CN110514750B - Application of C-33.987 at position 385 of ACRBP protein in the preparation of diagnostic reagents for severe oligozoospermia - Google Patents

Abstract

The invention discloses an application of cysteine with-33.987 +/-0.005 mass shift at position 385 of ACRBP protein as a biomarker in preparing a diagnostic reagent for severe oligospermia and asthenospermia. The invention discovers that: the detection frequency of cysteine with mass shift of-33.987 +/-0.005 occurring at position 385 of ACRBP protein can be used for diagnosing severe oligozoospermia, and a new diagnosis and treatment target point is provided for severe oligozoospermia.

Description

The role of C-33.987 at position 385 of ACRBP protein in the preparation of diagnostic reagents for severe oligozoospermia application

technical field

The invention relates to the technical field of medicine and molecular diagnosis, in particular to the use of a cysteine with a mass offset of -33.987±0.005 at position 385 of ACRBP protein as a marker in the preparation of a diagnostic reagent for severe oligospermia.

Background technique

According to a survey by the World Health Organization, 15% of couples of reproductive age have infertility 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 testicular dysfunction. J Androl, 2008. 29(5): p. 488-98). Asthenozoospermia can be diagnosed if the sperm count of grade a is <25%, the sperm count of grade (a+b) is <50%, and the sperm motility rate is less than 60%. Oligospermia is a condition in which the number of sperm in the semen is lower than that of normal fertile men. When a man's sperm is less than 20 million per milliliter, it is oligospermia. Although there are many studies on the pathogenesis of oligoasthenozoospermia, the exact mechanism is still unclear, which also hinders the development of new treatments. Since the transcription and translation of sperm are stalled after maturation, it is convenient for researchers to study the physiological and pathological mechanisms of oligoasthenozoospermia at the level of proteome and its post-translational modifications.

There are currently many studies on the sperm proteome, with a total of approximately 6238 non-redundant proteins identified (Semenproteomics and male infertility, Meritxell Jodar, Ada Soler-Ventura, Rafael Oliva, Molecular Biology of Reproduction and Development Research Group, Journal of Proteomics 162 (2017) 125 –134). The latest updated human sperm proteome has been completed by Amaral et al. A total of 6198 proteins have been identified (Amaral A, Castillo J, Ramalho-Santos J, Oliva R. The combined human sperm proteome: cellular pathways and implications for basic and clinical science. Human reproduction update, 20(1), 40-62(2014)). Mayank et al. used differential proteomics to quantify 667 proteins in sperm cells of 5 groups of healthy men and 8 groups of oligoasthenospermia patients, 447 proteins in seminal plasma, and 8 significantly down-regulated proteins. , and pathway analysis was performed (Human Spermatozoa Quantitative Proteomic Signature Classifies Normo-and Asthenozoospermia, Mayank Saraswat, Sakari Joenvaara, Tushar Jain, Anil KumarTomar, Ashima Sinha, Sarman Singh, Savita Yadav, and Risto Renkonen, Mol Cell Proteomics. 2017 Jan; 16( 1):57-72). To study the molecular mechanism of azoospermia, Mehdi et al. used label-free quantitative proteomics to find 520 significantly altered proteins in testicular tissues of human obstructive and non-obstructive azoospermia, including several key transcription factors, This also lays the foundation for studying the molecular regulatory mechanisms of spermatogenesis and human reproduction (Quantitative proteomic analysis offhuman testis reveals system-widemolecular and cellular pathways associated with non-obstructive azoospermia, MehdiAlikhani, MehdiMirzaei, Marjan Sabbaghian, PouriaParsamatin, RaziehKaramzadeh, SamaneAdib, NiloofarSodeifi, Mohammad Ali Sadighi Gilani, Masoud Zabet-Moghaddam, Lindsay Parker, Yunqi Wu, Vivek Gupta, Paul A. Haynes, Hamid Gourabi, Hossein Baharvand, Ghasem Hosseini Salekdeh, Journal of Proteomics, Volume 162, 6 June 2017, Pages 141-154). The silencing of translational and transcriptional activity in mature sperm also makes it an ideal cellular model to study post-translational modifications, but mass spectrometry-based large-scale studies of sperm post-translational modifications are rare. Research on modification has mainly focused on phosphorylation, glycosylation, acetylation and ubiquitination (The Challenge of Human Spermatozoa Proteome: A Systematic Review, Kambiz Gilany, Arash Minai-Tehrani, Mehdi Amini, Niloofar Agharezaee, Babak Arjmand, J Reprod Infertil. 2017 Jul-Sep;18(3):267–279.). Phosphorylation of tyrosine plays an important role in sperm motility, capacitation, and hyperexcited motility. Chying-Chyuan Chan et al. found that 12 proteins including TUBGCP2 were hyperphosphorylated by proteomic analysis of sperm from 20 groups of normal and asthenozoospermia patients. Non-coding amino acids, including post-translational modifications and amino acid mutations, are important ways to regulate protein function and structure. Therefore, non-coding amino acids that are abnormal in disease states or whose quantities vary greatly are used as biomarkers of disease and then used to diagnose the process of disease. important meaning.

The acrosomal protein is the main protein of the acrosomal matrix protein and is a serine protease that exists in the form of an inactive protease. Acrosome plays an important role in physiological processes such as the disassembly of acrosome matrix proteins, the binding of sperm to the zona pellucida, and the acrosome reaction (Modes of acrosin functioning during fertilization). Studies have shown that sperm fertilization rate is closely related to the activity of acrosin (Sperm acrosin activity and fluorescencemicroscopic assessment of proacrosin/acrosin in ejaculates of infertile and fertile men).

SUMMARY OF THE INVENTION

At present, medical researchers are not clear about the pathogenesis of severe oligoasthenospermia. The inventors of this case chose sperm protein as the research object and analyzed the mutation status of non-coding amino acids, which is helpful to analyze the pathogenesis of severe oligoasthenospermia from the genetic level. mechanism. Most of the treatment drugs for severe oligoasthenospermia are Chinese patent medicines and hormone drugs, and the cure rate is low. Carrying out research on non-coding amino acid mutation sites is beneficial to provide targets for the treatment of severe oligo-asthenospermia and provide more basis for drug research and development.

Regarding the research on biomarkers of severe oligoasthenozoospermia, the inventors of this case have achieved certain results in previous researches, which have been published in patents CN106872630A, CN106932597A, CN106990177A, CN106996981A, CN106996979A, CN106996980A, CN107015005A, CN107024777A. It is well known that there are limited non-amino acid sites. The inventors of this case chose to study the relationship between these mutation sites and the onset of severe oligoasthenozoospermia. However, in fact, mutations at these sites may be associated with the occurrence of various diseases in the human body. The screening of mutated non-coding amino acid sites is of great significance for disease diagnosis and medical development. Therefore, the inventor of the present case conducted a more in-depth study on the mutation status of non-coding amino acids in sperm proteins. In the follow-up research process, the inventor carried out meticulous and arduous research work, and obtained 21 by constantly identifying the mutation sites. The inventors once again obtained significant research results by performing statistical analysis on the potentially meaningful mass spectrometry protamine data and the correlation between the screened mutation sites and the disease.

In view of the above prior art, the purpose of the present invention is to provide a screening and application of biomarkers related to severe oligoasthenozoospermia. The present invention firstly uses NanoHPLC-MS/MS mass spectrometry system and non-label quantitative proteomics method to carry out in-depth mass spectrometry analysis of sperm protein non-coding amino acids of multiple groups of severe oligoastomospermia diseases; The mass spectrometry data was searched, and then multivariate Gaussian mixture distribution cluster analysis was performed to identify as many non-coding amino acids in the sperm proteome as possible; The non-coding amino acid sites of spermatozoa-related proteins can be used as molecular markers for severe oligoasthenospermia.

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

Equal amounts of severe oligoasthenozoospermia and normal sperm samples were washed three times with DPBS respectively, added with the same amount of RIPA lysis buffer and sonicated for 1-2 min, incubated on ice for 30 min to lyse, and centrifuged at 4°C for 14,000g × 20 min to take the supernatant. Protein concentration was determined using the Bradford method.

Approximately 150 μg of sperm protein was taken from the samples of severe oligospermia and normal sperm, and the proteins were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE). Peptides were desalted using ziptip.

Nanoflow liquid chromatography separation: Phase A: water with 0.1% formic acid; Phase B: acetonitrile with 0.1% formic acid.

)，分析柱(30cm×75μm I.D.,C18填料填充，粒径3μm,

Dr.Maisch GmbH,Germany)。平衡之后样品在A相的带动下首先载样于预柱，然后在不同梯度下进行液相分离。150min色谱梯度变化如下：5-32％流动相B，100min；32-80％流动相B，20min；80％流动相B，30min。流速始终保持在300nL/min。经过纳流液相分离的样品直接进入ESI离子喷雾源并进入Orbitrap Elite质谱仪中进行质谱检测。Each sample was dissolved with 13.5 μL phase A, the sample injection volume was 4 μL, and the nanoflow liquid chromatography mass spectrometry system was Orbitrap Elite (Thermo Scientific). The self-made pre-column and analytical column were equilibrated with 4 μL of phase A before sample separation. The specifications of the pre-column and the analytical column are: pre-column (4cm×150μm ID, C18 filler particle size 5μm,

), analytical column (30cm×75μm ID, C18 packing, particle size 3μm,

Dr. Maisch GmbH, Germany). After equilibration, the samples were first loaded on the pre-column under the driving of phase A, and then liquid-phase separation was carried out under different gradients. The 150min chromatographic gradient changes are as follows: 5-32% mobile phase B, 100min; 32-80% mobile phase B, 20min; 80% mobile phase B, 30min. The flow rate was always maintained at 300 nL/min. The sample after nanoflow liquid phase separation directly enters the ESI ion spray source and enters the Orbitrap Elite mass spectrometer for mass detection.

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

In order to identify the non-coding amino acids of sperm protein, the present invention adopts Byonic ^TM to analyze the sperm protein profile data of 21 pairs of normal and severe oligoasthenozoospermia patients. The search parameters are as follows: the protease is trypsin, the missed cleavage site is set to 2, the mass deviation of parent ions is 10ppm, the mass deviation of fragment ions is 0.6Da, the upper limit of blind search is set to 1000, and the lower limit of blind search is set to -200. Protein FDR is 0.01.

Select the unknown modified peptide data searched by Byonic Wildcard Search with FDR<0.01 to form a one-dimensional data matrix. The delta mass range of the data is -200Da-400Da, and then the data is divided according to the change interval of 1Da, and 0.5Da is the interval limit. into 601 data windows. For each data window, use the mclust package in R language to do Gaussian mixture distribution clustering analysis, take the optimal value according to BIC, then merge and analyze each peak, and then fit each peak with Gaussian distribution to determine the peak value . Each peak after clustering contains amino acid information. According to the distribution ratio of unknown modification on 20 amino acids, with 10% as the selection parameter, the iterative model of RUP is used to select non-coding amino acids.

The non-coding amino acids of normal and diseased groups were screened according to their detection frequency T test (p<0.01), ratio (ratio>2) and detection frequency (>100) to obtain differential non-coding amino acids. Then use SPSS software to make ROC curve of differential non-coding amino acid, and calculate its area under the curve (AUC), and then judge its diagnostic value.

The above screening method is used to obtain biomarkers, not for the purpose of obtaining the results of diagnosis and treatment of diseases; the biomarkers obtained by the above screening method can be used for theoretical research of severe oligoasthenozoospermia or development of new drugs.

The first aspect of the present invention provides biomarkers related to severe oligoasthenospermia screened according to the above-mentioned screening method, the biomarkers include but are not limited to:

The cysteine at position 385 of ACRBP protein with a mass offset of -33.987±0.005 (marked as C-33.987, according to the mass offset, it is determined that this site is cysteine to remove H ₂ S and convert to dehydroalanine) ;

The second aspect of the present invention provides the use of a cysteine (marked as C-33.987) with a mass shift of -33.987±0.005 at position 385 of ACRBP protein as a biomarker in preparing a diagnostic reagent for severe oligospermia.

The present invention also provides a kit for diagnosing severe oligozoospermia, the kit includes a specific detection method for the above-mentioned biomarker (cysteine with a mass shift of -33.987±0.005 at position 385 of ACRBP protein). reagents.

Preferably, the cysteine with a mass offset of -33.987±0.005 at position 385 of ACRBP protein can also be used as a target for the treatment of severe oligoasthenospermia, so as to be used for the treatment of severe oligoasthenospermia.

The invention also provides the use of the cysteine with a mass shift of -33.987±0.005 at the 385th position of the ACRBP protein as a biomarker in the preparation of a therapeutic drug for severe oligospermia.

The present invention also provides a medicament for treating severe oligoasthenoma, which contains a component capable of converting the cysteine at position 385 of ACRBP protein into dehydroalanine.

The invention also provides a method for diagnosing severe oligozoospermia, comprising the steps of: detecting the frequency of -33.987±0.005 mass shift of the cysteine at position 385 of the ACRBP protein of the sample to be tested; Average results, if the average detection frequency is less than 4.5, it is judged as oligoastrosis patients.

Beneficial effects of the present invention:

The present invention further studies the biomarkers obtained by the above-mentioned screening method, and finds that severe oligoasthenospermia can be diagnosed by the test frequency of the above-mentioned biomarkers, which provides a new diagnosis and treatment target for severe oligoasthenospermia.

Description of drawings

The accompanying drawings that form a part of the present application are used to provide further understanding of the present application, and the schematic embodiments and descriptions of the present application are used to explain the present application and do not constitute improper limitations on the present application.

Figure 1: ROC curve diagram of the detection frequency of the non-coding amino acid C-33.987 at position 385 of ACRBP protein;

Figure 2: Comparison of the detection frequencies of the non-coding amino acid C-33.987 at position 385 of ACRBP protein in healthy and oligoastrotic samples.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, 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 should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components, and/or combinations thereof.

Terminology Explanation:

Detection frequency: After the sample to be tested is processed according to the method described in Example 1 of the present invention, and then analyzed by mass spectrometry, the number of times that the non-coding amino acid C-33.987 in the injected sample shifts is called the detection frequency.

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

A -33.987±0.005 mass-shifted cysteine at position 385 of ACRBP protein (marked as C-33.987);

In another embodiment of the present application, a kit for diagnosing severe oligozoospermia is proposed, wherein the kit includes reagents for specifically detecting the above-mentioned biomarkers.

By detecting the above-mentioned biomarkers, the diagnosis of severe oligoasthenospermia can be achieved.

In order to enable those skilled in the art to understand the technical solutions of the present application more clearly, 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 test materials in the art, and can be purchased through commercial channels.

Example 1: Screening of biomarkers associated with severe oligoasthenospermia

The specific filtering methods are as follows:

1. Sample processing and experimental analysis

1. Extraction of sperm cell whole protein: equal amounts of severe oligoasthenospermia and normal sperm samples were washed three times with DPBS respectively, added with an equal amount of RIPA lysis buffer and sonicated for 1-2 minutes, incubated on ice for 30 minutes to lyse, and centrifuged at 14,000g at 4°C ×20min to take the supernatant. Protein concentration was determined using the Bradford method.

2. Proteolysis: Take about 150 μg sperm protein from each of the severe oligospermia and normal sperm samples, separate the proteins by 10% polyacrylamide gel electrophoresis (SDS-PAGE), and divide each into 5 parts for gel cutting and enzymatic hydrolysis. Peptides were desalted using ziptip.

3. Mass spectrometry analysis: Nanoflow liquid chromatography separation: Phase A: water containing 0.1% formic acid; Phase B: acetonitrile containing 0.1% formic acid.

)，分析柱(30cm×75μm I.D.,C18填料填充，粒径3μm,

Dr.Maisch GmbH,Germany)。平衡之后样品在A相的带动下首先载样于预柱，然后在不同梯度下进行液相分离。150min色谱梯度变化如下：5-32％流动相B100min；32-80％流动相B，20min；80％流动相B，30min。流速始终保持在300nL/min。经过纳流液相分离的样品直接进入ESI离子喷雾源并进入Orbitrap Elite质谱仪中进行质谱检测。Each sample was dissolved with 13.5 μL phase A, the sample injection volume was 4 μL, and the nanoflow liquid chromatography mass spectrometry system was Orbitrap Elite (Thermo Scientific). The self-made pre-column and analytical column were equilibrated with 4 μL of phase A before sample separation. The specifications of the pre-column and the analytical column are: pre-column (4cm×150μm ID, C18 filler particle size 5μm,

), analytical column (30cm×75μm ID, C18 packing, particle size 3μm,

Dr. Maisch GmbH, Germany). After equilibration, the samples were first loaded on the pre-column under the driving of phase A, and then liquid-phase separation was carried out under different gradients. The 150min chromatographic gradient changes are as follows: 5-32% mobile phase B, 100min; 32-80% mobile phase B, 20min; 80% mobile phase B, 30min. The flow rate was always maintained at 300 nL/min. The sample after nanoflow liquid phase separation directly enters the ESI ion spray source and enters the Orbitrap Elite mass spectrometer for mass detection.

Mass Spectrometry Data Acquisition: Full scan from 350-1800 m/z with a resolution of 60,000 (m/z 200). During secondary mapping, the activation time was 10 ms and the isolation width was 2 m/z. The fragmentation method was collision-induced dissociation (CID), the normalized collision energy was set to 35%, and the dynamic discharge time was 90 s.

2. Mass Spectrometry Data Analysis

Byonic Analysis: In order to identify non-coding amino acids of sperm proteins, the sperm protein profile data of 21 pairs of normal and severe oligoasthenospermia patients were analyzed with Byonic ^™ . The search parameters are as follows: the protease is trypsin, the missed cleavage site is set to 2, the mass deviation of the precursor ion is 10 ppm, the mass deviation of the fragment ion is 0.6 Da, the upper limit of the blind search is set to 1000, and the lower limit of the blind search is set to -200. Protein FDR is 0.01.

Select the unknown modified peptide data searched by Byonic Wildcard Search with FDR<0.01 to form a one-dimensional data matrix. The delta mass range of the data is -200Da-400Da, and then the data is divided into 601 data windows. For each data window, use the mclust package in R language to do Gaussian mixture distribution clustering analysis, take the optimal value according to BIC, and then merge and analyze each peak, and then use Gaussian distribution to fit each peak to determine the peak value . Each peak after clustering contains amino acid information. According to the distribution ratio of unknown modification on 20 amino acids, with 10% as the selection parameter, the iterative model of RUP is used to select non-coding amino acids.

The non-coding amino acids of normal and diseased groups were screened according to their detection frequency T test (p<0.01), ratio (ratio>2) and detection frequency (>100) to obtain differential non-coding amino acids. Then use SPSS software to make ROC curve of differential non-coding amino acid, and calculate its area under the curve (AUC), and then judge its diagnostic value.

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

Pos TotalCount ave_c ave_b ratio Ttest AUC pValue 382 370 4.507937 0.507937 -8.875 5.6E-20 0.922 4.33E-22

3. Experimental results:

Through mass spectrometry data analysis and comparison of non-coding amino acids between normal and diseased groups, the following differential non-coding amino acids were obtained, which can be used as biomarkers associated with severe oligoasthenozoospermia, as follows:

A -33.987±0.005 mass-shifted cysteine at position 385 of ACRBP protein (labeled as C-33.987)

After mass spectrometry data analysis, it was found that the cysteine at position 385 of ACRBP protein had a mass shift of -33.987±0.005 (C-33.987). After comparison, it was found that this non-coding amino acid C-33.987 was significantly down-regulated in severe oligospermia samples 8.8 times, p-value 5.60E-20<<0.01.

Figure 1 shows the ROC curve of the detection frequency of the non-coding amino acid C-33.987 at position 385 of ACRBP protein. The ROC analysis shows that the AUC of this non-coding amino acid C-33.987 is 0.992>0.7, indicating that it has a good diagnostic effect.

A comparison of the detection frequency of the non-coding amino acid C-33.987 at position 385 of ACRBP protein in healthy and oligoastrotic samples is shown in Figure 2. It can be seen from Figure 2 that this non-coding amino acid occurs 4.5 times on average in healthy samples and 4.5 times in pathological samples. Happened 0.5 times.

In view of the above results, the non-coding amino acid cysteine C-33.987 at position 385 of ACRBP protein can be used as a potential biomarker for oligoasthenozoospermia to predict this disorder.

Example 2: Clinical Test Validation

3 healthy samples and 3 clinically diagnosed severe oligospermia samples were used as the research objects for verification, and the detection frequency of cysteine with a mass shift of -33.987±0.005 in the ACRBP protein 385 of the above samples was detected, and the The sample to be tested is diagnosed according to the judgment criteria for individual detection of each biomarker in Example 1.

The results showed that when the above biomarkers were independently diagnosed, the detection frequency of non-coding amino acid C-33.987 in 3 healthy samples was 5 times or more, while this non-coding amino acid in 3 severe oligospermia samples was not detected. Offset detected. The diagnosis was consistent with known results. This shows that the biomarkers screened in the present invention can be used as diagnostic markers for severe oligoasthenosis.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims (1)

1. Use of cysteine with a mass shift of-33.987 + -0.005 at position 385 in an ACRBP protein in a sperm sample as a biomarker in the preparation of a diagnostic agent for severe oligozoospermia.

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