CN106153754A

CN106153754A - The differentially expressed protein of Alport syndrome patient's inductive pluripotent stem cells and analysis methods and applications

Info

Publication number: CN106153754A
Application number: CN201510188185.8A
Authority: CN
Inventors: 戴勇; 黄建溶; 陈文标
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-04-20
Filing date: 2015-04-20
Publication date: 2016-11-23

Abstract

The present invention relates to differentially expressed protein and the analysis methods and applications of Alport syndrome patient's inductive pluripotent stem cells of a kind of study of pathogenesis that can be used for AS.By using the differentially expressed protein between iTRAQ technical tie-up liquid phase tandem mass spectrum technical Analysis Alport syndrome patient's inductive pluripotent stem cells (iPSCs) and the iPSCs of normal person, the biomarker of screening early diagnosis Alport syndrome and possible therapy target.Owing to Alport syndrome is a kind of multiple organ concurrency syndrome, analyzing the differentially expressed protein obtaining can provide intermediate result information for diagnosis Alport syndrome, provides technical support for studying the pathogenesis of Alport syndrome and potential therapy target further.

Description

The differentially expressed protein of Alport syndrome patient's inductive pluripotent stem cells and analysis methods and applications

Technical field

The present invention relates to disease protein research field, especially relate to a kind of Alport syndrome patient's inductivity The differentially expressed protein of multipotential stem cell and analysis methods and applications.

Background technology

Alport syndrome (Alport syndrome, AS), is that one mainly shows as blood urine, renal function enters The heredity GBM that row goes down, nerve deafness and eye are abnormal (glomerularbasementmembrane, GBM) disease.By coding type Ⅳ collagen α the 3rd, α the 4th, AS is A kind of basilar memebrane disease that encoding gene COL4A3, COL4A4 and COL4A5 sudden change of α 5 chain causes. The mutational site of hitherto reported said gene is more than 500, and the AS case of about 85% is X sex-kink AS, Remaining 15% is autosomal recessive inheritance AS, and autosomal dominant inheritance AS is very rare.Kidney damages Evil is the topmost symptom of AS, almost all can show the kidney damage such as blood urine, albuminuria all patients Symptom, AS accounts for the 0.3%～2.3% of whole patient's ESRD sum.

The generation of the kidney damage of AS is drawn mainly due to the COL4An gene unconventionality sudden change of coding type Ⅳ collagen Rise.The triple-helix structure albumen that type Ⅳ collagen is made up of three α chains.Have now been found that 6 kinds of different α Chain (α 1～α 6), 6 α chains can form three kinds of triple-helix structure amyloid protein precursors, respectively α 1 α 1 α 2 (IV), α 3 α 4 α 5 (IV) and α 5 α 5 α 6 (IV).In glomerulus, α 1 α 1 α 2 (IV) isomers can by sertoli cell, Glomerular endothelial cells, mesangial cell produce.But, GBM upper α 3 α 4 α 5 (IV) isomers can only be by foot Cell produces, and the wherein generation of α 3 chain is the key factor that isomers is formed.Therefore, sertoli cell is also AS One of key cells of pathogenesis.Owing to α 3 α 4 α 5 (IV) isomers of AS does not normally produce, lead The mechanical stability causing GBM declines and tear.GBM is as the filtration barrier of kidney, the albumen of AS patient Urine is relevant with the change of the change of sertoli cell positioning, the fusion of podocytic process and slit diaphragm.There are some researches show, sick The sertoli cell becoming can transmit infringement to neighbouring normal sertoli cell.Change over time, ultimately results in thin Extracellular matrix deposits and glomerulosclerosis in a large number.

It is found to have the abnormal deposition of laminin α 1 β 1 γ 1 and α 2 β 1 γ 1 in mankind AS and animal model, On the especially wavy GBM thickening.Studies have found that on the GBM of AS patient extracellular matrix and Laminin α 5 deposits and matrix metalloproteinase (matrixmetalloproteinases, MMPs) Raising, the rise with MMP-9 and MMP-14 that thickens of basement membrane is proportionate.The GBM of AS patient Jejune laminin α 1 β 1 γ 1 and α 5 β 1 γ 1 isomers instead of laminin α 5 β 2 γ 1 of maturation Isomers.The interphase interaction of integrin and laminin α 5 β 2 γ 1 isomers can destroy actin cell Skeleton and exciting signal transmission.Between different types of cell, regulation is to have been come by integrin and other acceptors Becoming, specific binding between type Ⅳ collagen chain is i.e. such.Discoidin domain receptor (discoidindomainreceptor1, DDR1) is distributed in glomerulus and inner ear, DDR1 and type Ⅳ collagen it Between interaction and type Ⅳ collagen and α 2 β 1 between complete to GMB and sertoli cell structure of interaction Whole and filter ability most important.The mouse model knocking out above-mentioned acceptor can cause mild proteinuria, limitation Property renal interstitial fibrosis and GBM is wavy thickens.Additionally, above-mentioned mouse be also found that part ceasma every Film disappears and podocytic process merges.

Therefore, studying the pathogenesis of AS, especially protein expression difference has great importance.

Content of the invention

Based on this, it is necessary to provide the Alport syndrome patient of the study of pathogenesis of a kind of AS of can be used for lure The differentially expressed protein of the property led multipotential stem cell and analysis methods and applications.

A kind of analysis method of the differentially expressed protein of Alport syndrome patient's inductive pluripotent stem cells, its It is characterised by, comprise the steps:

Step one, extracts the inductivity of Alport syndrome patient's inductive pluripotent stem cells and Healthy People respectively The total protein of multipotential stem cell, obtains two histone samples；

Step 2, described in two groups, albumen sample carries out reductive alkylation process respectively, obtains two histone samples This solution；

Step 3, adds trypsase to carry out enzymolysis processing respectively in two histone sample solutions；

Step 4, uses different labelled reagents, carries out iTRAQ to two groups of samples after enzymolysis processing respectively Mark process, and the Alport syndrome patient group after mark process is mixed with the sample solution of Healthy People；

Step 5, carries out strong cation exchange separating treatment to mixed sample；

Step 6, carries out liquid chromatogram separation to the sample after strong cation exchange separating treatment, then by liquid phase The peptide fragment of chromatographic isolation carries out Tandem Mass Spectrometry Analysis；

Step 7, uses protein identification software to carry out identification of proteins to mass spectrometry results, according to albumen Matter abundance level, analyzes the difference obtaining Alport syndrome patient and the inductive pluripotent stem cells of Healthy People Expressing protein.

The analysis method of the differentially expressed protein of above-mentioned Alport syndrome patient's inductive pluripotent stem cells obtains Differentially expressed protein, including the NEFL of up-regulated, PSME4, UTF1, RBM14, AIF1L, STOM, FGF2、SF3B14、DNMT3A、IK、Keratin-18、CRYZ、RG9MTD1、MTHFD2、 HIST1H1A, CPT1A, UBXN4 and SEMA6A, and express lower Keratin-14, Keratin-10、SRSF2、TUBA1A、Keratin-9、Keratin-5、MRPL27、Keratin13、APLP2、 PAPSS1, SURF4, RCN3, EIF1AY, APEX1, ACBD3, UBA2 and NLGN4X.

Above-mentioned differentially expressed protein answering in the detection chip or detection reagent of preparation diagnosis Alport syndrome With.

By using iTRAQ technical tie-up liquid phase tandem mass spectrum technical Analysis Alport syndrome patient's inductivity Differentially expressed protein between multipotential stem cell (iPSCs) and the iPSCs of normal person, screening early diagnosis Alport The biomarker of syndrome and possible therapy target.Owing to Alport syndrome is concurrent for a kind of multiple organ Property syndrome, analyzing the differentially expressed protein that obtains can be that diagnosis Alport syndrome provides intermediate object program Information, provides technical support for studying the pathogenesis of Alport syndrome and potential therapy target further.

Brief description

Fig. 1 is Alport syndrome patient's inductive pluripotent stem cells form；

Fig. 2 is Alport syndrome patient's inductive pluripotent stem cells karyotype characteristic figure of induction；

Fig. 3 is the stem cell surface molecular marker schematic diagram of flow cytomery；

Fig. 4 is that PCR detection endogenous OCT4 gene is in primary cell and inductive pluripotent stem cells strain Expression of results figure；

Fig. 5 is sample protein calibration curve；

Fig. 6 is iPSCs sample protein SDS-PAGE collection of illustrative plates；

Fig. 7 is sample peptide fragment spectrogram quality of match error map；

Fig. 8 is for identifying protein relative molecular mass distribution figure；

Fig. 9 is peptide section sequence distribution of lengths schematic diagram；

Figure 10 is peptide section sequence coverage distribution schematic diagram；

Figure 11 is for identifying peptide fragment distributed number figure；

Figure 12 is protein abundance distribution schematic diagram；

Figure 13 is GO classification schematic diagram；

Figure 14 is the COG function classification schematic diagram of protein.

Detailed description of the invention

Below mainly in combination with Alport syndrome patient's inductive pluripotent to the present invention for the drawings and the specific embodiments The differentially expressed protein of stem cell and analysis methods and applications are described in further detail.

1. experimental subjects

The research object of the present embodiment is inductive pluripotent stem cells (iPSCs), including experimental group and control group, Wherein, the iPSCs of experimental group can be prepared as follows, and the iPSCs sample of control group comes from section of China The iPSCs of institute's Guangzhou biological medicine and health research Yuan Panguangjin seminar present Healthy People.Patient knows the inside story simultaneously And agree to participate in the present embodiment, Ji'nan University's the second Clinical Medical Institute (Shenzhen people's hospital) has been passed through in experiment The examination & verification of Ethics Committee.

The structure of 2.Alport syndrome patient's inductive pluripotent stem cells

2.1 experiment material

By research, finding an Alport syndrome family, it is chain that its existing 38 people are diagnosed as X-through Electronic Speculum Dominant inheritance (X-linkedAlportsyndrome, XLAS) Alport syndrome, in this XLAS family Find that c.1517-1G the splice site of a new COL4A5 gene suddenlys change > T.The present embodiment collects this family One now 39 years old female patient be research object, eliminate this patient and suffer from other systemic diseases.

Morals, the ethic principle that World Medical Association's Declaration of Helsinki is formulated is followed in this research with China GPC, All experimenters, before participating in research, all understand research contents, voluntary participation, sign written in the know same simultaneously Meaning book.This research obtains attached second clinical institute's (Shenzhen people's hospital) the ethics committee member of Ji'nan University The approval of meeting.

2.2 main experiment reagents and instrument

2.3 experimental technique

2.3.1Alport the collection of syndrome patient's urine kidney tubular fossils

(1) urine is gathered: with concentration 5% iodophor disinfection Alport Urinary road junction skin 2 times, with adding Enter the dual anti-aseptic wide mouth glass bottle of 5ml and collect patient midstream urine in early morning about 300ml, urine specimen put to 4 DEG C of Refrigerator stores.

(2) collect urine cell: 75% alcohol disinfecting wide mouth glass bottle, in super bacterium platform, dispense urine to 50ml Centrifuge tube, 400g centrifuges 10 minutes.

(3) centrifuge tube upper strata urine is abandoned in suction, is drawn to bottom cell suspension, among a 50ml centrifuge tube, add PBS liquid is to 50ml, and 400g centrifuges 10 minutes.

(4) (3) step is repeated once.

(5) bed board: by 1%Gelatin paving to 6 orifice plates, put to 37 DEG C of incubators more than 30 minutes.

(6) cultured cells: take out centrifuge tube, inhales and abandons supernatant liquid, and indwelling lower floor suspension adds 2mlREGM Urine culture medium is resuspended, re-suspension liquid is drawn to a hole of 6 orifice plates in (5) afterwards, puts into 37 DEG C after shaking up In incubator.

(7) cell changes liquid and adherent: within about 2-3 days, change liquid once, about about one week it is observed that cell in Colonisation.

(8) passing on: the speed of growth of root observation of cell and form, cell density is more than 90%, general first quarter moon Left and right can be passed on or frozen.

2.3.2Alport the induction of syndrome patient iPSCs

Plasmid PEP4-E02S-ET2K and PCEP4-miR302-367 by Chinese Academy of Sciences's Guangzhou biological medicine with Health research Yuan Panguangjin seminar provides.

(1) the urine kidney tubular fossils of electrotransfection is prepared: 37 DEG C of Matrigel are coated culture dish more than 30 minutes, For improving the efficiency of transfection, the generation in P3 generation is selected preferably to urinate cell, after PBS washes twice, 0.25% pancreas Cell dissociation is disperseed by enzyme.Prepare Tissue Culture Dish (cell density 1 × 10⁶/10cm²)。

(2) electrotransfection

A: draw 1 × 10⁶Individual urine cell, 200g centrifuges 5 minutes, abandons supernatant.

B: press Amaxa^TMBasicNucleofector^TMKitforprimarymammalianEpithelialcells tries Agent box illustrates, adds BasicNucleofector^TMSolutionforMammalianEpithelialCells 82 μ l, Supplement 18 μ l, re-suspended cell.

C: respin cell is added oriP/EBNAl episomal carrier (episomalvectors) plasmid PEP4-E02S-ET2K and PCEP4-miR302-367, mixing process.

D: homomixture is added to electricity revolving cup, notes liquid feeding speed, prevent bubble.Electricity revolving cup is put into electricity Turning instrument, electricity turns procedure Selection T-020.

E: after electricity turns, takes out electricity revolving cup, adds appropriate REGM culture medium to be coated cultivation to 37 DEG C of Matrigel More than 30 minutes 10cm of ware²Culture dish, the mixed liquor taking a turn for the better electricity adds culture dish, puts into 37 DEG C, 5%CO₂ In incubator.

(3) changing liquid: the growing state of observation of cell, after general 24h, cell attachment is good, density ＞ 70% Left and right, inhales and abandons REGM culture medium, and PBS washes twice, adds appropriate mTeSRl culture medium, puts into 37 DEG C, 5%CO₂In incubator.

(4) picked clones: change mTeSRl culture medium every day, general about 20 days, can see under the microscope Examine with the close clone of form and human embryo stem cell, clone is picked out, is primary iPSCs.

2.3.3 the qualification of Alport syndrome patient iPSCs

(1) iPSCs colony morphology and observation of characteristics.

(2) nucleus type analysis.

(3) flow cytometry stem-cell marker antigen presentation.

(4) RT-PCR detection people's endogenous stem cells significant gene OCT4 gene expression dose.

2.4 result

2.4.1 inductive pluripotent stem cells (iPSCs) form

Basis of microscopic observation inductive pluripotent stem cells is flat, the smooth of the edge, nucleus/cytoplasmic ratios Higher, similar human embryo stem cell, see Fig. 1.

2.4.2Alport syndrome patient's inductive pluripotent stem cells karyotyping.

Women Alport syndrome patient's inductive pluripotent stem cells normal karyotype characteristic pattern of above-mentioned induction such as figure Shown in 2.

2.4.3 flow cytometry stem-cell marker antigen presentation

The stem cell surface molecular marker analysis result of flow cytomery as it is shown on figure 3, wherein, A:Oct4；B:SSEA4,C:TRA-1–6；D:TRA-1–8.

2.4.4PCR endogenous OCT4 gene is detected in primary cell and induced pluripotent stem cells cell line Express

Expression in primary cell and induced pluripotent stem cells cell line for the endogenous OCT4 gene of PCR detection Result as shown in Figure 4, wherein, 1:maker2000；2:Oct4；3:SV40LT；4:Sox2；5: Klf4；6:oriP；7:EBNA1；8:Oct4；9:maker2000；10:Oct4；: 11:SV40LT； 12:Sox2；13:Klf4；14:oriP；15:EBNA1；16:Oct4endo；17:maker2000； 2-8 is Alport syndrome patient iPSCs；10-16 is primary cell.

The differentially expressed protein research of 3.Alport syndrome patient's inductive pluripotent stem cells

3.1 major experimental instruments and reagent

The recovery of 3.2 Alport syndrome groups iPSCs and control group iPSCs and process

(1) wear protective gear from liquid nitrogen, take out iPSCs cryopreservation tube, among both hands, rub 10-15 back and forth with the hands Second, until the frost disappearance on cryopreservation tube surface.

(2) place cryopreservation tube in 37 DEG C of water-baths rapidly, jiggle, note observing inside cryopreservation tube thin The state of cellular lysis, must not allow water logging there be not cryopreservation tube lid.

(3) when cryopreservation tube only remains a small amount of ice (about 2 minutes), by cryopreservation tube from water-bath Middle taking-up.Cell Name on the good frozen tube wall of record, algebraically, with the surface of 75% alcohol wipe cryopreservation tube, After vaporized alcohol, put into super bacterium platform.

(3) cryopreservation tube lid is opened, with iPSCs to the 15ml centrifuge tube that pasteur pipet transfer 1ml is frozen In, it is slowly added dropwise the fresh mTeSr1 culture medium of 4ml, limit piping and druming, limit shake up so that it is mix.

(4), after mixing, put into centrifuge 200g, 3-5 minute, inhale and abandon supernatant.By 2.5mlmTeSr1 Culture medium adds to each sky of 6 orifice plates, blows and beats resuspended gently, is inoculated in feeder orifice plate, 6 orifice plates All recover the iPSCs of a pipe cryopreservation tube in each hole.Put into containing 5% carbon dioxide, in 37 DEG C of incubators.

(5) recover latter second day observation of cell growing state, if cell attachment is few, then add 1ml fresh MTeSr1 culture medium；If cell attachment is more, then add the culture medium of 2ml.

(6) observation of cell every day growing state, changes culture medium according to growing state.

(7) when cell density reaches 95%, taking out 6 orifice plates, discarding culture medium, every hole is used 1mlDMEM/F12 culture medium is washed one time, inhales and abandons culture medium, and every hole adds 1ml Dispase enzyme, and placement contains 5% carbon dioxide, in 37 DEG C of incubators about 3 minutes, takes out 6 orifice plates, examines under a microscope, if Clone starts crimping and i.e. stops digestion.Dispase, every hole 1mlDMEM/F12 culture medium wash-out three are abandoned in suction Time.

(8) pasteur pipet is burnt till looper shape, cell clone is scraped to 15ml centrifuge tube from 6 orifice plates In, add a small amount of mTeSr1 culture medium to rinse at the bottom of plate after having scraped, centrifuge tube is put into centrifuge, 1130r/min, Centrifugal 5 minutes, inhale and abandon supernatant, add 1ml frozen stock solution, blow and beat 2-3 time gently, be transferred in cryopreservation tube, Put in-80 DEG C of refrigerators standby.

3.3 protein samples prepare

(1) by above-mentioned frozen iPSCs recovery, cultivate as stated above to cell density and reach 95%.

(2) culture medium is abandoned in suction, is washed 2 times by PBS, inhales and abandons PBS.The 6 every holes of orifice plate add Enter the albumen extract of 100 μ l, add the EDTA of PMSF, 2mM of final concentration of 1mM afterwards respectively, After 5 minutes, add the DTT of final concentration 10mM.Ice-bath ultrasonic 15 minutes, 2sec/3sec, then 25,000 × g Centrifugal 20 minutes, take supernatant.

(3) supernatant adds final concentration 10mMDTT to process 1 hour at 56 DEG C, again adds IAM extremely Final concentration 55mM, is put in dark place 45 minutes under normal temperature.

(4) add the ice acetone of 5 times of volumes of supernatant in supernatant, be positioned over-20 DEG C of refrigerators 2 hours, Then 25,000 × g centrifuges 20 minutes, inhales and abandons supernatant.

(5) ultrasonic dissolution 15 minutes in 200 μ l0.5M tetraethylammonium bromide (TEAB) will be deposited in. 25,000 × g centrifuges 20 minutes, takes supernatant, is i.e. total protein of cell solution.

(6) Bradford method is used to carry out quantification of protein.Take the centrifuge tube of 10 1.5ml, once add Enter the bovine serum albumin(BSA) that concentration is 0.2 μ g/ μ l the 0th, the 2nd, the 4th, the 6th, the 8th, the 10th, the 12nd, the 14th, the 16th, 18 μ l.Again Add the pure water of 20 μ l to often pipe, i.e. often pipe is containing albumen the 0th, the 0.4th, the 0.8th, the 1.2nd, the 1.6th, the 2nd, the 2.4th, the 2.8th, 3.2nd, 3.6 μ g albumen (wherein the first pipe is blank group).

(7) each centrifuge tube adds 20 μ l protein dissolution liquid, sequentially adds 180 μ l Proteinassay Reagent, shakes up gently, hatches 10 minutes under normal temperature.

(8) arranging ELIASA absorbance is 595nm, using the first pipe as object of reference, reads each sample cell Absorptivity.Calculate the concentration of each tubulin according to calibration curve.

3.4 SDS electrophoresis

(1) 12%SDS polyacrylamide gel (SDS-PAGE) formula

(2) each albumen sample mixes with 4 × loading buffer respectively, 95 DEG C of heating 5min.Each sample Product applied sample amount is 30 μ g, Marker applied sample amount 10 μ g.120V constant voltage electrophoresis 2 hours.

(3) gel is taken off, coomassie brilliant blue staining 2 hours, then decolour 3～5 times with destainer, every time 30 Minute.

3.5 protein digestion

(1) every part of protein sample takes 100 μ g.

(2) (5 μ g trypsase add 40 μ lDissolutionbuffer to be separately added into 40 μ l trypsase buffer solutions Preparation), place in 37 DEG C of incubators and digest 4 hours.

(3), after taking out, (5 μ g trypsase add again to add 40 μ l trypsase buffer solutions 40 μ lDissolutionbuffer preparations), place in 37 DEG C of incubators and digest 8 hours.

3.6iTRAQ (isobarictagsforrelativeandabsolutequantitation, relative and absolute quantitation Equivalent dystopy label) mark

(1), after Trypsin Induced, vaccum centrifugal pump is used to drain peptide fragment freezing.

(2) with 0.5M TEAB redissolution peptide fragment to the solubility required for iTRAQ mark, iTRAQ marks behaviour Making to carry out by the handbook of 8-plexiTRAQ (AppliedBiosystems), wherein, labelled reagent 116 is used for Mark Alport syndrome patient's iPSCs group, labelled reagent 118 is used for marking control group.

(3) 2 hours are at room temperature hatched, subsequently by two pipe sample mixing.By mixing sample traditional vacuum Pump is freezing dries.

3.7 strong cation exchange (Strongcationexchange, SCX) separate

(1) ShimadzuLC-20AB liquid phase systems, splitter is used to be 4.6 × 250mm model UltremexSCX post carries out SCX separation to sample.

The mixing peptide fragment 4mLbufferA that (2) 3.3.5 obtained (25mMNaH2P04in25%CAN, PH2.7) redissolve.

(3) sample after redissolving is loaded to the 4.6x250mmUltremexSCX cylinder equipped with 5-10 μm In.

(4) flow of gradient elution is 1mL/min.By measurement 214nm absorbance monitoring elution process, Obtain 20 components through screening.Using StrataX desalination post desalination respectively, freezing afterwards is drained.

3.8 based on the LC-MS analysis of QE

The present embodiment liquid phase Tandem Mass Spectrometry Analysis is provided by Shenzhen Hua Da genome company.

(1) redissolve each group component bufferA (containing 5%CAN and 0.1%FA) drained to about 0.5 μ g/ μ l Concentration, 20000 × g centrifuges 10 minutes, discards insoluble matter.

(2) each component loading 5 μ l (about 2.5 μ g albumen), by Shimadzu company LC-20AD type Number nanoliter liquid chromatograph carry out standard separation.

(3) peptide fragment through liquid phase separation enters into series connection ESI mass spectrograph: Q-EXACTIVE (ThermoFisherScientific,SanJose,CA).First mass spectrometric resolution ratio is set to 70000, two grades of resolutions Rate is 17500.Selecting electric charge in parent ion is 2⁺～5⁺, 15 parent ions more than 20000 for the peak intensity enter Row secondary analysis, carries out fragmentation by the HCD pattern that collision energy is 27 (± 12%) to peptide fragment, and fragment exists Detecting in Orbi, the dynamic eliminating time is set as: chromatogram half-peak breadth duration.Ion source voltage is set to 1.6 Kilovolt.AGC is realized by orbi, and it is set to: one-level 3E6 to two grades 1E5.The mass-to-charge ratio of scanning Scope is one-level 350～2000, two grade 100～1800.

4. bioinformatic analysis

4.1 information analysis flow processs

(1) raw mass spectrum data: download obtains mass spectrum original document, are converted into mgf form (sample.mgf), carry out peak identification, obtain peak list.

(2) selection of database: the database that the present embodiment selects: IPIhumanv3.87 (91464Seuqences)。

(3) Mascot search: Mascot is a protein identification software, and the version used by the present embodiment is Mascot2.3.02, with mgf file as original document, selects the database having built up, then enters line number According to library searching.Search parameter is shown in Table 4-1.

Table 4-1 Mascot identifies related parameter choosing

(4) identification of proteins Information Statistics: according to protein abundance level, when fold differences reaches 1.2 times Above, it, when and being less than 0.05 through its p-value value of statistical check, is considered as differentially expressed protein.

(5) protein function annotation and differentially expressed protein analysis: including that GO annotates, COG annotates, Pathway Metabolic pathway annotates, and the GO enrichment of differentially expressed protein is analyzed, the Pathway enrichment point of differentially expressed protein Analysis, expression pattern cluster between Multi-example.

5. result and analysis

5.1 protein detection

(1) sample protein calibration curve

As it is shown in figure 5, with BSA standard liquid make obtain calibration curve be y=1.9748x+0.0012, Phase relation R2=0.9972, description standard sample concentration gradient meets requirement of experiment, can carry out next step test.

(2) sample protein matter is quantitative

When Bradford working solution (Coomassie brilliant G-250) with when albumen combines in acid condition, one In fixed scope, protein content becomes linear positive dependency relation with the absorbance of 595nm.Quantitative information is shown in Table 5-1。

Table 5-1 each sample extracts quantification of protein testing result

(3) each sample Protein S DS electrophoresis

The concentration of separation gel is 12%, and Marker applied sample amount is 12 μ g, in Alport-iPSCs group and control group Sample amount is 20 μ g, and SDS electrophoresis result is shown in Fig. 6, and wherein, M is marker, and 1 is Alport-iPSCs, 2 is normal healthy controls group iPSCs.

5.2 identification of proteins

(1) spectrogram quality of match error distribution

Mass spectrometer uses Q-Exactive, has high resolution and pinpoint accuracy advantage, is widely used in big point The analysis of sub and little molecule, particularly suitable for the complicated proteomics research of height of specimen.For prevention Omitting the result identified, therefore the present embodiment controls based on the peptide fragment matching error of database search strategy Below 0.02Da.Fig. 7 is spectrogram quality of match error map.Wherein, abscissa: mass discrepancy, number Magnitude ppm；Ordinate: Mascot database ion is marked.Fig. 7 shows relatively dividing of the peptide fragment matching Error distribution between the actual value of son amount and theoretical value, error is concentrated mainly within 10/1000000ths, i.e. Error qualification result is preferable.

(2) basic authentication information

Identify that essential information statistics is as follows: there are 280267 two grades spectrograms (TotalSpectra), pass through Identify 38088 spectrograms (Spectra) after quality control, wherein match peculiar peptide fragment spectrogram (UniqueSpectra) quantity be 34727, peculiar peptide fragment (Peptide) quantity that identifies be 13188 Individual, wherein distinctive peptide section sequence (UniquePeptide) be 12600, identify protein (Protein) 3470。

(3) protein relative molecular mass distribution is identified

As shown in Figure 8, polymolecular quality size is thought according to protein, the albumen obtained by identifying above Matter carries out statistic of classification, wherein, abscissa be identify protein molecular quality (unit: kilodalton, KDa), ordinate is that the protein of this Range molecular weight accounts for the percentage identifying protein quantity.As shown in Figure 8, Major part protein relative molecular mass concentrates on 20-80KDa.

(4) peptide section sequence distribution of lengths

It is illustrated in figure 9 peptide section sequence distribution of lengths, the figure shows different length peptide fragment and account for the hundred of all peptide fragments Proportion by subtraction.Abscissa is peptide fragment total number of atnino acid, and ordinate is the percentage that this length peptide fragment accounts for whole peptide fragment Value.

(5) peptide section sequence coverage

Figure 10 is shown in the distribution of peptide section sequence coverage, show difference peptide section sequence coverage and include in Figure 10 Protein quantity.Coverage has 1334 at the protein quantity of 0%-10%, accounts for the 38% of total number；5%-10% Albumen have 820, account for total number 24%；447, the albumen of 10%-15%, accounts for the 13% of total amount；15%-20% 309, albumen, account for the 9% of total amount；The protein 20 of 20%-25% 8, accounts for the 6% of total amount；25%-30% 130, albumen, account for sum 4%；86, the albumen of 30%-35%, accounts for sum 2%；The egg of 35%-40% White 58, account for sum 2%；78, the albumen of 40%-100%, accounts for sum 2%.

(6) Unique peptide fragment distributed number

Unique peptide fragment distributed number as shown in figure 11, shows the distributed number of peptide fragment contained by the albumen identifying Situation, abscissa is for identifying the peptide fragment quantitative range of albumen, and ordinate is protein quantity.Display in Figure 11 Trend shows, the identified albumen arriving of major part, its contained peptide fragment quantity is within 10, and albumen number Amount reduces with the increase of coupling peptide fragment quantity.

5.2 quantification of protein

(1) differentially expressed protein statistics

According to protein abundance level, when fold differences reaches more than 1.2 times, and through its p-value of statistical check When value is less than 0.05, then it represents that be differentially expressed protein.The differentially expressed protein sum obtaining in the present embodiment Being 383, the protein wherein raising has 156, and the protein of downward has 227.

The present embodiment is picked out fold differences and is more than more than 2.0 times, and representative differentially expressed protein is shown in Table 5-2.The albumen wherein raising has 17, the protein 18 of downward.

Table 5-2 Alport-iPSCs compares with normal person iPSCs the protein of notable differential expression

(2) protein abundance compares

When relative quantification, if the amount of same protein does not has significant change at two sample rooms, that Its protein abundance ratio is close to 1.When the abundance ratio i.e. fold differences of albumen reaches more than 1.2 times, p-value When value is less than 0.05, regard the differentially expressed protein as different sample rooms for this albumen.To each protein fold differences Distribution is made with 2 as shown in figure 12 after taking the logarithm the end of for.The albumen that expression raises occupy abscissa 0 position Right side, the albumen that expression is lowered occupy the left side of abscissa 0 position.

Figure 12 shows the distribution situation of the fold differences of all proteins that can be quantitative, and wherein abscissa represents poor Value after logarithmic transformed with 2 as the truth of a matter for the different multiple.Lowering for expression more than 0, less than 0 Raise for expression.Wherein the point more than 1.2 for the fold differences is red and green marks (red under expression Adjust, decline 227；Green raises for expression, rises 156).

(3) GO analyzes

Carry out GO functional annotation analysis for all albumen identifying, for three ontology GO entry involved in (cellularcomponent, biologicalprocess, molecularfunction), List ID and the albumen number of all corresponding albumen, remove the GO entry not having corresponding albumen.

As shown in figure 13, in cellularcomponent (cellular component) protein expression most be cellpart (accounting for 18.31%) and organelle (accounting for 16.27%).Egg in biologicalprocess (biological process) That white expression is most is cellularprocess (13.34%) and metabolicprocess (11.02%), What in molecularfunction (molecular function), protein expression was the highest is binding (49.39%), expresses relatively Many is catalyticactivity (28.37%), and chemoattractantactivity accounts for 0.08%.

(4) COG annotation

COG (ClusterofOrthologousGroupsofproteins) is the abbreviation clustering of the adjacent class of albumen, It is the database carrying out ortholog classification to protein.The albumen constituting each COG is to be assumed It from ancestors' original protein, and is that orthologs or paralogs.Orthologs refers to come from not The infraspecific albumen being come by vertical family (species are formed) evolution, and retain and original egg specifically The function of Bai Xiangtong.Paralogs is those albumen deriving from gene duplication in certain species, may Evolve that make new advances with originally relevant function.This analysis by the protein that identifies and COG database comparison, Predict the possible function of these protein and do function statistic of classification to it.

As shown in figure 14, the protein having identified out is compared with COG database, altogether obtain 2795, the albumen of annotation, belongs to 24 kinds of FunctionClass.What albumen number was most is Generalfunctionpredictiononly (R, 592), more is Posttranslationalmodification, proteinturnover, chaperones (O, 286), Translation, ribosomalstructureandbiogenesis (J, 283), Replication, recombinationandrepair (L, 211), Transcription (K, 205).

(5) the GO enrichment of differentially expressed protein is analyzed

The protein that the GO enrichment analysis of differentially expressed protein can represent difference is bright with which biological function Aobvious related.Utilize GeneOntology database, differential expression protein is carried out to each entry of this database Map, calculate protein number, application hypergeometry inspection, find out the GO of differential expression protein significant enrichment Entry.P-value≤0.05 is defined as threshold value, and the GO representing differentially expressed protein significant enrichment analyzes.Table 5-3 Being GO-CellularComponent significant enrichment analysis result, table 5-4 is differentially expressed protein GO-MolecularFunction significant enrichment analysis result, table 5-5 is differentially expressed protein GO-BiologicalProcess significant enrichment analysis result.

Table 5-3 differentially expressed protein GO-CellularComponent significant enrichment analysis result

Table 5-4 differentially expressed protein GO-MolecularFunction significant enrichment analysis result

Table 5-5 differentially expressed protein GO-BiologicalProcess significant enrichment analysis result

(6) the Pathway enrichment of differentially expressed protein is analyzed

Main biochemical metabolism approach and signal transduction pathway that differentially expressed protein participates in can pass through Pathway Conspicuousness enrichment determines.By contrasting the egg of Normal-iPSCs and Alport-iPSCs differential expression In vain≤0.05 is threshold value, it is stipulated that be Normal-iPSCs and Alport-iPSCs differentially expressed protein significant enrichment Analyze, be shown in Table 5-6.Result shows, the path that Alport-iPSCs expresses with Normal iPSCs significant difference It is concentrated mainly on the infection of staphylococcus aureus, the biosynthesis of aminoacyl-tRNA, pathogenic large intestine bar The infection of bacterium, FcgammaR-mediatedphagocytosis, glycolysis, gluconeogenesis, DCM, Right ventricular cardiomyopathy, Onecarbonpoolbyfolate, Selenocompoundmetabolism, the viral heart In the approach such as myositis.

The Pathway significant enrichment analysis result of table 5-6 differential gene

The present embodiment uses iTRAQ technical tie-up liquid phase tandem mass spectrum technical Analysis Alport syndrome patient Differentially expressed protein between iPSCs and the iPSCs of normal person, the life of screening early diagnosis Alport syndrome Substance markers thing and possible therapy target.By iPSCs and the Alport syndrome patient iPSCs to normal person Being analyzed, fold differences reaches more than 1.2 times, and is less than 0.05 through its p-value value of statistical check When, it is differentially expressed protein.The differentially expressed protein sum obtaining in the present embodiment is 383, Qi Zhongshang The protein adjusted has 156, and the protein of downward has 227.Obtained differentially expressed protein is entered Row GO function and KEGGpathway analyze, and respectively obtain the biological mistake of the participation of differentially expressed protein gene Journey (BiologicalProcess), residing cell position (CellularComponent), molecular function And main biochemical metabolism approach and signal transduction pathway (MolecularFunction).

Alport syndrome patient iPSCs compares with the iPSCs of normal person, and wherein differentially expressed protein is expressed Substantially lower have Keratin-14, Keratin-10, SRSF2, TUBA1A, Keratin-9, Keratin-5, MRPL27、Keratin13、APLP2、PAPSS1、SURF4、RCN3、EIF1AY、APEX1、 ACBD3, UBA2 and NLGN4X etc..Wherein, Keratin 14 (Keratin-14) belongs to I type angle egg The intermediate filament protein class of white family, Keratin 14 and 2 type keratin 5s form heterodimer and express at epidermis The cytoskeleton of cell.Keratin 10 (Keratin-10) falls within I type keratin family intermediate filament protein class, Participate in constituting the cytoskeleton of epithelial cell.The gene mutation of coding SRSF2 and chrooic myelomonocytic Leukaemic's gene mutation situation is relevant.TUBA1A belongs to tubulin family, participates in constituting aixs cylinder Cytoskeleton, can regulate neural remote growth and regeneration, and the gene of coding TUBA1A is undergone mutation, can To cause the generation of multiple the nervous system disease, such as congenital agyria, the exception in CA3 region.Keratin-9 Being I type keratin, it is relevant with the gene mutation of encoded K eratin-9 that epidermolytic slaps seborrheic keratosis of hesitating. Keratin-5 is II type keratin, participates in constituting the basalis of epithelial cell, the gene mutation of encoded K eratin-5 May result in Dowling-Degos sick.Keratin13 belongs to I type keratin family, is formed different with Keratin4 Matter dimer participates in constituting, and the gene mutation of encoded K eratin13 may be relevant with white sponge naevus.

The iPSCs differentially expressed protein of Alport syndrome patient iPSCs and normal person expresses having of substantially rise NEFL、PSME4、UTF1、RBM14、AIF1L、STOM、FGF2、SF3B14、DNMT3A、 IK、Keratin-18、CRYZ、RG9MTD1、MTHFD2、HIST1H1A、CPT1A、UBXN4 And SEMA6A etc..

NEFL is neurofilament light gene coding, and the gene mutation of coding NEFL is hard with amyotrophic lateral sclerosis funiculus lateralis medullae spinalis The generation changing disease is relevant, NEFL albumen and the growth of SCCHN and attack relevant.UTF1 starts The aberrant methylation of son is relevant with the generation of cervical carcinoma, and the versatility to multipotential stem cell for the UTF1 has an impact. AIF1L is allograft inflammatory factor gene code, also known as ionization Ca²⁺In conjunction with adjuster, at testis Higher with spleen tissue is expressed, relatively low in kidney, brain tissue, lung, expression, there are some researches show in Japan When sea cucumber receives injury and bacterium infection, AIF1 expresses and raises, and AIF1 also can manage β catenin as lung cancer Independent prognostic indicator.FGF2 albumen is a member of fiber mother cell growth factor family, can promote cell Division, can promote the multiplication capacity of external mescenchymal stem cell, maintains the differentiation capability of mescenchymal stem cell, It is immature Gegenbaur's cell that FGF-2 can make Derived from Mesenchymal Stem Cells.DNMT3A is the transfer of DNA methyl The one of enzyme, participates in the methylation procedure of DNA, DNMT3A lifting in the induction and silence of tumour cell The effect wanted.Keratin-18 belongs to I type keratin family intermediate filament protein class, and Main Function is that protection liver is thin Born of the same parents' cell can promote hepatocellular seepage and necrosis from damage, the gene mutation of encoded K eratin-18.

Alport syndrome is by encoding gene COL4A3, COL4A4 of coding type Ⅳ collagen α the 3rd, α the 4th, α 5 chain A kind of basilar memebrane disease causing with COL4A5 sudden change.Alport-iPSCs contrasts with control group, Keratin-14, The expression of Keratin-10, Keratin-9, Keratin-5 and Keratin13 is significantly to lower, may be with Alport The pathogenesis of syndrome is relevant.FGF2 protein expression is significantly lowered, and FGF2 albumen can promote stem cell Differentiation, analyzes a lot of protein of discovery by GO enrichment relevant with the institutional framework composition of cell, needs into one The research of step.Discovery main biochemical metabolic pathway and signal transduction are analyzed in the Pathway enrichment of differentially expressed protein Approach includes the biosynthesis of aminoacyl-tRNA, the infection of bacterium, myocarditis, glycolysis, Glycogen synthesis etc. Relevant.

The differentially expressed protein that above-mentioned Analysis and Screening obtains can be widely used in preparation diagnosis Alport syndrome Detection chip or detection reagent in, as prepared the monoclonal antibody of corresponding albumen, and associated proteins chip Technology, the getting up early diagnosis for Alport syndrome provides intermediate object program to support, and is conducive to the Alport making a definite diagnosis The pathogenesis of syndrome patient launches research.

Each technical characteristic of embodiment described above can combine arbitrarily, for making description succinct, not right The all possible combination of each technical characteristic in above-described embodiment is all described, but, if these skills There is not contradiction in the combination of art feature, is all considered to be the scope that this specification is recorded.

Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, But can not therefore be construed as limiting the scope of the patent.It should be pointed out that, for this area For those of ordinary skill, without departing from the inventive concept of the premise, can also make and some deform and change Entering, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended power Profit requires to be as the criterion.

Claims

1. an analysis method for the differentially expressed protein of Alport syndrome patient inductive pluripotent stem cells, It is characterized in that, comprise the steps:

2. the differential expression egg of Alport syndrome patient's inductive pluripotent stem cells as claimed in claim 1 White analysis method, it is characterised in that in described step one, described Alport syndrome patient's inductivity is many Can stem cell be to use electrotransfection method that Oct4, Sox2, Kif4 and SV40LT gene is proceeded to Alport to combine In the urine kidney tubular fossils of simulator sickness patient, reprogram the inductive pluripotent stem cells obtaining.

3. the differential expression egg of Alport syndrome patient's inductive pluripotent stem cells as claimed in claim 1 White analysis method, it is characterised in that in described step 2, uses tetraethylammonium bromide to two groups extracting The precipitation of albumen sample carries out reductive alkylation process, obtains corresponding albumen sample solution.

4. the differential expression egg of Alport syndrome patient's inductive pluripotent stem cells as claimed in claim 1 White analysis method, it is characterised in that in described step 4, uses iTRAQ 8 labelling kit, uses Labelled reagent 116 marks the sample of Alport syndrome patient group, uses labelled reagent 118 to mark Healthy People Sample.

5. the differential expression egg of Alport syndrome patient's inductive pluripotent stem cells as claimed in claim 1 White analysis method, it is characterised in that in described step 5, described strong cation exchange separating treatment uses Shimadzu LC-20AB liquid phase systems, splitter is the Ultremex SCX post of 4.6 × 250mm model.

6. the differential expression egg of Alport syndrome patient's inductive pluripotent stem cells as claimed in claim 1 White analysis method, it is characterised in that in described step 6, carries out using LC-20AD when liquid chromatogram separates Nanoliter liquid chromatograph of model carries out standard separation；

The described Tandem Mass Spectrometry Analysis that carries out uses series connection ESI mass spectrograph, and a class resolution ratio is set to 70000, and two Class resolution ratio is 17500；Electric charge is selected to be 2 in parent ion⁺～5⁺, 15 mothers more than 20000 for the peak intensity Ion carries out secondary analysis, carries out fragmentation, fragment by the HCD pattern that collision energy is 27+12% to peptide fragment Detecting in Orbi, the dynamic eliminating time is set as: chromatogram half-peak breadth duration, ion source voltage is set to 1.6 Kilovolt；AGC is realized by Orbi, is set to: one-level 3*E⁶To two grades of 1*E⁵；The mass-to-charge ratio of scanning For one-level 350～2000, two grade 100～1800.

7. the differential expression egg of Alport syndrome patient's inductive pluripotent stem cells as claimed in claim 1 White analysis method, it is characterised in that in described step 7, described protein identification software is Mascot, During analysis, scan for IPI human v3.87 for database.

8. the differential expression egg of Alport syndrome patient's inductive pluripotent stem cells as claimed in claim 1 White analysis method, it is characterised in that in described step 7, is being analyzed according to protein abundance level During, when fold differences reaches more than 1.2 times, and when being less than 0.05 through statistical assumption value p-value, For differentially expressed protein.

9. Alport syndrome patient's inductive pluripotent stem cells as according to any one of claim 1～8 The analysis method of differentially expressed protein obtains differentially expressed protein, including the NEFL of up-regulated, PSME4, UTF1、RBM14、AIF1L、STOM、FGF2、SF3B14、DNMT3A、IK、Keratin-18、 CRYZ, RG9MTD1, MTHFD2, HIST1H1A, CPT1A, UBXN4 and SEMA6A, with And express lower Keratin-14, Keratin-10, SRSF2, TUBA1A, Keratin-9, Keratin-5, MRPL27、Keratin13、APLP2、PAPSS1、SURF4、RCN3、EIF1AY、APEX1、 ACBD3, UBA2 and NLGN4X.

10. differentially expressed protein as claimed in claim 9 is at the detection core of preparation diagnosis Alport syndrome Application in piece or detection reagent.