CN110514835B

CN110514835B - Application of C +47.985 at position 385 of ACRBP protein in preparation of severe oligozoospermia diagnostic reagent

Info

Publication number: CN110514835B
Application number: CN201810488386.3A
Authority: CN
Inventors: 杨静华; 孙胜楠; 吕鑫; 陈子江
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-05-21
Filing date: 2018-05-21
Publication date: 2020-06-23
Anticipated expiration: 2038-05-21
Also published as: CN110514835A

Abstract

The invention discloses application of cysteine with mass shift of +47.985 +/-0.005 Da at position 385 of ACRBP protein as a biomarker in preparation of a diagnostic reagent for severe oligozoospermia. The invention discovers that: the detection frequency of cysteine with mass shift of +47.985 +/-0.005 Da at position 385 of ACRBP protein can be used for diagnosing severe asthenospermia, and a new diagnosis and treatment target point is provided for severe asthenospermia.

Description

Application of C +47.985 at position 385 of ACRBP protein in preparation of severe oligozoospermia diagnostic reagent

Technical Field

The invention relates to the technical field of medicine and molecular diagnosis, in particular to application of cysteine with mass shift of +47.985 +/-0.005 Da at position 385 of ACRBP protein as a marker in preparation of a diagnostic reagent for severe oligozoospermia.

Background

According to the world health organization investigation, 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 stress: a common factor in physiological stress.J. Androl,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, and a total of 6198 proteins were identified (Amaral A, Castillo J, Ramalho-Santos J, Oliva R. the combined human sperm protein: cellular pathways and identities for basic research. human reproduction update,20(1),40-62 (2014)). Mayank et al used differential proteomics to quantify 667 proteins in sperm cells, 447 proteins in seminal plasma, and 8 significantly downregulated proteins in 5 groups of healthy and 8 groups of patients with oligospermia, and performed pathway analysis (HumanSpermatola Quantitative diagnostic Signature classes Normo-andAspartaozopermia, Mayank Sawat, 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, Hayaura, Hayayu, Jun, 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 same amount of severe oligozoospermia and normal sperm samples by using DPBS three times respectively, adding the same amount of RIPA lysate to perform ultrasonic treatment for 1-2 min, placing the mixture on ice to perform incubation for 30min for lysis, centrifuging the mixture at 4 ℃ for 14,000g × 20min, taking supernatant, and measuring the protein concentration by using a 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 with 13.5. mu. L A phases, the sample introduction volume was 4. mu.L, the nanoflow liquid mass spectrometry system was Orbitrap Elite (Thermo Scientific), the self-made pre-column and analytical column were separately equilibrated with 4. mu. L A phases before sample separation, the specifications of the pre-column and analytical column were, respectively, pre-column (4cm × 150. mu.m I.D., C18 filler particle size 5. mu.m,

) 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 subjected to nanoflow liquid phase separation directly enters an ESI ion spray source and enters an Orbitrap Elite 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 spectrum data analysis shows that the 385 th cysteine of the ACRBP protein has mass deviation (C +47.985) of +47.985 +/-0.005 Da, the cysteine is deeply oxidized into the cysteic acid, comparison shows that the non-coding amino acid C +47.985 is regulated by 65.5 times under the significance of severe asthenospermia samples, and the p value is 1.26E-14< < 0.01.

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:

cysteine with a mass shift of +47.985 ± 0.005Da at position 385 in the ACRBP protein (labeled C +47.985, which is determined by the mass shift to be deep oxidation of cysteine to cysteic acid);

in a second aspect of the invention, there is provided the use of cysteine (C +47.985) with a mass shift of +47.985 ± 0.005Da at position 385 of the ACRBP protein as a biomarker for the preparation of a diagnostic agent for severe oligozoospermia.

The invention also provides a kit for diagnosing severe asthenospermia, which comprises a reagent for specifically detecting the biomarker (cysteine with mass shift of +47.985 +/-0.005 Da at position 385 of ACRBP protein).

Preferably, the cysteine with a mass shift of +47.985 ± 0.005Da at position 385 of the ACRBP protein can also be used as a target for treatment of severe asthenospermia, and thus for treatment of severe asthenospermia.

The invention also provides the application of cysteine with mass shift of +47.985 +/-0.005 Da at position 385 of ACRBP protein as a biomarker in the preparation of a medicine for treating severe asthenospermia.

The invention also provides a medicine for treating severe asthenospermia, which contains a component capable of converting cysteine at the 385 th position of the ACRBP protein into cysteic acid.

The invention also provides a diagnosis method of severe oligozoospermia, which comprises the following steps: detecting the frequency of +47.985 +/-0.005 Da mass shift of the 385 site cysteine of the ACRBP protein of the sample to be detected, detecting for 3 times in parallel, and judging as a patient with less and weak sperm if the average detection frequency is less than 2.1 times.

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 curve diagram of the detection frequency of non-coding amino acid C +47.985 at position 385 of ACRBP protein;

FIG. 2: a graph comparing the frequency of detection of non-encoded amino acid C +47.985 at position 385 in ACRBP protein of healthy and oligozoospermia 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 C +47.985 in the sample injection is called as 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:

cysteine with a mass shift of +47.985 + -0.005 Da at position 385 in the ACRBP protein (labeled C + 47.985; determining the deep oxidation of cysteine at this position to cysteic acid based on mass shift values);

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. And (3) extracting the whole protein of the sperm cell, namely washing an equivalent amount of severe oligozoospermia and an equivalent amount of normal sperm sample by using DPBS (deep plasma-assisted sperm) for three times respectively, adding an equivalent amount of RIPA (Ribose nucleic acid) lysate for ultrasonic treatment for 1-2 min, incubating on ice for 30min for lysis, centrifuging at 4 ℃ for 14,000g × 20min, taking supernatant, and measuring the protein concentration by using a 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.

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

382

143

2.079365

0.031746

-65.5

1.26E-14

0.882

3.49E-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:

a cysteine with a mass shift of +47.985 + -0.005 Da (labeled C +47.985) at position 385 of the ACRBP protein.

Mass spectrum data analysis shows that the 385 th cysteine of the ACRBP protein has mass deviation of +47.985 +/-0.005 Da (C +47.985), and comparison shows that the non-coding amino acid C +47.985 is remarkably down-regulated by 65.5 times in a sample with less severe and weak sperm, and the p value is 1.26E-14< < 0.01.

FIG. 1 is a ROC curve of the detection frequency of the non-coding amino acid C +47.985 at position 385 of ACRBP protein, and ROC analysis shows that the AUC of the non-coding amino acid C +47.985 is 0.882 to 0.7, which shows that the ACRBP protein has better diagnostic effect.

FIG. 2 compares the frequency of detection of the non-coding amino acid C +47.985 in healthy and oligozootic samples, and it can be seen that this non-coding amino acid occurred on average 2.1 times in healthy human samples and 0 times in pathological samples.

In view of the above results, cysteine (C +47.985) with a mass shift of +47.985 ± 0.005Da at position 385 of the ACRBP protein could be a potential biomarker for oligoasthenospermia and thus predict this disorder.

Example 2: clinical examination and verification

3 healthy samples and 3 clinically confirmed samples of severe asthenospermia were used as subjects to be examined, the frequency of detecting cysteine which causes a mass shift of +47.985 + -0.005 Da at position 385 in the ACRBP protein of the above samples was respectively detected, 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 biomarkers are individually diagnosed, the detection frequency of the non-coded amino acid C +47.985 in 3 healthy samples is respectively 4 times or more, and the detection frequency of the non-coded amino acid in 3 severe oligozoospermia samples is 0 time. The diagnostic results are consistent with known results. 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 cysteine with a mass shift of +47.985 + -0.005 Da at position 385 of the ACRBP protein in a sperm sample as a biomarker for the preparation of a diagnostic agent for severe oligozoospermia.