significance for the diagnosis of diseases and the development of medicines. Therefore, the inventors conducted more intensive research on the mutation status of non-coding amino acids in sperm protein, and in the subsequent research process, the inventors conducted intensive and heavy research work to obtain 21 pairs of sperm protein data with potential significance by continuously identifying mutation sites, and conducted statistical analysis on the correlation between the screened mutation sites and diseases, and the inventors obtained significant research results again.

Aiming at the prior art, the invention aims to provide a method for screening and applying biomarkers related to severe asthenospermia. The invention firstly carries out deep mass spectrometric analysis on a plurality of groups of sperm protein non-coding amino acids of severe oligozoospermia diseases by utilizing a NanoHPLC-MS/MS mass spectrometric system and a non-labeled quantitative proteomics method; then, mass spectrum data are searched by using a non-limited amino acid protein modification analysis method, and a large amount of non-coded amino acids in the sperm protein group are identified as much as possible through multivariate Gaussian mixture distribution clustering analysis; and finally, comparing non-coding amino acids in normal and patient sperm proteomes to obtain a protein non-coding amino acid site related to severe oligozoospermia, so that the protein non-coding amino acid site is used as a molecular marker of severe oligozoospermia.

In order to achieve the above purpose, the present invention provides the following technical solutions:

and washing the equivalent severe oligozoospermia and normal sperm samples with DPBS for three times respectively, adding equivalent RIPA lysate for ultrasonic treatment for 1-2 min, placing on ice for incubation for 30min for lysis, and centrifuging at 4 ℃ for 14,000g multiplied by 20min to obtain supernatant. Protein concentration was determined using the Bradford method.

About 150. mu.g of sperm protein was taken from each of the low-gravity, weak sperm and normal sperm samples, and the proteins were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE) and fractionated into 5 portions for gel-cutting enzymolysis. The peptide fragments were desalted using ziptip.

Nano-flow liquid chromatography separation: phase A: water containing 0.1% formic acid; phase B: acetonitrile containing 0.1% formic acid.

Each sample was separately dissolved in 13.5. mu. L A phases, and the sample size was 4. mu.L, NaThe flow-liquid mass spectrometry system was the Orbitrap Elite (Thermo Scientific). The home-made pre-column and analytical column were equilibrated with 4 μ L A phase, respectively, prior to sample isolation. The specifications of the pre-column and the analytical column are respectively as follows: a pre-column (4cm x 150 μm i.d., C18 filler particle size 5 μm,

) Analytical column (30cm × 75 μm I.D., packed with C18 packing, particle size 3 μm,

dr. maisch GmbH, Germany). After the equilibrium, the sample is loaded on a pre-column under the drive of the phase A, and then the liquid phase separation is carried out under different gradients. The 150min chromatographic gradient varied as follows: 5-32% of mobile phase B for 100 min; 32-80% of mobile phase B for 20 min; 80% mobile phase B, 30 min. The flow rate was maintained at 300nL/min at all times. The sample after nanofluid phase separation directly enters an ESI ion spray source and enters an OrbitrapE mass spectrometer for mass spectrum detection.

Mass spectral data acquisition conditions were 350-1800m/z full scan with a resolution of 60,000(m/z 200). In secondary atlas scanning, the activation time was 10ms and the isolation width was 2 m/z. The fragmentation mode was induced-collision-induced dissociation (CID), the normalized collision energy was set at 35%, and the dynamic discharge time was 90 s.

To identify non-coding amino acids of sperm proteins, the present invention employs Byonic^TMProtamine mass spectral data of normal and severe oligozoospermic patients were analyzed 21. The search parameters are as follows: the protease is trypsin, the missed cutting site is set to be 2, the mass deviation of the parent ion is 10ppm, the mass deviation of the fragment ion is 0.6Da, the upper limit of the blind search is set to be 1000, the lower limit of the blind search is set to be-200, and the protein FDR is 0.01.

Selecting unknown modified peptide segment data searched by Byonic Wildcard Search with FDR <0.01 to form a one-dimensional data matrix, selecting a delta mass range of the data to be-200 Da-400Da, and dividing the data into 601 data windows according to a 1Da variation interval and 0.5Da as an interval limit. And aiming at each data window, carrying out Gaussian mixture distribution clustering analysis by adopting an mclust program package in the R language, obtaining an optimal value according to BIC, carrying out combination analysis on each peak, fitting each peak by using Gaussian distribution, and determining a peak value. Each peak after clustering contains information of amino acids, and non-coding amino acids are selected by an iterative model of RUP with 10% as a selection parameter according to the distribution ratio of unknown modifications on 20 amino acids.

Screening the non-coding amino acids of the normal and disease groups according to the T test (p <0.01), the ratio (ratio >2) and the detection frequency (>100) of the detection frequency of the non-coding amino acids, thereby obtaining the differential non-coding amino acids. Then, SPSS software is used for making a difference non-coding amino acid ROC curve, and the area under the curve (AUC) is calculated, so that the diagnostic value of the difference non-coding amino acid ROC curve is judged.

Mass spectrometry data analysis shows that the asparagine at the 184 position of the AKAP4 protein has mass shift of +22.968 +/-0.005 (N +22.968), and comparison shows that the non-coding amino acid N +22.968 is down-regulated by 5.1 times in the significance of samples with severe asthenospermia, and the p value is 2.63E-16< < 0.01.

The above screening methods are used to obtain biomarkers and are not aimed at obtaining diagnostic and therapeutic results for disease; the biomarker obtained by the screening method can be used for theoretical research of severe asthenospermia or development of new medicaments.

In a first aspect of the present invention, there is provided biomarkers related to severe asthenospermia, which are screened according to the above screening method, including but not limited to:

asparagine with a mass shift of +22.968 ± 0.005 at position 184 of AKAP4 protein (labeled N + 22.968);

in a second aspect of the invention, there is provided the use of asparagine (labeled N +22.968) with a mass shift of +22.968 ± 0.005 at position 184 of AKAP4 protein as a biomarker for the preparation of a diagnostic reagent for the treatment of severe asthenospermia.

The invention also provides a kit for diagnosing severe asthenospermia, which comprises a reagent for specifically detecting the biomarker (asparagine with mass shift of +22.968 +/-0.005 at position 184 of AKAP4 protein).

Preferably, asparagine with a mass shift of +22.968 ± 0.005 at position 184 of AKAP4 protein is also a target for treatment of severe asthenospermia and thus for treatment of severe asthenospermia.

The invention also provides application of asparagine with mass shift of +22.968 +/-0.005 at position 184 of AKAP4 protein as a biomarker in preparation of a medicine for treating severe asthenospermia.

The invention also provides a diagnosis method of severe oligozoospermia, which comprises the following steps: detecting the frequency of +22.968 +/-0.005 mass deviation of the 184-bit asparagine of the AKAP4 protein of the sample to be detected, carrying out parallel detection for 3 times, and averaging the detection results, wherein when the detection frequency is less than 4.4, the patient is judged to be a patient with less asthenospermia.

The invention has the beneficial effects that:

the invention further researches the biomarkers obtained by the screening method, finds that severe oligozoospermia can be diagnosed by the detection frequency of the biomarkers, and provides a new diagnosis and treatment target for severe oligozoospermia.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.

FIG. 1: ROC plot of the frequency of detection of the non-encoded amino acid N +22.968 at position 184 of the AKAP4 protein;

FIG. 2: comparison of the frequency of detection of the non-coding amino acid N +22.968 at position 184 of the AKAP4 protein in healthy and oligozoospermic samples.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Interpretation of terms:

detecting frequency: the frequency of the deviation of the non-coded amino acid N +22.968 in the sample injection sample is called the detection frequency by mass spectrometry after the sample to be detected is processed according to the method described in the embodiment 1 of the invention.

The invention obtains the biomarkers related to severe oligospermia by screening, and the biomarkers are as follows:

asparagine with a mass shift of +22.968 ± 0.005 at position 184 of AKAP4 protein (labeled N + 22.968).

In another embodiment of the present application, a kit for diagnosis of severe oligozoospermia is proposed, which comprises reagents specifically detecting the above biomarkers.

By detecting the biomarkers, diagnosis of severe oligoasthenospermia can be realized.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available.

Example 1: screening of biomarkers associated with severe oligoasthenospermia

The specific screening method is as follows:

first, sample treatment and experimental analysis

1. Extracting the whole protein of the spermatid: and washing the equivalent severe oligozoospermia and normal sperm samples with DPBS for three times respectively, adding equivalent RIPA lysate for ultrasonic treatment for 1-2 min, placing on ice for incubation for 30min for lysis, and centrifuging at 4 ℃ for 14,000g multiplied by 20min to obtain supernatant. Protein concentration was determined using the Bradford method.

2. And (3) proteolysis: about 150. mu.g of sperm protein was taken from each of the low-gravity, weak sperm and normal sperm samples, and the proteins were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE) and fractionated into 5 portions for gel-cutting enzymolysis. The peptide fragments were desalted using ziptip.

3. Mass spectrometry analysis: nano-flow liquid chromatography separation: phase A: water containing 0.1% formic acid; phase B: acetonitrile containing 0.1% formic acid.

Each sample was separately solubilized with 13.5. mu. L A phases, 4. mu.L sample size, and Orbitrap Elite (Thermo Scientific) as a nanoflow liquid mass spectrometry system. The home-made pre-column and analytical column were equilibrated with 4 μ L A phase, respectively, prior to sample isolation. The specifications of the pre-column and the analytical column are respectively as follows: a pre-column (4cm x 150 μm i.d., C18 filler particle size 5 μm,

Collecting mass spectrum data: 350-1800m/z full scan with a resolution of 60,000(m/z 200). In secondary atlas scanning, the activation time was 10ms and the isolation width was 2 m/z. The fragmentation mode is induced-induced dissociation (CID), the normalized collision energy is set to 35%, and the dynamic discharge time is 90 s.

Second, mass spectrometry data analysis

Byonic analysis: to identify non-coding amino acids of sperm proteins, Byonic was used^TMProtamine mass spectral data of normal and severe oligozoospermic patients were analyzed 21. The search parameters are as follows: the protease is trypsin, the missed cutting site is set to be 2, the mass deviation of the parent ion is 10ppm, the mass deviation of the fragment ion is 0.6Da, the upper limit of the blind search is set to be 1000, the lower limit of the blind search is set to be-200, and the protein FDR is 0.01.

Selecting unknown modified peptide segment data searched by Byonic Wildcard Search with FDR <0.01 to form a one-dimensional data matrix, selecting a delta mass range of the data from-200 Da to 400Da, and dividing the data into 601 data windows according to a variation range of 1Da and 0.5Da as a limit. And aiming at each data window, carrying out Gaussian mixture distribution clustering analysis by using an mclust program package in the R language, obtaining an optimal value according to BIC, carrying out combination analysis on each peak, fitting each peak by using Gaussian distribution, and determining a peak value. Each peak after clustering contains information of amino acids, and non-coding amino acids are selected by an iterative model of RUP with 10% as a selection parameter according to the distribution ratio of unknown modifications on 20 amino acids.

The classification algorithm accuracy results are shown in the following table:

Pos

TotalCount

ave_c

ave_b

ratio

Ttest

AUC

pValue

184

366

4.444444

0.873016

-5.09091

2.63E-16

0.898

4.55E-15

thirdly, experimental results:

through mass spectrum data analysis and comparison of non-coding amino acids of normal and diseased groups, the following differential non-coding amino acids can be obtained and can be used as biomarkers related to severe oligospermia, and the specific characteristics are as follows:

FIG. 1 is a ROC curve of the detection frequency of the non-coding amino acid N +22.968 at position 184 of the AKAP4 protein, and ROC analysis shows that the AUC of the non-coding amino acid N +22.968 is 0.898 to 0.7, which indicates that the protein has better diagnostic effect.

The comparison of the detection frequency of the non-coding amino acid N +22.968 in the healthy and oligozoospermia samples is shown in FIG. 2, and it can be seen from FIG. 2 that the non-coding amino acid occurs 4.4 times in the healthy human samples and 0.8 times in the pathological samples on average.

In view of the above results, the non-coding amino acid asparagine N +22.968 at position 184 of AKAP4 protein can be used as a potential biomarker for asthenospermia, thereby predicting this disorder.

Example 2: clinical examination and verification

3 healthy samples and 3 clinically confirmed samples of severe asthenospermia were used as subjects for examination, the frequency of detecting asparagine with mass shift of +22.968 + -0.005 at position 184 of AKAP4 protein in the samples was respectively determined, and the samples to be tested were diagnosed according to the criteria for individual detection of the biomarkers in example 1.

The results show that: when the biomarker is used for independent diagnosis, the detection frequency of the non-coded amino acid N +22.968 in 3 healthy samples is more than 6 times, the detection frequency of 3 samples of patients with severe oligozoospermia is 1 time at most, and the diagnosis result is consistent with the known result. The biological marker obtained by screening can be used as a diagnosis marker of severe asthenospermia.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of asparagine with a mass shift of +22.968 ± 0.005 at position 184 of AKAP4 protein in a sperm sample as a biomarker for the preparation of a diagnostic agent for severe oligozoospermia.