CN110514837A - Application of 3 S+79.967 of AKAP3 protein 20 in the few weak smart diagnostic reagent of preparation severe - Google Patents
Application of 3 S+79.967 of AKAP3 protein 20 in the few weak smart diagnostic reagent of preparation severe Download PDFInfo
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Abstract
The invention discloses AKAP3 protein 20 3 to occur purposes of the serine of+79.967 ± 0.005 mass shift as biomarker in the diagnostic reagent that preparation severe lacks weak essence.Present invention discover that: the inspection frequency for the serine that+79.967 ± 0.005 mass shift occurs by AKAP3 protein 20 3, which can be used to diagnose severe, lacks azoospermia, lacks azoospermia for severe and provides new diagnosing and treating target spot.
Description
Technical field
The present invention relates to medicine and molecular diagnostic techniques field, and in particular to it is a kind of AKAP3 protein 20 3 occur+
The serine (undoded amino acid AKAP3@203S+79.967) of 79.967 ± 0.005 mass shifts is being prepared as marker
Purposes in the few weak smart diagnostic reagent of severe.
Background technique
It is investigated according to the World Health Organization, 15% couple at child-bearing age has sterile problem, and it is strong that sterility has become the influence mankind
A global sex medicine and social concern (Turner, T.T.and J.J.Lysiak, Oxidative for health and social development
stress:a commonfactor in testicular dysfunction.J Androl,2008.29(5):p.488-
98).If a grades of sperm count < 25%, (a+b) grade sperm count < 50%, and can be diagnosed as if sperm motility rate is lower than 60% weak
Sperm disease.Oligospermatism refers to that the sperm number in sperm lower than a kind of normal illness with fecundity male, works as male
Sperm every milliliter be lower than 2,000 ten thousand when, just be oligospermatism.Although the research about the pathogenesis of teen bra now
Very much, but its precise mechanism is still uncertain, this also counteracts the development of its new treatment means.Due to its turn after spermioteleosis
Record and translation stay cool, this is just that researcher studies few weak sperm in protein group and its posttranslational modification level
The physiological and pathological mechanism of disease provides convenience.
The research about sperm protein matter group has much at present, about identifies 6238 non-redundant proteins (Semen in total
Proteomics and male infertility, Meritxell Jodar, Ada Soler-Ventura, Rafael
Oliva, Molecular Biology of Reproductionand Development ResearchGroup,
JournalofProteomics 162(2017)125–134).Current last updated human sperm's protein group Amaral etc.
It completes, identifies 6198 albumen (Amaral A, Castillo J, Ramalho-Santos J, Oliva R.The altogether
combined humanspermproteome:cellular pathways and implications for basic and
clinical science.Human reproduction update,20(1),40-62(2014)).It is poor that Mayank etc. is utilized
The method of M-band matter group has quantified 667 albumen in 5 groups of Healthy Peoples and the spermatoblast of 8 groups of teen bra patients,
447 albumen have been quantified in refining, have gone out 8 significant down-regulation proteins about point, and path analysis has been carried out to it
(HumanSpermatozoa Quantitative Proteomic Signature Classifies Normo-and
Asthenozoospermia, Mayank Saraswat, Sakari Joenvaara, Tushar Jain, Anil Kumar
Tomar, Ashima Sinha, Sarman Singh, Savita Yadav, and Risto Renkonen, Mol Cell
Proteomics.2017Jan；16(1):57-72).In order to study the molecular mechanism of azoospermatism, Mehdi etc. is fixed using non-marked
Amount proteomics method has found 520 conspicuousnesses variation eggs in the obstructive testis tissue with nonobstructive azoospermatism of people
White, including several key transcription factors, this is also to study spermatogenesis and the molecular regulation mechanism of human reproduction is laid a good foundation
(Quantitative proteomic analysis of human testis reveals system-wide
Molecular and cellular pathways associated withnon-obstructive azoospermia,
MehdiAlikhani, MehdiMirzaei, MarjanSabbaghian, PouriaParsamatin,
RaziehKaramzadeh, SamaneAdib, NiloofarSodeifi, Mohammad Ali SadighiGilani,
MasoudZabet-Moghaddam, LindsayParker, YunqiWu, VivekGupta, PaulA.Haynes,
HamidGourabi, HosseinBaharvand, GhasemHosseiniSalekdeh, Journal ofProteomics,
Volume 162,6June 2017,Pages 141-154).The silencing of mature sperm translation transcription activity also becomes research
The ideal cell model of posttranslational modification, but about based on mass spectrographic sperm posttranslational modification broad scale research or seldom.
Research about modification focuses primarily upon phosphorylation, glycosylation, acetylation and ubiquitination (The Challenge ofHuman
Spermatozoa Proteome:A Systematic Review, Kambiz Gilany, Arash Minai-Tehrani,
MehdiAmini, Niloofar Agharezaee, Babak Arjmand, J Reprod Infertil.2017Jul-Sep；18
(3):267–279.).Tyrosine phosphorylation is for processes important function such as the movement of sperm, capacitation, super sharp movements.Chying-
ChyuanChan etc., which carries out proteome analysis discovery by the sperm to 20 groups of normal persons and azoospermia patient, 12 kinds of packets
It includes the albumen including TUBGCP2 and peroxophosphoric acid has occurred.Undoded amino acid includes posttranslational modification and amino acid mutation is to adjust
The important way of protein function and structure is controlled, therefore abnormal under morbid state or quantity is changed into great non-coding amino
Acid is of great significance as the process that the biomarker of disease is used to diagnose the illness in turn.
Acrosin is the major protein of acrosome stromatin, is a kind of serine protease, with inactive protein proenzyme
Form exist.Acrosin disintegrates in acrosome stromatin, and sperm rises in the physiology courses such as acrosome reaction in conjunction with oolemma
Important function (Modes ofacrosinfunctioning during fertilization).Research shows that sperm fertilization
Closely related (the Sperm acrosin activity and fluorescence of the activity of rate and acrosin
microscopic assessment of proacrosin/acrosin in ejaculates of infertile and
fertile men)。
Summary of the invention
Medical researchers lack the pathogenesis of azoospermia and indefinite to severe at present, and inventor selects sperm egg
It is white to be used as research object, the mutation situation of wherein undoded amino acid is analyzed, helps to lack weak essence from gene level parsing severe
The pathogenesis of disease.The therapeutic agent for lacking azoospermia about severe is controlled mostly based on the Chinese patent drug of qi-restoratives and hormone medicine
More rate is low.Carry out the therapeutic agent for being conducive to lack for severe weak essence about the research in undoded amino acid mutational site and target is provided
Mark, provides more foundations for the research and development of drug.
Lack the research of the biomarker of azoospermia about severe, inventor achieves centainly in previous research
Achievement, and be published in patent CN106872630A, CN106932597A, CN106990177A, CN106996981A,
In CN106996979A, CN106996980A, CN107015005A, CN107024553A, CN107037172A.It is well known that
The site of non-amino acid is limited, these mutational sites of inventor's Selecting research and severe lack the relationship that azoospermia is fallen ill, but
It is more as far as possible to filter out mutation in fact, the mutation in these sites may be associated with the generation of a variety of diseases of human body
Undoded amino acid site has great importance for the diagnosis and medicine development of disease.Therefore, inventor is to sperm
In albumen undoded amino acid mutation situation carried out deeper into research, in subsequent research process, inventor carry out
Careful and heavy research work has obtained 21 pairs of mass spectrum essence eggs for being possible to significant by constantly identifying mutational site
White data, and statistical analysis is carried out to the mutational site screened and the correlation of disease, inventor is had again
The research achievement of significance.
For the above-mentioned prior art, it is an object of the invention to provide a kind of biomarkers relevant to severe teen bra
Screening and application.The invention firstly uses NanoHPLC-MS/MS mass spectrometer systems and label-free proteomics method pair
The sperm protein undoded amino acid of the few weak smart disease of multiple groups severe has carried out the mass spectral analysis of depth；Then non-limiting ammonia is utilized
Base acid protein modification analysis method scans for mass spectrometric data, using multivariate Gaussian mixed distribution clustering, to the greatest extent
Undoded amino acid in sperm protein group may largely be identified；Finally by non-coding in normal and patient's sperm protein group
The comparison of amino acid obtains lacking azoospermia relevant albumen undoded amino acid site to severe, thus few as severe
The molecular marker of azoospermia.
In order to achieve the goal above, the present invention the following technical schemes are provided:
The few weak essence of the severe of equivalent and eupyrene sperm sample are washed three times with DPBS respectively, and equivalent RIPA lysate ultrasound is added
1-2min, is placed in and is incubated for 30min cracking on ice, and 4 DEG C of centrifugation 14,000g × 20min take supernatant.It is measured using Bradford method
Protein concentration.
The about few weak essence of severe and each 150 μ g sperm protein of eupyrene sperm sample are taken, 10% polyacrylamide gel electricity is used
Swimming (SDS-PAGE) separates albumen, is respectively divided into 5 parts and carries out cutting glue enzymatic hydrolysis.Desalination is carried out to peptide fragment using ziptip.
Receive flow liquid phase chromatographic isolation: A phase: the water containing 0.1% formic acid；B phase: the acetonitrile containing 0.1% formic acid.
Each sample uses 13.5 μ L A phased solns respectively, and sample injection volume is 4 μ L, and flow liquid phase mass spectrometry system of receiving is
Orbitrap Elite(Thermo Scientific).Sample separation before respectively with 4 μ L A balance each other homemade pre-column and divide
Analyse column.The specification of pre-column and analytical column be respectively as follows: pre-column (5 μm of 4cm × 150 μm I.D., C18 packing material size,), analysis
Column (filling of 30cm × 75 μm I.D., C18 filler, 3 μm of partial size, Dr.Maisch GmbH,Germany).After balance
Then sample load sample first under the drive of A phase carries out liquid phase separation in pre-column under different gradients.150min chromatography gradient becomes
Change as follows: 5-32% Mobile phase B, 100min；32-80% Mobile phase B, 20min；80% Mobile phase B, 30min.Flow velocity is protected always
It holds in 300nL/min.Through receiving stream liquid phase separation sample be directly entered ESI ionspray source and enter Orbitrap Elite
Mass Spectrometer Method is carried out in mass spectrograph.
Mass spectrometric data acquisition condition is the full scan of 350-1800m/z, resolution ratio 60,000 (m/z 200).Second level figure
When spectrum scanning, activation time 10ms, isolation width is 2m/z.Fragmentation pattern is that induction is collisionally dissociated (collision-
Induced dissociation, CID), normalization collision energy is set as 35%, and dynamic efflux time is 90s.
In order to identify the undoded amino acid of sperm protein, the present invention uses ByonicTM21 pairs of analysis is normally and severe
The protamine mass spectrometric data of few weak smart patient.Search parameter is as follows: protease is trypsase, and leakage enzyme site is set as 2, it is female from
Protonatomic mass deviation is 10ppm, and the mass deviation of fragment ion is 0.6Da, and the blind upper limit of searching is set as 1000, it is blind search lower limit be set as-
200. albumen FDR are 0.01.
The unknown modifications peptide segment data that the Byonic Wildcard Search of selection FDR < 0.01 is searched, forms one-dimensional
Data matrix, the delta mass range selection -200Da-400Da of data, then by data according to the constant interval of 1Da, 0.5Da
For interval limit, it is divided into 601 data windows.For each data window, done using the mclust program bag in R language
Gaussian Mixture distributed clustering analysis takes optimal value according to BIC, then merges analysis to each peak, then uses Gaussian Profile
It is fitted each peak, determines peak value.It include the information of amino acid in each peak after cluster, according to unknown modifications 20
Distribution proportion on kind amino acid, is selection parameter with 10%, selects undoded amino acid with the iterative model of RUP.
By normally with the undoded amino acid of illness group according to its detect frequency T examine (p<0.01), ratio (ratio>
2) it is screened with detection frequency (> 100), to obtain difference undoded amino acid.Then difference is made using SPSS software
Undoded amino acid ROC curve, and its area under the curve (AUC) is calculated, and then judge its diagnostic value.
203 serines by MASS SPECTRAL DATA ANALYSIS discovery AKAP3 albumen have+79.967 ± 0.005 quality inclined
It moves (S+79.967), phosphorylation modification occurs for serine, by it was found that this undoded amino acid S+79.967 is being weighed
The few weak smart sample significance of degree has lowered 10.7 times, p value be 5.92E-13 < < 0.01.
Above-mentioned screening technique is for obtaining biomarker, and is not to obtain the diagnosing and treating result of disease as mesh
's；The biomarker obtained through above-mentioned screening technique can be used for the theoretical research of severe teen bra or opening for novel drugs
Hair.
The first aspect of the present invention, provide screened according to above-mentioned screening technique it is relevant to severe teen bra
Biomarker, the biomarker includes but is not limited to:
The serine of 203+79.967 ± 0.005 mass shifts of generation of AKAP3 albumen (is labeled as S+79.967；Very
According to mass shift, determine that phosphorylation modification has occurred in the serine of the position)；
The second aspect of the present invention provides the silk ammonia of 203+79.967 ± 0.005 mass shifts of generation of AKAP3 albumen
Purposes of the acid (being labeled as S+79.967) as biomarker in the diagnostic reagent that preparation severe lacks weak essence.
It include specific detection in the kit the present invention also provides a kind of kit for the few weak essence diagnosis of severe
The reagent of above-mentioned biomarker (serine of 203+79.967 ± 0.005 mass shifts of generation of AKAP3 albumen).
Preferably, the serine of 203+79.967 ± 0.005 mass shifts of generation of AKAP3 albumen is also used as weight
The target of the few weak essence treatment of degree, for severe lacks the treatment of weak essence.
The present invention also provides the serines of 203+79.967 ± 0.005 mass shifts of generation of AKAP3 albumen as life
Purposes of the object marker in the therapeutic agent that preparation severe lacks weak essence.
The present invention also provides a kind of drug treated severe and lack weak essence, contain 203 silks to AKAP3 albumen in the drug
The component of propylhomoserin progress phosphorylation modification.
The present invention also provides the diagnostic method that a kind of severe lacks weak essence, steps are as follows: detection sample to be tested AKAP3 albumen
The frequency of+79.967 ± 0.005 mass shift occurs for 203 serines, Parallel testing 3 times, takes average inspection as a result, if average
When detecting the frequency less than 4.6, it is judged to less weak smart patient.
Beneficial effects of the present invention:
The biomarker that the present invention further obtains above-mentioned screening technique is studied, and discovery can be by above-mentioned
The inspection frequency of biomarker lacks azoospermia to diagnose severe, lacks azoospermia for severe and provides new diagnosing and treating target
Point.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation are not constituted an undue limitation on the present application for explaining the application.
The ROC curve figure of 203 undoded amino acid S+79.967 detection frequency of Fig. 1: AKAP3 albumen；
Fig. 2: 203 undoded amino acid S+79.967 of the AKAP3 albumen of healthy and few weak smart sample detect frequency pair
Than figure.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
Term is explained:
The detection frequency: sample to be tested passes through mass spectral analysis, sample introduction after being handled according to 1 recording mode of the embodiment of the present invention
The number that undoded amino acid S+79.967 shifts in sample, referred to as the detection frequency.
The present invention screens to obtain biomarker relevant to severe teen bra, specific as follows:
The serine of AKAP3 protein 20 3+79.967 ± 0.005 mass shifts of generation (is labeled as S+79.967；According to
Quality offset value determines that phosphorylation modification occurs for the serine of the position).
In the another embodiment of the application, a kind of kit for the few weak essence diagnosis of severe is proposed, it is described
It include the reagent of the above-mentioned biomarker of specific detection in kit.
By detecting to above-mentioned biomarker, the diagnosis for lacking azoospermia to severe may be implemented.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.
Embodiment 1: the screening of biomarker relevant to severe teen bra
Specific screening technique is as follows:
One, sample process and experimental analysis
1. the extraction of spermatoblast holoprotein: the few weak essence of the severe of equivalent and eupyrene sperm sample wash three with DPBS respectively
It is secondary, equivalent RIPA lysate ultrasound 1-2min is added, is placed in and is incubated for 30min cracking on ice, 4 DEG C of centrifugation 14,000g × 20min take
Supernatant.Protein concentration is measured using Bradford method.
2. proteolysis: taking the about few weak essence of severe and each 150 μ g sperm protein of eupyrene sperm sample, use 10% polypropylene
Acyl ammonia gel electrophoresis (SDS-PAGE) separates albumen, is respectively divided into 5 parts and carries out cutting glue enzymatic hydrolysis.Using ziptip to peptide fragment into
Row desalination.
3. mass spectral analysis: receiving flow liquid phase chromatographic isolation: A phase: the water containing 0.1% formic acid；B phase: contain 0.1% formic acid
Acetonitrile.
Each sample uses 13.5 μ L A phased solns respectively, and sample injection volume is 4 μ L, and flow liquid phase mass spectrometry system of receiving is
Orbitrap Elite(Thermo Scientific).Sample separation before respectively with 4 μ L A balance each other homemade pre-column and divide
Analyse column.The specification of pre-column and analytical column be respectively as follows: pre-column (5 μm of 4cm × 150 μm I.D., C18 packing material size,), analysis
Column (filling of 30cm × 75 μm I.D., C18 filler, 3 μm of partial size, Dr.Maisch GmbH,Germany).After balance
Then sample load sample first under the drive of A phase carries out liquid phase separation in pre-column under different gradients.150min chromatography gradient becomes
Change as follows: 5-32% Mobile phase B 100min；32-80% Mobile phase B, 20min；80% Mobile phase B, 30min.Flow velocity is protected always
It holds in 300nL/min.Through receiving stream liquid phase separation sample be directly entered ESI ionspray source and enter Orbitrap Elite
Mass Spectrometer Method is carried out in mass spectrograph.
Mass spectrometric data acquisition: the full scan of 350-1800m/z, resolution ratio 60,000 (m/z 200).Second order spectrum scanning
When, activation time 10ms, isolation width is 2m/z.Fragmentation pattern is that induction is collisionally dissociated (collision-induced
Dissociation, CID), normalization collision energy is set as 35%, and dynamic efflux time is 90s.
Two, MASS SPECTRAL DATA ANALYSIS
Byonic analysis: the undoded amino acid in order to identify sperm protein uses ByonicTM21 pairs of analysis is normally and again
The protamine mass spectrometric data of the few weak smart patient of degree.Search parameter is as follows: protease is trypsase, and leakage enzyme site is set as 2, mother
Mass of ion deviation is 10ppm, and the mass deviation of fragment ion is 0.6Da, and the blind upper limit of searching is set as 1000, it is blind search lower limit be set as-
200. albumen FDR are 0.01.
The unknown modifications peptide segment data that the Byonic Wildcard Search of selection FDR < 0.01 is searched, forms one-dimensional
Data matrix, the delta mass range selection -200Da-400Da of data, then by data according to the variation range of 1Da, 0.5Da
For boundary, it is divided into 601 data windows.For each data window, it is mixed that Gauss is with the mclust program bag in R language
Distributed clustering analysis is closed, optimal value is taken according to BIC, then analysis is merged to each peak, it is then every with Gauss Distribution Fitting
One peak, determines peak value.It include the information of amino acid in each peak after cluster, according to unknown modifications in 20 kinds of amino
Distribution proportion on acid is selection parameter with 10%, selects undoded amino acid with the iterative model of RUP.
By normally with the undoded amino acid of illness group according to its detect frequency T examine (p<0.01), ratio (ratio>
2) it is screened with detection frequency (> 100), to obtain difference undoded amino acid.Then difference is made using SPSS software
Undoded amino acid ROC curve, and its area under the curve (AUC) is calculated, and then judge its diagnostic value.
Sorting algorithm accuracy result is as shown in the table:
| Pos | TotalCount | ave_c | ave_b | ratio | Ttest | AUC | pValue |
| 202 | 395 | 4.587302 | 0.428571 | -10.7037 | 5.92E-13 | 0.856 | 3.05E-15 |
Three, experimental result:
Through MASS SPECTRAL DATA ANALYSIS and by normally compared with the undoded amino acid of illness group, to obtain the non-volume of following difference
Code amino acid, can be used as biomarker relevant to severe teen bra, specific as follows:
The serine (being labeled as S+79.967) of 203+79.967 ± 0.005 mass shifts of generation of AKAP3 albumen
By MASS SPECTRAL DATA ANALYSIS, it is found that 203 serines of AKAP3 albumen have+79.967 ± 0.005 quality inclined
It moves (S+79.967), by it was found that this undoded amino acid S+79.967 is lowered in the few weak smart sample significance of severe
10.7 times, p value be 5.92E-13 < < 0.01.
Fig. 1 is the ROC curve that 3 undoded amino acid S+79.967 of AKAP3 protein 20 detect frequency, ROC analysis shows that
The AUC of this undoded amino acid S+79.967 is 0.856 > 0.7, illustrates there is preferable diagnosis effect.
3 undoded amino acid S+79.967 detection frequencies of AKAP3 protein 20 of healthy and few weak smart sample, which compare, sees figure
2, this undoded amino acid averagely has occurred 4.6 times in Healthy People sample and sends out in pathology sample as seen from Figure 2
It has given birth to 0.4 time.
In view of the above results, this undoded amino acid of the S+79.967 in 3 site of AKAP3 protein 20 can be used as less weak essence
The potential source biomolecule marker of sub- disease, to predict this illness.
Embodiment 2: clinical detection verifying
It is verified, is distinguished as research object using the few weak smart sample of severe of 3 healthy samples, 3 clinical definites
The detection frequency of the serine of AKAP3 protein 20 3+79.967 ± 0.005 mass shifts of generation of above-mentioned sample is detected, and is pressed
Judgment criteria when biomarker carries out individual detection in embodiment 1 diagnoses sample to be tested.
The result shows that: when above-mentioned biomarker is individually diagnosed, undoded amino acid S+ in 3 healthy samples
The 79.967 detection frequency is 6 times or more, and this undoded amino acid in the few weak smart sample of severe is not detected partially
It moves.Diagnostic result is consistent with known results, illustrates that the biomarker that present invention screening obtains can be used as the few weak essence of severe
Diagnosis marker.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (6)
- The serine of 1.AKAP3 protein 20 3+79.967 ± 0.005 mass shifts of generation is as biomarker in preparation weight Spend the application in the diagnostic reagent of few weak essence.
- The serine of 2.AKAP3 protein 20 3+79.967 ± 0.005 mass shifts of generation is as biomarker in preparation weight Spend the purposes in the therapeutic agent of few weak essence.
- 3. a kind of drug treated severe and lack weak essence, phosphoric acid is carried out containing 203 serines to AKAP3 albumen in the drug Change the component of modification.
- It include specific detection AKAP3 protein 20 3 in the kit 4. a kind of lack the kit that weak essence diagnoses for severe The reagent of the serine of+79.967 ± 0.005 mass shift occurs.
- 5. a kind of biomarker, it is characterised in that the serine of ± 0.005 mass shift of the generation+79.967 is located at AKAP3 Protein 20 3.
- 6. a kind of severe lacks the diagnostic method of weak essence, step are as follows: 203 serines hair of detection sample to be tested AKAP3 albumen The frequency of raw+79.967 ± 0.005 mass shift Parallel testing 3 times, takes average inspection as a result, if the average detected frequency is less than 4.6 When, it is judged to less weak smart patient.
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