CN110514749B

CN110514749B - Application of cysteine with mass shift at 73 th site of ACR protein in preparation of diagnostic reagent for severe oligospermia and asthenospermia

Info

Publication number: CN110514749B
Application number: CN201810486951.2A
Authority: CN
Inventors: 杨静华; 孙胜楠; 陈新骏; 陈子江
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-05-21
Filing date: 2018-05-21
Publication date: 2020-09-22
Anticipated expiration: 2038-05-21
Also published as: CN110514749A

Abstract

The invention discloses an application of cysteine with mass shift of-33.987 +/-0.005 occurring at 73 th site of ACR protein as a biomarker in preparing a diagnostic reagent for severe oligozoospermia. The invention discovers that: the detection frequency of cysteine with mass shift of-33.987 +/-0.005 occurring at the 73 th site of ACR protein can be used for diagnosing severe oligozoospermia, and a new diagnosis and treatment target point is provided for severe oligozoospermia.

Description

Application of cysteine with mass shift at 73 th site of ACR protein in preparation of diagnostic reagent for severe oligospermia and asthenospermia

Technical Field

The invention relates to the technical field of medicine and molecular diagnosis, in particular to application of cysteine (novel non-coding amino acid ACR @73C-33.987) with mass shift of-33.987 +/-0.005 Da at 73 th site of ACR protein as a marker in preparation of a diagnostic reagent for severe oligospermia and weak sperm.

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 (Semenproteomics and male fertility, MeritellJodar, Ada Soler-Venturia, RafaelOliva, 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 of 5 groups of healthy and 8 groups of patients with oligospermia, 447 proteins in seminal plasma, and 8 significantly down-regulated proteins were identified and subjected to pathway analysis (HumanSpermatozoa Quantitative diagnostic Signature Classification Normo-andAsdaenozopermia, MayankSaraswat, Sakari Joenvarara, Tushar Jain, oil KumarTomar, AshimaSinha, Sarman Singh, SavitaYadavav, and RistoRenkon, Mol cell Proteimics.2017; 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, Hayahoo, Hayayu, Guikayama, 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, KambizGilany, ArashMinai-Tehrani, Mehdi Amini, Niloofargarezaee, Babak Arjmand, J reprodInfertil.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 sites of non-coding amino acids are limited, and the inventors of the present invention have selected and studied the relationship between these mutation sites and the onset of severe asthenospermia, but in fact, the mutation at these sites may be associated with the occurrence of various diseases in the human body, and the selection of the non-coding amino acid sites with mutations as many 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:

washing the samples of severe oligozoospermia and normal spermatozoa with equal amount with DPBS for three times, adding equal amount of RIPA lysate, performing ultrasound for 1-2min, incubating on ice for 30min for lysis, and centrifuging at 4 deg.C for 14,000g × 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 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 is induced-induced dissociation (CID), the normalized collision energy is set to 35%, and the dynamic discharge time is 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, and the cleavage site is missedSet to 2, parent ion mass deviation to 10ppm, fragment ion mass deviation to 0.6Da, blind search upper limit to 1000, blind search lower limit to-200, protein FDR to 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 cysteine at 73 th position of the ACR protein has mass deviation of-33.987 +/-0.005 Da (C-33.987), and H is removed from the cysteine₂S is changed into dehydroalanine, and the comparison shows that the non-coding amino acid C-33.987 is regulated by 35.6 times under the significance of a severe asthenospermia sample, and the p value is 3.49E-12<<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-33.987 + -0.005 Da at position 73 of ACR protein (labeled C-33.987; determination of cysteine removal H at this position based on mass shift values₂S to dehydroalanine);

in a second aspect of the invention, there is provided the use of cysteine (labeled C-33.987) having a mass shift of-33.987 + -0.005 Da at position 73 of ACR 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-33.987 at the 73 th position of ACR protein).

Preferably, the cysteine with the mass shift of-33.987 at the 73 th position of the ACR protein can also be used as a target for treating severe asthenospermia, thereby being used for treating severe asthenospermia.

The invention also provides application of cysteine with-33.987 mass shift at 73 th site of ACR protein as a biomarker in preparation of a therapeutic drug for severe oligospermia.

The invention also provides a medicine for treating severe oligospermia, which contains a component for converting the 73 th cysteine of the ACR protein into dehydroalanine.

The invention also provides a diagnosis method of severe oligozoospermia, which comprises the following steps: detecting the frequency of-33.987 +/-0.05 Da mass shift (ACR @73C-33.987) of cysteine at the 73 th site of the ACR protein of the sample to be detected, parallelly detecting each sample for 3 times, and judging the patient as a patient with less asthenospermia if the average detection frequency is less than 1.7.

The invention also provides a biomarker, wherein the cysteine generating-33.987 +/-0.005 mass shift is positioned at the 73 th position of ACR protein.

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 detection frequency of non-coding amino acid C-33.987 at position 73 of ACR protein;

FIG. 2: a graph comparing the frequency of detection of the non-encoded amino acid C-33.987 at position 73 of ACR 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 C-33.987 in the sample injection 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:

the-33.98733.987 + -0.005 mass shifted cysteine (labeled C-33.987; determining the cysteine removal H at this position based on the mass shift value) occurred at position 73 of ACR protein (₂S to dehydroalanine);

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: washing the samples of severe oligozoospermia and normal spermatozoa with equal amount with DPBS for three times, adding equal amount of RIPA lysate, performing ultrasound for 1-2min, incubating on ice for 30min for lysis, and centrifuging at 4 deg.C for 14,000g × 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.

dr. maisch GmbH, Germany). After balancing, the sample is loaded on the pre-column under the drive of the phase A, and then the sample is loaded on the pre-column without the drive of the phase AThe liquid phase separation was carried out under the same gradient. 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.

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

70

138

1.698413

0.047619

-35.6667

3.49E-12

0.861

7.60E-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:

the-33.98733.987 + -0.005 mass-shifted cysteine (labeled C-33.987) occurred at position 73 of ACR protein (determination of the removal of H from the cysteine at this position was made based on the mass shift value)₂S to dehydroalanine);

through mass spectrometry data analysis, mass shift C-33.987 is found on the 73 th cysteine of ACR protein, and through comparison, the non-coding amino acid is found to be adjusted by 35.6 times in the significance of samples with severe asthenospermia, and the p value is 3.49E-12< < 0.01.

In order to evaluate the diagnosis efficiency of the mass deviation C-33.987 detection frequency on the 73 th cysteine of the ACR protein on the severe weakness, the invention adopts ROC curve analysis, and the AUC is the area under the ROC curve, is the most commonly used parameter for evaluating the characteristics of the ROC curve, and is an important test accuracy index. If the AUC is below 0.7, the diagnosis accuracy is low; the AUC is more than 0.7, so that the requirement of clinical diagnosis can be met.

FIG. 1 is a ROC curve showing the detection frequency of mass shift of-33.987 + -0.005 occurring in cysteine at position 73 of ACR protein, and ROC analysis shows that the AUC of this non-coding amino acid C-33.987 is 0.861>0.7, which indicates that cysteine occurring in mass shift of-33.98710 at position 73 of ACR protein can be used as a diagnostic marker for severe asthenospermia.

The results of comparing the frequency of cysteine detection with-33.987. + -. 0.005 mass shift at position 73 of ACR protein in healthy and oligozoospermic samples are shown in FIG. 2. it can be seen from FIG. 2 that this non-encoded amino acid occurs 1.7 times on average in healthy human samples and 0 times in pathological samples.

In view of the above results, cysteine C-33.987 with a mass shift of-33.987 + -0.005 at position 73 of ACR protein could be used as a potential biomarker for oligoasthenospermia, 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 to be examined for verification, the detection frequency of cysteine causing mass shift of-33.987 + -0.005 at position 73 of ACR protein in the samples was respectively detected, and the samples to be detected were diagnosed according to the judgment criteria in the individual detection of the biomarkers in example 1.

The results show that: when the biomarkers are used for independent diagnosis, the non-coded amino acid C-33.987 in 3 healthy samples is detected at 2 times and more than 2 times, and no deviation is detected in the non-coded amino acid in 3 samples with severe oligospermia. The diagnosis result is consistent with the known result, which indicates that the biomarker obtained by screening of the invention 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. The application of cysteine with mass shift of-33.987 +/-0.005 Da at 73 site of ACR protein in sperm sample as biomarker in preparing diagnostic reagent for severe asthenospermia.