CN110514839A - Application of 616 N+12.000 of AKAP4 albumen in the few weak smart diagnostic reagent of preparation severe - Google Patents
Application of 616 N+12.000 of AKAP4 albumen in the few weak smart diagnostic reagent of preparation severe Download PDFInfo
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The asparagine for the mass shift for occurring+12.000 ± 0.005 the invention discloses 616, AKAP4 albumen is preparing the purposes in the diagnostic reagent that severe lacks weak essence as biomarker.Present invention discover that: the inspection frequency that the asparagine of+12.000 ± 0.005 mass shift occurs by 616, AKAP4 albumen, 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 616, AKAP4 albumen occur+
The asparagine (undoded amino acid AKAP4@616N+12.000) of 12.000 ± 0.005 mass shift exists as marker
Prepare the 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 common factor 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 Reproduction and Development Research Group,
Journal of Proteomics 162(2017)125–134).Current last updated human sperm's protein group Amaral
Deng completion, 6198 albumen (Amaral A, Castillo J, Ramalho-Santos J, Oliva R.The is identified altogether
combined human sperm proteome:cellular pathways and implications for basic
and clinical science.Human reproduction update,20(1),40-62(2014)).The benefits such as Mayank
667 eggs have been quantified in 5 groups of Healthy Peoples and the spermatoblast of 8 groups of teen bra patients with the method for Different Proteomics
It is white, 447 albumen have been quantified in refining, have gone out 8 significant down-regulation proteins about point, and path analysis (Human has been carried out to it
Spermatozoa 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 with non-obstructive azoospermia,
MehdiAlikhani, MehdiMirzaei, MarjanSabbaghian, PouriaParsamatin,
RaziehKaramzadeh, SamaneAdib, NiloofarSodeifi, Mohammad Ali SadighiGilani,
MasoudZabet-Moghaddam, LindsayParker, YunqiWu, VivekGupta, Paul A.Haynes,
HamidGourabi, HosseinBaharvand, Ghasem HosseiniSalekdeh, Journal of Proteomics,
Volume 162,6 June 2017,Pages 141-154).The silencing of mature sperm translation transcription activity, which also becomes, grinds
Study carefully the ideal cell model of posttranslational modification, but about based on mass spectrographic sperm posttranslational modification broad scale research still very
It is few.Research about modification focuses primarily upon 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.).Tyrosine phosphorylation is for important works of processes such as the movement of sperm, capacitation, super sharp movements
With.Chying-Chyuan Chan etc. carries out proteome analysis hair by the sperm to 20 groups of normal persons and azoospermia patient
Peroxophosphoric acid has occurred in existing 12 kinds of albumen including TUBGCP2.Undoded amino acid includes posttranslational modification and amino
Acid mutation is the important way of modulin function and structure, therefore abnormal under morbid state or quantity is changed greatly
Undoded 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 of acrosin functioning during fertilization).Research shows that sperm by
Closely related (the Sperm acrosin activity and fluorescence of the activity of smart 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 OrbitrapElite
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/z200).Second order spectrum
When 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.
There is+12.000 ± 0.005 quality by 616 asparagines of MASS SPECTRAL DATA ANALYSIS discovery AKAP4 albumen
It deviates (N+12.000), formylated modification occurs for asparagine, by it was found that this undoded amino acid N+12.000
Lowered 6.3 times in the few weak smart sample significance of severe, p value be 6.87E-09 < < 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 asparagine of 616, the AKAP4 albumen mass shifts for occurring+12.000 ± 0.005 (is labeled as N+12.000；
According to quality offset value, determine that formylated modification occurs for the asparagine of the position)；
The second aspect of the present invention provides the day of the mass shift of 616 generations+12.000 ± 0.005 of AKAP4 albumen
Purposes of the winter amide (being labeled as N+12.000) as biomarker in the diagnostic reagent that preparation severe lacks weak essence.
Preferably, the asparagine that+12.000 ± 0.005 mass shift occurs for 616 of AKAP4 albumen can also be made
Lack the target of weak essence treatment, for severe for severe lacks the treatment of weak essence.
Further, the day of+12.000 ± 0.005 mass shift occurs for 616 the present invention also provides AKAP4 albumen
Purposes of the winter amide as biomarker 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, containing in the drug can be to the 616 of AKAP4 albumen
Position aspartic acid carries out the component of formylated modification.
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 (asparagine that+12.000 ± 0.005 mass shift occurs for 616 of AKAP4 albumen).
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 AKAP4 albumen
The frequency of+12.000 ± 0.005 mass shift occurs for 616 asparagines, and each sample Parallel testing 3 times is averaged
Testing result is judged to less weak smart patient if the detection frequency is less than 4.4.
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 616 asparagine N+12.000 detection frequency of Fig. 1: AKAP4 albumen；
Fig. 2: 616 asparagine N+12.000 of the AKAP4 albumen of healthy and few weak smart sample detect frequency comparison 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 N+12.000 shifts in sample, referred to as the detection frequency.
The present invention obtains biomarker relevant to severe teen bra by screening, specific as follows:
The asparagine of 616, the AKAP4 albumen mass shifts for occurring+12.000 ± 0.005 (is labeled as N+12.000；
According to positional shift value, determine that formylated modification occurs for the asparagine 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 OrbitrapElite
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 |
| 615 | 199 | 2.285714 | 0.365079 | -6.26087 | 6.87E-09 | 0.795 | 1.41E-10 |
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 asparagine (being labeled as N+12.000) of the mass shift of 616, AKAP4 albumen generations+12.000 ± 0.005；
By MASS SPECTRAL DATA ANALYSIS ,+12.000 ± 0.005 mass shift occurs for the asparagine of discovery 616, AKAP4 albumen, warp
It crosses it was found that this undoded amino acid N+12.000 has lowered 6.3 times in the few weak smart sample significance of severe, p value is
6.87E-09<<0.01。
Fig. 1 is the ROC curve that 616 asparagine N+12.000 of AKAP4 albumen detect frequency, ROC analysis shows that this
The AUC of undoded amino acid is 0.795 > 0.7, illustrates there is preferable diagnosis effect.
616 asparagine N+12.000 detection frequencies of AKAP4 albumen of healthy and few weak smart sample, which compare, sees Fig. 2, can
To find out that this undoded amino acid averagely has occurred 4.4 times in Healthy People sample and has occurred in pathology sample 0.8 time.
In view of the above results, 616 of the AKAP4 albumen asparagines that+12.000 ± 0.005 mass shift occurs can
Using the potential source biomolecule marker as teen bra, 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 asparagine of the mass shift of 616, AKAP4 albumen generations+12.000 ± 0.005 of above-mentioned sample is detected,
And judgment criteria when carrying out individual detection by biomarker in embodiment 1 diagnoses sample to be tested.
The result shows that: when above-mentioned biomarker is diagnosed, undoded amino acid N+12.000 inspection in 3 healthy samples
Frequency measurement time is 5 times or more, and the detection frequency is 0 time in the few weak smart clinical samples of 3 severes, diagnostic result and known results one
It causes.Illustrate that present invention screening obtains biomarker and can be used as the diagnosis marker that severe lacks weak essence.
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 asparagine of 616, the 1.AKAP4 albumen mass shifts for occurring+12.000 ± 0.005 is being made as biomarker Standby severe lacks the application in the diagnostic reagent of weak essence.
- The asparagine of 616, the 2.AKAP4 albumen mass shifts for occurring+12.000 ± 0.005 is being made as biomarker Standby severe lacks the purposes in the therapeutic agent of weak essence.
- 3. a kind of drug treated severe and lack weak essence, containing carrying out formyls to 616 aspartic acids of AKAP4 albumen in the drug Change the component of modification.
- It include 616, specific detection AKAP4 albumen in the kit 4. a kind of lack the kit that weak essence diagnoses for severe The reagent of the asparagine of+12.000 ± 0.005 mass shift occurs.
- 5. a kind of biomarker, it is characterised in that the asparagine of ± 0.005 mass shift of the generation+12.000 is located at 616, AKAP4 albumen.
- 6. a kind of severe lacks the diagnostic method of weak essence, step are as follows: 616 asparagines of detection sample to be tested AKAP4 albumen The frequency of+12.000 ± 0.005 mass shift occurs, each sample Parallel testing 3 times takes average testing result, if detection When the frequency is less than 4.4, it is judged to less weak smart patient.
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