CN106596967B - A kind of quantitative approach of stable isotope phosphorylated labelled protein - Google Patents
A kind of quantitative approach of stable isotope phosphorylated labelled protein Download PDFInfo
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Abstract
A kind of quantitative approach of stable isotope phosphorylated labelled protein, is related to Protein quantitative analysis.Using the commercially available small molecule containing labels such as carbon 13, oxygen 18 or deuteriums 2 as raw material, pass through hydrolysis of the halogenated hydrocarbons under silver salt promotion, the alcohol of the isotope labellings such as carbon containing 13 and oxygen 18 is selectively synthesized, then the phosphite ester labelled reagent of the stable isotope labelings such as carbon containing 13, oxygen 18 or deuterium 2 is obtained by the reaction by base exchange.By combining different stable isotope labeling phosphite esters, more flux albumen relative quantitative assay such as two marks, three marks, four marks or five marks are obtained, the molecular weight difference between each label is greater than or equal to 3 dalton.Stable isotope N phosphorylateds labelled protein quantitatively has preferable accuracy, high sensitivity, the interference of No Parity element effect and wide applicability.
Description
Technical field
The present invention relates to Protein quantitative analysis, more particularly, to a kind of determining for stable isotope phosphorylated labelled protein
Amount method.
Background technology
The Large scale identification of protein and quantitative analysis are the key that proteomics researches.The new skill of Protein quantitative analysis
Art plays crucial impetus in the discovery of life fundamental mechanism and disease marker.Wherein, based on biological mass spectrometry
Stable isotope chemical labeling techniques play increasingly important role in the relative quantification of protein and absolute quantitation.[1]
Plurality of stable isotope-labelled protein composes quantitative approach and reagent is successfully developed in succession, such as iTRAQ[2],ICAT[3]And TMT[4]It is widely used in life science and clinical research Deng as commercial reagents.However, conventional tag strategy is based on
Classical protein chemistry, mark the selectivity of chemistry, the sensitivity of mass spectrometry of labelled reagent and chromatographic isolation, isotope effect and
Reagent price is very expensive etc., seriously restricts large-scale protein matter group Quantitative Study.New labelling strategies and label
The research and development of reagent have great importance.
Bibliography:
1.Ong,S.E.;Mann,M.Mass spectrometry-based proteomics turns
quantitative.Nat.Chem.Biol.2005,1,252-262.
2.Ross,P.L.;Huang,Y.N.;Marchese,J.N.;Williamson,B.;Parker,K.;Hattan,
S.;Khainovski,N.;Pillai,S.;Dey,S.;Daniels,S.;Purkayastha,S.;Juhasz,P.;Martin,
S.;Bartlet-Jones,M.;He,F.;Jacobson,A.;Pappin,D.J.Multiplexed protein
quantitation in Saccharomyces cerevisiae using amine-reactive isobaric
tagging reagents.Mol.Cell.Proteomics 2004,3,1154-1169.
3.Gygi,S.P.;Rist,B.;Gerber,S.A.;Turecek,F.;Gelb,M.H.;Aebersold,
R.Quantitative analysis of comlex protein mixtures using isotope-coded
affinity tags.Nat.Biotechnol.1999,17,994-999.
4.Thompson,A.;Schafer,J.;Kuhn,K.;Kienle,S.;Schwarz,J.;Schmidt,G.;
Neumann,T.;Hamon,C.Tandem mass tags:a novel quantification strategy for
comparative analysis of complex protein mixtures by MS/MS.Anal.Chem.2003,75,
1895-1904.
Invention content
The organophosphor protein labeling examination containing carbon -13, oxygen -18 or deuterium -2 label that the purpose of the present invention is to provide a kind of
Agent and preparation method thereof.
Another object of the present invention is to provide a kind of quantitative approach of stable isotope phosphorylated labelled protein.
The structural formula of the organophosphor protein labeling reagent containing carbon -13, oxygen -18 or deuterium -2 label is as follows:
Wherein, superscript notation a is 1 or 2;Superscript notation b is 16 or 18;Superscript notation c is 12 or 13;Subfix n is
1,2,3,4 or 5, preferably 1 and 2.
The preparation method of the organophosphor protein labeling reagent containing carbon -13, oxygen -18 or deuterium -2 label, including with
Lower step:
Step 1:Carbon containing -13, the preparation of the alcohol of -18 isotope labelling of oxygen, specific preparation route are as follows:
It is as follows:Within the temperature range of 10~60 DEG C, by halogenated alkane and water containing silver salt with it is organic molten
It is stirred to react in the mixed system of agent, the alcohol of the isotope labellings such as product carbon -13, oxygen -18 or deuterium -2 is obtained after reacting 6~72h;
In step 1, the optional self-contained C of the halogenated alkane1~C6Linear chain or branched chain chloro, bromo or alkane iodide,
It is preferred that the alkane of bromo or iodo;C in halogenated hydrocarbons is carbon -12 or carbon -13, H is hydrogen -1 or deuterium -2;Oxygen in water is oxygen -16
Or oxygen -18;Silver salt is the silver salt of inorganic acid, contains C1~C6Carboxylic acid silver salt, preferably silver acetate, silver tetrafluoroborate or silver nitrate;
Organic solvent is ether solvent, preferably ether or tetrahydrofuran.
Step 2:Carbon containing -13, the preparation of the diphosphites of the isotope labellings such as oxygen -18 or deuterium -2, specific preparation route
It is as follows:
It is as follows:The isotope labellings such as the alcohol of the isotope labellings such as carbon -13, oxygen -18 and oxygen -18, deuterium -2 are sub-
Diphenyl phosphate reacts in organic solvent, and the alkali of 0.1~10 equivalent is added in reaction, and the reaction time is 1~12h, has been reacted
After remove organic solvent, analyze to obtain product stable isotope labeling phosphite ester through column chromatography.
In step 2, the isotope labellings phosphorous such as alcohol and oxygen -18, deuterium -2 of the isotope labellings such as the carbon -13, oxygen -18
The molar ratio of diphenyl phthalate can be 1 ︰ 1;Inorganic base or organic base can be used in the alkali, and the alkali can be selected from sodium hydroxide, hydrogen-oxygen
Change potassium, pyridine, one kind in triethylamine etc., preferably pyridine or triethylamine;Ether solvent can be used in the organic solvent, described to have
Solvent can be selected from one kind in ether, tetrahydrofuran, Isosorbide-5-Nitrae-dioxane etc., preferably ether or tetrahydrofuran.
A kind of quantitative approach of stable isotope phosphorylated labelled protein is as follows:
1) compared with the H-Phosphonate of selection stable isotope labeling carries out molecular weight with unlabelled phosphite ester and match
It is right, the stable isotope labeling phosphite ester composition two that selection molecular weight difference is greater than or equal to 3 dalton is marked, three mark, four mark,
Five marks or six scalar quantity reagent sets;
2) chemical mark is carried out respectively to reference polypeptide and protein digestion polypeptide sample under identical label reaction condition
Note, mixed mark sample carry out removing salt treatment, and obtained labeling polypeptide sample carries out mass spectrum sequencing identification and analysis, and by not
Relative quantification mass spectral analysis is carried out to corresponding albumen with stable isotope labeling characteristic mass difference and ionic strength;
The label reaction of stable isotope phosphorylated has the reaction selectivity of height, only the ends N- to peptide fragment and lysine side
Chain amino is marked, as follows:
3) under the conditions of ice-water bath, by 10~50 μ g polypeptide samples (synthesis polypeptide or protein digestion polypeptide etc.) and molecular weight
Difference is greater than or equal to the stable isotope labeling such as isotope labellings phosphorous acid such as carbon -13, oxygen -18 or deuterium -2 of 3 dalton
1 ︰ (5~10) reacts ester in the in the mixed solvent of water and organic solvent in molar ratio, and the alkali of 0.1~10 equivalent is added in reaction,
Reaction time is 0.5~2h, removes organic solvent and salt after the completion of reaction, carries out mass spectrum quantitative analysis.
The halogenating agent can be selected from carbon tetrachloride, iodine/hydrogen peroxide, sodium hypochlorite, hydrogen peroxide/potassium iodide, cupric iodide etc.
In one kind, preferably sodium hypochlorite or carbon tetrachloride.
The organic solvent can be selected from one kind in ethyl alcohol, methanol, acetone, tetrahydrofuran, acetonitrile etc., preferably acetonitrile or second
Alcohol.
The alkali can be selected from sodium hydroxide, triethylamine, sodium bicarbonate, sodium carbonate, potassium hydroxide, tri-n-butylamine, in pyridine etc.
One kind, preferably triethylamine or sodium bicarbonate.
Protein quantification mass spectral analysis condition is:
Mass spectrograph is:Electron spray ionisation-flight time mass spectrum (ESI-TOF MS), spraying ionization-Orbitrap mass (ESI-
Orbitrap MS), electron spray ionisation-ion trap mass spectrometry (ESI-IT MS), ground substance assistant laser induction desorption ionization-flight when
Between mass spectrum (MALDI-TOF/TOF MS) etc., preferably electron spray ionisation-flight time mass spectrum (ESI-TOF MS) and electron spray electricity
From-Orbitrap mass (ESI-Orbitrap MS).
Ion ionizes pattern:Positive ion mode.
Cracking pattern is:Collision induced dissociation (CID), energetic encounter cracking (HCD) etc., preferably energetic encounter cracks
(HCD) pattern.
The present invention is based on organophosphor label chemistry, have synthesized a series of stable isotope labeling organophosphor examination of structure novels
Agent, and successful quantitation analysis is carried out to standard protein and biological sample albumen with the reagent.
Description of the drawings
Fig. 1 is stable isotope13C (4) and18O (2) label three is marked N- phosphorylated labelled protein quantitative analysis strategies and is shown
It is intended to.
Fig. 2 is stable isotope13C (4) and18Three mark diethoxy phosphite ester mass spectral analysis figures of O (2) labels.
Fig. 3 is three scalar quantity analytical standard synthesis polypeptide of stable isotope N- phosphorylateds label.
Fig. 4 is three scalar quantity analytical standard protein B SA of stable isotope N- phosphorylateds label.
Fig. 5 is three scalar quantity analytical standard albumen β-Casein of stable isotope N- phosphorylateds label.
Fig. 6 is three scalar quantity analytical standard albumen Myoglobin of stable isotope N- phosphorylateds label.
Fig. 7 is three scalar quantity analytical standard albumen Lysozyme of stable isotope N- phosphorylateds label.
Fig. 8 investigates for stable isotope N- phosphorylated labeling method isotope effects.
Fig. 9 is that three mark stable isotope N- phosphorylateds mark CDK9 protein-protein interactions in quantitative analysis cell.
Figure 10 is that the molecular biology of CDK9 protein-protein interaction quantitative analysis results is verified.
Specific implementation mode
Below by specific embodiment, the present invention will be further described.
Table 1 is stable isotope N- phosphorylated labelled protein quantitative analyses part related organophosphate reagent.
Embodiment one, carbon -13 (4), oxygen -18 (2) label diethyl phosphite (13C4 18O2- DEP-H, 1 compound of table
14) preparation
Step 1, carbon containing -13 (2), oxygen -18 (1) isotope labelling ethyl alcohol preparation
In the flask of 25mL, under nitrogen atmosphere, the iodoethane (6.4mmol) of 1g labelings is added.1.08g silver nitrate
(6.4mmol), the water and 5mL ether that 1mL oxygen -18 marks.After being stirred to react for 24 hours at room temperature, it is filtered to remove precipitation.It obtains
The diethyl ether solution of -18 isotope labelling ethyl alcohol of carbon -13 and oxygen.
The preparation of the diethyl phosphite of step 2, carbon containing -13 (4) and oxygen -18 (2) isotope labelling
In 25mL flasks, under nitrogen atmosphere, it is added into the diethyl ether solution of -18 isotope labelling ethyl alcohol of carbon -13 and oxygen
0.1mL triethylamines and 0.789g diphenyl phosphites (3.2mmol) after stirring 6h at room temperature, remove solvent, crude product is through silicon
The isolated product of plastic column chromatography.
1H NMR(CDCl3,500MHz):δ=6.82 (d, 1H), 4.39-4.30 (m, 2H), 4.02-3.93 (m, 2H),
1.56-1.51(m,3H),1.24-1.19(m,3H)ppm。
13C NMR(125MHz,CDCl3):δ=61.9 (dd), 16.3 (dd) ppm.
31P NMR(CDCl3,203MHz):δ=7.3ppm.
ESI-MS:M/z=147 [M+H]+,169[M+Na]+。
Embodiment two, carbon -13 (4) label diethyl phosphite (13C4- DEP-H, 1 compound 13 of table) preparation
As in the first embodiment, the water that the oxygen -18 of step 1 (1) marks only to be changed into the water of oxygen -16.
1H NMR(CDCl3, 500MHz):δ=6.82 (d, 1H), 4.34-4.27 (m, 2H), 4.04-3.98 (m, 2H),
1.52-1.48(m,3H),1.27-1.23(m,3H)ppm。
13C NMR(125MHz,CDCl3):δ=61.8 (dd), 16.3 (dd) ppm.
31P NMR(CDCl3,203MHz):δ=7.0ppm.
ESI-MS:M/z=143 [M+H]+,165[M+Na]+。
Embodiment three, oxygen -18 (2) label dimethoxy phosphite ester (18O2- DMP-H, 1 compound 1 of table) preparation
As in the first embodiment, the iodoethane of the labeling of step 1 only to be changed into the iodomethane of carbon -12.
1H NMR(CDCl3,500MHz):δ=6.67 (d, 1H), 3.98-3.32 (m, 6H) ppm.
13C NMR(125MHz,CDCl3):δ=58.4 (d) ppm.
31P NMR(CDCl3,203MHz):δ=6.7ppm.
ESI-MS:M/z=115 [M+H]+。
Example IV, oxygen -18 (3) label dimethoxy phosphite ester (18O3- DMP-H, 1 compound 2 of table) preparation
With embodiment three, only step 2 phosphorous acid diphenyl ester is changed into18The diphenyl phosphite of O (3) labels.
1H NMR(CDCl3,500MHz):δ=6.69 (d, 1H), 3.97-3.12 (m, 6H) ppm.
13C NMR(125MHz,CDCl3):δ=57.3 (d) ppm.
31P NMR(CDCl3,203MHz):δ=6.6ppm.
ESI-MS:M/z=117 [M+H]+。
Embodiment five, carbon -13 (2), oxygen -18 (2) label dimethoxy phosphite ester (13C2 18O2- DMP-H, 1 compound of table
3) preparation
As in the first embodiment, the iodoethane of step 1 labeling only to be changed into the iodomethane of labeling, silver nitrate changes into
Silver acetate.
1H NMR(CDCl3,500MHz):δ=7.01 (d, 1H), 3.76-3.15 (m, 6H) ppm.
13C NMR(125MHz,CDCl3):δ=62.1 (d) ppm.
31P NMR(CDCl3,203MHz):δ=6.9ppm.
ESI-MS:M/z=119 [M+H]+。
Embodiment six, oxygen -18 (2), deuterium -2 (1)-label dimethoxy phosphite ester (18O2- DMP-D, 1 compound 6 of table)
Preparation
With embodiment three into, hydrogen diphenyl phosphite in step 2 is only changed to the diphenyl phosphite of the label of deuterium -2, step 2
Triethylamine change pyridine into.
1H NMR(CDCl3,500MHz):δ=3.87-3.12 (m, 6H) ppm.
13C NMR(125MHz,CDCl3):δ=60.1 (d) ppm.
31P NMR(CDCl3,203MHz):δ=6.9ppm.
ESI-MS:M/z=116 [M+H]+。
Embodiment seven, carbon -13 (4), deuterium -2 (1)-label diethoxy phosphite ester (13C4- DEP-D, 1 compound of table
25) preparation
With embodiment two, the water that the oxygen -18 in step 1 marks only is changed to the water of oxygen -16 into, by step 2 phosphorous acid two
Phenyl ester changes deuterium-labeled diphenyl phosphite into.
1H NMR(CDCl3, 500MHz):δ=4.23-4.14 (m, 2H), 4.01-3.87 (m, 2H), 1.47-1.34 (m,
3H),1.29-1.21(m,3H)ppm。
13C NMR(125MHz,CDCl3):δ=60.4 (dd), 15.7 (dd) ppm.
31P NMR(CDCl3,203MHz):δ=7.3ppm.
ESI-MS:M/z=144 [M+H]+。
Embodiment eight to embodiment 14, stable isotope three marks N- phosphorylated labelled protein relative quantification mass spectral analyses
Method
Embodiment eight, three scalar quantity analytical standard peptide fragment of stable isotope N- phosphorylateds
Three part of 10 μ g human fibrin peptide fibrinopeptide (peptide sequence ADSGEGDFLAEGGGVR) is taken respectively
Sample is dissolved in water/ethyl alcohol (1 ︰ 1) in the mixed solvent of 20 μ L, is then respectively adding the triethylamine of 5 μ L, and vortex mixed 3~
5min makes peptide sample be completely dissolved.Under the conditions of ice-water bath, 10 μ L diethoxy hydrogen phosphorous are added in above-mentioned label solution respectively
Acid esters, carbon -13 (4) label hydrogen diethyl phosphite (13C4- DEP-H, 1 compound 13 of table) and carbon -13 (4), oxygen -18 (2)
Label diethyl phosphite (13C4 18O2- DEP-H, 1 compound 14 of table) carbon tetrachloride mixed solution (ratio be 1 ︰ 4), three
A independent marking reaction is respectively designated as:Light mark-, acceptance of the bid-, again mark-, vortex mixed reaction 30min, concentration are tried except dereaction
Agent, three parallel samples are sufficiently mixed, and are carried out desalination using -18 solid phase extraction column of reverse phase carbon, are then carried out liquid chromatogram-matter
Analysis is used in conjunction in spectrum, and (light mark-︰ acceptances of the bid-︰ marks -=1 ︰, 1 ︰ to the relative quantification result measured to fibrinopeptide again with theoretical proportions
1) it compares and analyzes.If attached drawing 3 is quantitative result, experiment measures light mark-︰ acceptances of the bid-︰ and marks -0.98 ︰ 1.04 of=1 ︰ again, with reason
It is sufficiently close to by value, the accuracy of three scalar quantity polypeptide of display stable isotope N- phosphorylateds label.
Salt-removal steps are:Acidified sample is to acidity between pH 2~4;Wash column:Add 0.1~1mL acetonitriles, is forced into liquid level
Close to filler;Balance:Add 0.1~1mL, 0.1% aqueous formic acids, is forced into liquid level close to filler, is repeated once;Loading:It will
Sample is fully transferred in column;Desalination:Add 0.1~1mL, 0.1% aqueous formic acids to wash column, is repeated twice;Elution:Add 100 μ L
Eluent (water of 70% acetonitrile/30%/0.1% formic acid) in the sample access albumen low adsorption pipe of elution, makes for mass spectral analysis
With.
Chromatograph mass spectrum analysis condition is:Use the nano-UHPLC Proxeon of Thermo Scientific companies
Easy-nLC1000 is coupled LTQ-Orbitrap Q-Exactive mass spectrums.Sample injects column captured first in liquid chromatogram
(C18,3 μm, 2cm × 100 μm) is captured, enter afterwards analytical column (Acclaim PepMap RSLC-C18,2.0 μm, 150mm × 75
μm separation) in separation, total sample size be 1 μ g polypeptides.Chromatographic condition is:5%~45% contains the acetonihile gradient elution of 0.1% formic acid
120min;Flow velocity:250nl/min.Column efflux is directly entered mass spectrographic electron spray ionisation source.HCD fragments are used for Q-
Exactive platforms.20 ion carries out broken and dynamic and excludes 20s before selection abundance.By Xcalibur softwares (Thermo
Scientific raw data file) is generated through Proteome Discoverer V2.0 processing, and is searched for using SEQUEST soft
Part retrieves the human protein database of Swiss-Prot, completes peptide fragment identification and quantitative analysis.
Embodiment nine, three scalar quantity analytical standard protein B SA of stable isotope N- phosphorylateds
With embodiment eight, only label sample is changed into standard protein enzymolysis polypeptide, it is 1h that the reaction time is increased by 30min.
19 peptide fragments are detected altogether, as by taking peptide hydrolysis QEPERNECFLSHK as an example, standard protein quantitative ratio is 1 ︰, 0.97 ︰ in Fig. 4
0.91, it is consistent with 1 ︰ of theoretical ratio, 1 ︰ 1.
Standard protein enzymolysis step:By molten 8.0 (8M urea) buffer solutions of 0.1M Tris/HCl pH of 250 μ gBSA albumen
In, it is added in the concentration tube of 10KD, 14,000g centrifuge to 30 μ L or so;It is added 100 μ L DTT solution, after mixing, is stored at room temperature
30min;Concentration tube 14,000g is centrifuged into 30min;It is added 100 μ L IAA solution, after mixing, is stored at room temperature 5min;By concentration tube
14,000g centrifuges 30min;100 μ L 0.1M Tris/HCl pH, 8.0 (8M urea) solution, 14000g are added into concentration tube
40min is centrifuged, is repeated twice;160 μ L are added into concentration tube, and dissolved with the ammonium bicarbonate solution of pancreatin, (enzyme and protein ratio are 1 ︰
50), mixing, 37 DEG C of incubation 16h;Collecting pipe is replaced, concentration tube is centrifuged into 40min at 14,000g;The 0.5M of 50 μ L is added
NaCl, by concentration tube 14,000g centrifuges 20min;After being acidified with formic acid (pH is 2~4), removed according to eight desalination method of embodiment
Salt.
Embodiment ten, three scalar quantity analytical standard albumen β-Casein of stable isotope N- phosphorylateds
With embodiment nine, quantitative analysis results such as attached drawing 5, detection altogether quantifies peptide fragment 5, with peptide hydrolysis EAMAPK standards
Protein quantification ratio is 1 ︰, 1.05 ︰ 1.02, is consistent with 1 ︰ of theoretical ratio, 1 ︰ 1.
Embodiment 11, three scalar quantity analytical standard albumen Myoglobin of stable isotope N- phosphorylateds
With embodiment nine, quantitative analysis results such as attached drawing 6, detection altogether quantifies attitude 7, with peptide hydrolysis
For LFTGHPETLEK, standard protein quantitative ratio is 1 ︰, 0.56 ︰ 0.13, is consistent with 1 ︰ of theoretical ratio, 0.5 ︰ 0.17.
Embodiment 12, three scalar quantity analytical standard albumen Lysozyme of stable isotope N- phosphorylateds
With embodiment nine, quantitative analysis results such as attached drawing 7, detection altogether quantifies peptide fragment 9, with peptide hydrolysis GTDVQAWIR
For, standard protein quantitative ratio is 1 ︰, 2.23 ︰ 1.03, is consistent with 1 ︰ of theoretical ratio, 2 ︰ 1.
Embodiment 13, three scalar quantity of stable isotope N- phosphorylateds analysis isotope effect are investigated
By taking lysozyme Lysozyme peptide hydrolysis CELAAAMK as an example, respectively with gently mark-, acceptance of the bid-, mark-corresponding matter again
Lotus ratio, including m/z 583.24, m/z 587.25, m/z 591.26 carry out ion stream extraction, and three mark peptide fragment ion stream
Retention time is in 23.4min or so, as shown in Figure 8.Show diethoxy phosphite ester, the phosphorous acid two of carbon -13 (4) label
Ethyl ester (13C4- DEP-H, 1 compound 13 of table), with carbon -13 (4), oxygen -18 (2) mark diethyl phosphite (13C4 18O2-DEP-
H, 1 compound 14 of table) etc. the stable isotope effects of three mark composite marking reagents do not influence the quantitative analysis of protein.
Albumen-egg in cell under the conditions of embodiment 14, three scalar quantity of stable isotope N- phosphorylateds analysis drug-treated
White interaction
With embodiment nine, only label sample is changed into cell extraction proteolysis peptide fragment sample, the reaction time is by 30min
It is 1.5h to increase.
Quantitative analysis results such as Fig. 9, total identification and CDK9 interaction proteins 145, wherein 127 have three scalar quantities
Information, quantitative rate reach 87% or so.Quantitative analysis is successfully made to the key protein compound and key protein of CDK9, such as
LARP7(1:0.11:0.06)、HEXIM2(1:0.36:0.07)、CDK9(1:0.93:0.9)、Cyclin T1(1:0.74:
0.99)、AFF4(1:0.67:And BRD4 (1 ︰, 1.34 ︰ 1.34) 0.82).
Molecular biology Western blotting verification analyses have been carried out to quantitative result, have shown stable isotope N- phosphorus
Acylated three gauged accuracys, the results are shown in Figure 10.
Cell drug processing method is:It is thin when drug-treated in F1C2 cell inoculations to 150mm Tissue Culture Dish, will be made
Born of the same parents' density about 80%-90%;It respectively takes 6 150mm Tissue Culture Dish respectively, the kinase inhibitor of DMSO, 0.5 μM and 5 μM is added
I-CDK9 handles 8h;Collect cell.
Cell protein extraction step is:Cell lysis buffer (0.1M KCl/20mM Hepes-KOH pH 7.8/
15% (v/v) Glycerol/0.2mM EDTA/1.0%NP 40/0.5mM PMSF/1mM DTT) it is handled, it is transferred to
In the centrifuge tube of 1.5mL, it is placed on rotor and shakes 30min for 4 DEG C;It is centrifuged with the maximum (top) speed of Eppendorf 5417R centrifuges
10min goes to supernatant spare in new centrifuge tube;
It draws in 60 μ L anti-Flag beads (the 60 pure beads of μ L) to the centrifuge tube of 1.5mL, with 1mL Buffer
D0.3 washes beads twice, and centrifugal rotational speed is about 10000rpm 1min, is placed on for use on ice;
It draws in the cell protein extract to the centrifuge tube containing 60 μ L anti-Flag beads after drug-treated, 4
DEG C be incubated 8h;Supernatant is sopped up, first washes beads three times with 1mL buffer solutions D0.15, then is washed 2 times with 1mL Buffer D0.1;It sops up
Whole supernatants is added and contains the 150 μ L buffer solution D0.1 that 1 ︰ 10 dilutes Flag peptide (0.5mg/mL), mixing, on ice
20min is placed, is centrifuged within 30 seconds with same centrifuge 5000-6000rpm, supernatant solution is taken to carry out enzymolysis or Western
Blotting verification analyses.
Solution formula:
Buffer solution D0.15:0.15M KCl/20mM Hepes-KOH (pH 7.8)/15% (v/v) Glycerol/0.2mM
EDTA/0.2%NP 40/0.5mM PMSF/1mM DTT;
Buffer solution D0.1:0.1M KCl/20mM Hepes-KOH (pH 7.8)/15% (v/v) Glycerol/0.2mM
EDTA/0.2%NP 40/0.5mM PMSF/1mM DTT.
Table 1
The present invention is using the commercially available small molecule containing labels such as carbon -13, oxygen -18 or deuteriums -2 as raw material, by halogenated hydrocarbons in silver
Hydrolysis under salt promotion, selectively synthesizes the alcohol of the isotope labellings such as carbon containing -13 and oxygen -18, then pass through base exchange
The phosphite ester labelled reagent of the stable isotope labelings such as carbon containing -13, oxygen -18 or deuterium -2 is obtained by the reaction.It is different by combining
Stable isotope labeling phosphite ester obtains more flux albumen relative quantitative assay such as two marks, three marks, four marks or five marks, each to mark
Molecular weight difference between note is greater than or equal to 3 dalton.Stable isotope N- phosphorylated labelled proteins are quantitative have compared with
Good accuracy, high sensitivity, the interference of No Parity element effect and wide applicability.
Claims (17)
1. the organophosphor protein labeling reagent containing carbon -13, oxygen -18 or deuterium -2 label, it is characterised in that its structural formula is as follows:
Wherein, superscript notation a is 1 or 2;Superscript notation b is 12 or 13;Superscript notation c is 16 or 18;Subfix n be 1,2,
3,4 or 5, and as n=1, labelled reagent structure does not include a=2, the corresponding chemical constitution of b=12, c=16.
2. the organophosphor protein labeling reagent as described in claim 1 containing carbon -13, oxygen -18 or deuterium -2 label, feature exist
In the subfix n be 1 and 2.
3. the preparation side of the organophosphor protein labeling reagent as described in claim 1 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that include the following steps:
Step 1:Carbon containing -13, the preparation of the alcohol of -2 isotope labelling of oxygen -18 or deuterium, is as follows:In 10~60 DEG C of temperature
Spend range in, halogenated alkane is dissolved in organic solvent, the halogenated alkane be halogenated, bromo or alkane iodide, it is described organic
Solvent is material of organic ethers solvent, and water and inorganic silver salt or the mixed solution of simple organic acid silver salt is added, it is stirred to react 6~
The alcohol of -2 isotope labelling of product carbon -13, oxygen -18 or deuterium is obtained after 72h;
Step 2:Carbon containing -13, the preparation of the diphosphites of -2 isotope labelling of oxygen -18 or deuterium is as follows:By carbon-
13, alcohol and oxygen -18, -2 isotope labelling diphenyl phosphite of deuterium of -2 isotope labelling of oxygen -18 or deuterium are anti-in organic solvent
It answers, the alkali of 0.1~10 equivalent is added in reaction, the reaction time is 1~12h, organic solvent is removed after having reacted, through column chromatography point
Analysis obtains product stable isotope labeling phosphite ester.
4. the preparation side of the organophosphor protein labeling reagent as claimed in claim 3 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that in step 1, the halogenated alkane, which is selected from, contains C1~C6Linear chain or branched chain chloro, bromo or idoalkane
Hydrocarbon;C in halogenated hydrocarbons is carbon -12 or carbon -13, H is hydrogen -1 or deuterium -2;Oxygen in water is oxygen -16 or oxygen -18.
5. the preparation side of the organophosphor protein labeling reagent as claimed in claim 4 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that the halogenated alkane is selected from the alkane of bromo or iodo.
6. the preparation side of the organophosphor protein labeling reagent as claimed in claim 3 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that in step 1, the silver salt is the silver salt of inorganic acid, contains C1~C6Carboxylic acid silver salt;Organic solvent is
Ether solvent.
7. the preparation side of the organophosphor protein labeling reagent as claimed in claim 6 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that the silver salt is silver acetate, silver tetrafluoroborate or silver nitrate;Organic solvent is ether or tetrahydrofuran.
8. the preparation side of the organophosphor protein labeling reagent as claimed in claim 3 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that in step 2, the carbon -13, the alcohol of -18 isotope labelling of oxygen and oxygen -18, -2 isotope labelling of deuterium are sub-
The molar ratio of diphenyl phosphate is 1 ︰ 1.
9. the preparation side of the organophosphor protein labeling reagent as claimed in claim 3 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that in step 2, the alkali use inorganic base or organic base, the alkali be selected from sodium hydroxide, potassium hydroxide,
Pyridine, one kind in triethylamine.
10. the preparation side of the organophosphor protein labeling reagent as claimed in claim 9 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that the alkali is selected from pyridine or triethylamine.
11. the preparation side of the organophosphor protein labeling reagent as claimed in claim 3 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that in step 2, the organic solvent uses ether solvent, the organic solvent to be selected from ether, tetrahydrochysene furan
It mutters, one kind in 1,4- dioxane.
12. the preparation of the organophosphor protein labeling reagent as claimed in claim 11 containing carbon -13, oxygen -18 or deuterium -2 label
Method, it is characterised in that the organic solvent is selected from ether or tetrahydrofuran.
13. a kind of quantitative approach of stable isotope phosphorylated labelled protein, it is characterised in that be as follows:
1) it compared with the H-Phosphonate of selection stable isotope labeling carries out molecular weight with unlabelled phosphite ester and matches, selects
Molecular weight difference is taken to be greater than or equal to two mark of stable isotope labeling phosphite ester composition, three marks, four marks, five marks of 3 dalton
Or six scalar quantity reagent set;
2) chemical labeling is carried out respectively to reference polypeptide or protein digestion polypeptide sample under identical label reaction condition, mixed
Label sample is closed, carries out removing salt treatment, obtained labeling polypeptide sample carries out mass spectrum sequencing identification and analysis, and passes through different stabilizations
Isotope labelling characteristic mass difference and ionic strength carry out relative quantification mass spectral analysis to corresponding albumen;
The label reaction of stable isotope phosphorylated has the reaction selectivity of height, only the ends N- to peptide fragment and lysine side-chain ammonia
Base is marked, as follows:
3) under the conditions of ice-water bath, 10~50 μ g synthesis polypeptides or protein digestion polypeptide sample are more than or are waited with molecular weight difference
In the phosphite ester that the carbon stable isotope -13, oxygen -18 or deuterium -2 of 3 dalton mark in molar ratio 1 ︰ (5~10) in water and
The in the mixed solvent of organic solvent is reacted, and the alkali of 0.1~10 equivalent is added in reaction, and the reaction time is 0.5~2h, and reaction is completed
Organic solvent and salt are removed afterwards, carry out mass spectrum quantitative analysis.
14. a kind of quantitative approach of stable isotope phosphorylated labelled protein as claimed in claim 13, it is characterised in that institute
State the one kind of halogenating agent in carbon tetrachloride, iodine/hydrogen peroxide, sodium hypochlorite, hydrogen peroxide/potassium iodide, cupric iodide.
15. a kind of quantitative approach of stable isotope phosphorylated labelled protein as claimed in claim 14, it is characterised in that institute
It states halogenating agent and is selected from sodium hypochlorite or carbon tetrachloride.
16. a kind of quantitative approach of stable isotope phosphorylated labelled protein as claimed in claim 13, it is characterised in that institute
State the one kind of organic solvent in ethyl alcohol, methanol, acetone, tetrahydrofuran, acetonitrile;The alkali be selected from sodium hydroxide, triethylamine,
Sodium bicarbonate, sodium carbonate, potassium hydroxide, tri-n-butylamine, one kind in pyridine.
17. a kind of quantitative approach of stable isotope phosphorylated labelled protein as claimed in claim 16, it is characterised in that institute
It states organic solvent and is selected from acetonitrile or ethyl alcohol;The alkali is selected from triethylamine or sodium bicarbonate.
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Effective date of registration: 20200907 Address after: C3 11, 4th floor, Shunfeng industrial building, No.2 Hongliu Road, Fubao community, Fubao street, Futian District, Shenzhen City, Guangdong Province Patentee after: Guangdong phosphorus based Biotechnology Co., Ltd Address before: Xiamen City, Fujian Province, 361005 South Siming Road No. 422 Patentee before: XIAMEN University |