CN109535223A - A kind of method of double titanium functional magnetic nano material separating and enriching phosphated peptides - Google Patents
A kind of method of double titanium functional magnetic nano material separating and enriching phosphated peptides Download PDFInfo
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- CN109535223A CN109535223A CN201811254427.9A CN201811254427A CN109535223A CN 109535223 A CN109535223 A CN 109535223A CN 201811254427 A CN201811254427 A CN 201811254427A CN 109535223 A CN109535223 A CN 109535223A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 71
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 67
- 239000010936 titanium Substances 0.000 title claims abstract description 65
- 229910052719 titanium Inorganic materials 0.000 title claims abstract description 65
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 27
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 16
- 239000012488 sample solution Substances 0.000 claims abstract description 16
- 238000002955 isolation Methods 0.000 claims abstract description 15
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000908 ammonium hydroxide Substances 0.000 claims abstract description 14
- 239000003480 eluent Substances 0.000 claims abstract description 11
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 4
- 238000002525 ultrasonication Methods 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 25
- 239000008367 deionised water Substances 0.000 claims description 21
- 229910021641 deionized water Inorganic materials 0.000 claims description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 19
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 18
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 16
- 229960004756 ethanol Drugs 0.000 claims description 12
- 235000019441 ethanol Nutrition 0.000 claims description 12
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 9
- 229910052760 oxygen Inorganic materials 0.000 claims description 9
- 239000001301 oxygen Substances 0.000 claims description 9
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 8
- 229910010413 TiO 2 Inorganic materials 0.000 claims description 8
- 229960005305 adenosine Drugs 0.000 claims description 8
- 239000002243 precursor Substances 0.000 claims description 8
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- DCKVFVYPWDKYDN-UHFFFAOYSA-L oxygen(2-);titanium(4+);sulfate Chemical compound [O-2].[Ti+4].[O-]S([O-])(=O)=O DCKVFVYPWDKYDN-UHFFFAOYSA-L 0.000 claims description 7
- 235000011121 sodium hydroxide Nutrition 0.000 claims description 7
- 229910000348 titanium sulfate Inorganic materials 0.000 claims description 7
- 238000001027 hydrothermal synthesis Methods 0.000 claims description 6
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 6
- 238000002604 ultrasonography Methods 0.000 claims description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- XJDNKRIXUMDJCW-UHFFFAOYSA-J titanium tetrachloride Chemical compound Cl[Ti](Cl)(Cl)Cl XJDNKRIXUMDJCW-UHFFFAOYSA-J 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 150000003608 titanium Chemical class 0.000 claims description 4
- QDZRBIRIPNZRSG-UHFFFAOYSA-N titanium nitrate Chemical compound [O-][N+](=O)O[Ti](O[N+]([O-])=O)(O[N+]([O-])=O)O[N+]([O-])=O QDZRBIRIPNZRSG-UHFFFAOYSA-N 0.000 claims description 4
- VXUYXOFXAQZZMF-UHFFFAOYSA-N titanium(IV) isopropoxide Chemical group CC(C)O[Ti](OC(C)C)(OC(C)C)OC(C)C VXUYXOFXAQZZMF-UHFFFAOYSA-N 0.000 claims description 4
- YHWCPXVTRSHPNY-UHFFFAOYSA-N butan-1-olate;titanium(4+) Chemical compound [Ti+4].CCCC[O-].CCCC[O-].CCCC[O-].CCCC[O-] YHWCPXVTRSHPNY-UHFFFAOYSA-N 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 238000010189 synthetic method Methods 0.000 claims description 2
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 230000026731 phosphorylation Effects 0.000 abstract description 18
- 238000006366 phosphorylation reaction Methods 0.000 abstract description 18
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 12
- 108010033276 Peptide Fragments Proteins 0.000 abstract description 9
- 102000007079 Peptide Fragments Human genes 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 239000004408 titanium dioxide Substances 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 230000009822 protein phosphorylation Effects 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 3
- LCKIEQZJEYYRIY-UHFFFAOYSA-N Titanium ion Chemical compound [Ti+4] LCKIEQZJEYYRIY-UHFFFAOYSA-N 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 2
- 238000007306 functionalization reaction Methods 0.000 abstract 1
- 102000011632 Caseins Human genes 0.000 description 15
- 108010076119 Caseins Proteins 0.000 description 15
- 235000021247 β-casein Nutrition 0.000 description 13
- 239000000523 sample Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 8
- 238000000926 separation method Methods 0.000 description 8
- 238000001819 mass spectrum Methods 0.000 description 7
- 235000013336 milk Nutrition 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 210000003296 saliva Anatomy 0.000 description 7
- 229910021645 metal ion Inorganic materials 0.000 description 6
- 230000000865 phosphorylative effect Effects 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000010183 spectrum analysis Methods 0.000 description 6
- 239000003463 adsorbent Substances 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 239000007790 solid phase Substances 0.000 description 5
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 4
- 238000013019 agitation Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000005352 clarification Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000009977 dual effect Effects 0.000 description 4
- 238000004503 metal oxide affinity chromatography Methods 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108010042653 IgA receptor Proteins 0.000 description 3
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 108091005981 phosphorylated proteins Proteins 0.000 description 2
- 229920000137 polyphosphoric acid Polymers 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000004257 Potassium Channel Human genes 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- MQRJBSHKWOFOGF-UHFFFAOYSA-L disodium;carbonate;hydrate Chemical compound O.[Na+].[Na+].[O-]C([O-])=O MQRJBSHKWOFOGF-UHFFFAOYSA-L 0.000 description 1
- IYAMYECUTLASCU-UHFFFAOYSA-L disodium;ethanol;carbonate Chemical compound [Na+].[Na+].CCO.[O-]C([O-])=O IYAMYECUTLASCU-UHFFFAOYSA-L 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- LSDSZZKMMNCFMR-UHFFFAOYSA-N nitric acid titanium Chemical compound [Ti].O[N+]([O-])=O LSDSZZKMMNCFMR-UHFFFAOYSA-N 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 108020001213 potassium channel Proteins 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Compounds Of Iron (AREA)
- Inorganic Compounds Of Heavy Metals (AREA)
Abstract
A kind of method that the present invention proposes double titanium functional magnetic nano material specific isolation enriching phosphated peptides, double titanium functional magnetic nano materials are scattered in Phosphorylated Peptide sample solution by ultrasonication, it is incubated for 10-60 minutes at 37 DEG C, it is sufficiently washed with Phosphorylated Peptide sample solution, using volume ratio 0.5-20% ammonium hydroxide as eluent, it is eluted 10-60 minutes at 37 DEG C, takes eluent point target, be analyzed by mass spectrometry.The present invention obtains good effect to the enrichment of mono-phosphorylated peptide fragment and multi-phosphopeptide section by control magnetic titanium dioxide/titanium ion functionalization material synthesis condition realization, it can be realized Large scale identification protein phosphorylation site in conjunction with chromatographic mass spectrometry technology, to establish the phosphorylation site database of specified disease, data support is provided for numerous disease researches relevant to protein phosphorylation and thinking inspires.
Description
Technical field
The invention belongs to Phosphorylated Peptide method for separating and concentrating, specific more particularly to a kind of double titanium functional magnetic nano materials
The method of specific isolation enriching phosphated peptide.
Background technique
The reversible phosphorylation of protein plays highly important effect in various bioprocess, for cell signal
It is particularly important for conductive process.Research shows that the phosphorylation (mono-phosphorylated or polyphosphoric acid) that protein is different degrees of is in biology
Different effects is played in the process: the mono-phosphorylated activator through frequently as tumor suppressor, and polyphosphoric acidization is then usually negative
The classification for blaming potassium channel adjusts or the adjusting etc. of cell cycle conversion.Therefore, it identifies more fully hereinafter and analysis phosphorylation egg
White matter group is to understanding that protein phosphorylation role in bioprocess is particularly important.In the past few decades, with
Mass spectrographic fast development, the phosphorylating protein based on mass spectrum strategy/peptide fragment analysis means are increasingly by the extensive of people
Concern.However, a major challenge that this analysis method faces is exactly that there is a large amount of non-phosphorylating protein/peptides in actual sample
Duan Yanchong inhibits mass signal.Therefore, before mass spectral analysis using efficiently separating enrichment strategy to more different degrees of
Protein phosphorylation progress assay value obtains people and explores for it.
In the method for separating and concentrating of all phosphated peptide sections, metal oxide affinity chromatography and fixing metal ions parent
It is two kinds of more commonly used enrichment methods with chromatography, mainly relies on phosphate group and metal ion/metal on peptide fragment
Different coordinations is formed between oxide and is realized and sample system separation and concentration.The phosphorus that both methods is enriched with
Being acidified peptide fragment has respective preference, for example fixing metal ions affinity chromatography preference is enriched with more alkaline hydrophilic polyphosphoric acid
Change peptide, and metal oxide affinity chromatography rule compares preference to mono-phosphorylated peptide fragment.Although currently, having developed substep repeatedly
Elution or it is the methods of chromatograph joint used analyzed with the separation for reinforcing to phosphated peptide section, but these methods usually have operation numerous
Trivial, the disadvantages such as flux is low develop both a kind of gathering metal oxide affinity chromatography and fixing metal ions affinity chromatography
The multifunction nano material of advantage analyzes extensive phosphorylation proteomics particularly important.
Summary of the invention
It is an object of the invention to propose a kind of double titanium functional magnetic nano material specific isolation enriching phosphated peptides
Method, synthesized double titanium functional magnetic nano materials with strong magnetic responsiveness for the first time and be used for phosphated peptide section
In isolating and purifying.Under the action of externally-applied magnetic field, the double titanium functional magnetic nano materials for capturing phosphated peptide section can be fast
Fast ground is separated from complex sample solution, is made entirely to be enriched with, be greatly shortened the time required to elution process.Meanwhile having both
The peptide of fixing metal ions affinity chromatography and this material of metal oxide affinity chromatography advantage to different degrees of phosphorylation
Duan Jun has good concentration effect.
A kind of method of double titanium functional magnetic nano material specific isolation enriching phosphated peptides provided by the invention, tool
Steps are as follows for body, disperses double titanium functional magnetic nano materials in Phosphorylated Peptide sample solution by ultrasonication, at 37 DEG C
It is incubated for 10-60 minutes, is sufficiently washed with Phosphorylated Peptide sample solution, using volume ratio 0.5-20% ammonium hydroxide as eluent, at 37 DEG C
Elution 10-60 minutes, takes eluent point target, is analyzed by mass spectrometry.
In the present invention, the synthetic method of double titanium functional magnetic nano materials, the specific steps are as follows:
(1) ferric trichloride is dissolved in ethylene glycol, after completely dissolution, anhydrous sodium acetate is added, turns after ultrasound is sufficiently stirred
It moves in hydrothermal reaction kettle, is heated 8-16 hours at 100-350 DEG C, after being cooled to room temperature, respectively with deionized water and anhydrous
Ethyl alcohol sufficiently washs products therefrom, is dried in vacuo at 50-100 DEG C；
(2) step (1) products therefrom is dispersed in solvent, TiO 2 precursor is added, gained mixed solution is existed
20-80 DEG C is stirred to react 12-48 hours, is sufficiently washed with deionized water and dehydrated alcohol respectively；
(3) adenosine triphyosphate disodium salt is dissolved in basic solvent, is stirred 5-30 minutes at 0-20 DEG C, is added
γ-glycidyl ether oxygen propyl trimethoxy silicane is stirred to react 6-18 hours at 10-80 DEG C；
(4) step (3) acquired solution is adjusted with acid pH value between 3-7, step (2) products therefrom is added, 50-95 DEG C it
Between stir 1-6 hours, sufficiently washed with deionized water and dehydrated alcohol；
(5) step (4) products therefrom is dispersed in the solvent containing titanium salt, is reacted 6-24 hours at 20-80 DEG C, gained
Product is dried in vacuo after sufficiently being washed with deionized water at 40-75 DEG C to get target product.
In the present invention, solvent in step (2) be one of methanol, ethyl alcohol or ethyl alcohol/deionized water mixed solution or
It is several.
In the present invention, TiO 2 precursor is tetraisopropyl titanate, tetrabutyl titanate, titanium sulfate or four in step (2)
One or more of titanium chloride.
In the present invention, it is additionally added in sodium hydroxide, sodium carbonate or ammonium hydroxide before TiO 2 precursor is added in step (2)
One or more.
In the present invention, the mass ratio of step (1) products therefrom and TiO 2 precursor is 30:1-15:1 in step (2).
In the present invention, step (3) neutral and alkali solvent is one of sodium hydrate aqueous solution, aqueous sodium carbonate or ammonium hydroxide
Or it is several.
In the present invention, γ-glycidyl ether oxygen propyl trimethoxy silicane and adenosine triphyosphate two in step (3)
The mass ratio of sodium salt is 1:5-1:1.
In the present invention, acid is one or more of concentrated hydrochloric acid, sulfuric acid or nitric acid in step (4).
In the present invention, titanium salt is one or more of titanium sulfate, titanium tetrachloride or Titanium Nitrate in step (5).
The method of specific isolation enriching phosphated peptide of the present invention has the advantages that
1. pair titanium functional magnetic nano material has good magnetic responsiveness, there is strong interaction with Phosphorylated Peptide, it can
With sensitiveer, more efficiently separating and enriching phosphated peptide.
2. the titanium dioxide and titanium ion in pair titanium functional magnetic nano material are combined with beneficial to different degrees of phosphorylation
Peptide peptide fragment capture, show the material to the good accumulation ability of Phosphorylated Peptide in complicated biological sample.
3. pair titanium functional magnetic Application of micron is in protein post-translational modification research, by combining metal oxygen
It the advantages of both compound affinity chromatography and fixing metal ions affinity chromatography, can be to this posttranslational modification of Phosphorylated Peptide
Peptide fragment realizes depth covering, can be with Large scale identification phosphorylating protein and determining phosphorylation site in conjunction with nano-LC MS/MS.
Detailed description of the invention
Fig. 1 is the electron scanning micrograph of double titanium functional magnetic nano materials of embodiment 1；
Fig. 2 is the transmission electron microscope photo of double titanium functional magnetic nano materials of embodiment 1；
Fig. 3 is the x-ray photoelectron spectroscopy of double titanium functional magnetic nano materials of embodiment 1；
Fig. 4 is enrichment of double titanium functional magnetic nano materials to mono-phosphorylated mark peptide and dual phosphorylation mark peptide in embodiment 2
Mass spectrogram.Figure A is the mass spectrogram for the phosphorylation mark peptide being enriched with without double titanium functional magnetic nano materials, and figure B is through magnetism two
The mass spectrogram of phosphorylation mark peptide after titanium dioxide nano material enrichment, figure C are after double titanium functional magnetic nano materials are enriched with
The mass spectrogram of phosphorylation mark peptide；
Fig. 5 is double titanium functional magnetic nano materials of embodiment 3 to phosphorus in standard phosphorylation albumen β-casein enzymolysis product
It is acidified the mass spectrogram of peptide separation and concentration.Scheme the mass spectrogram that A is not enriched β-casein Phosphorylated Peptide, figure B is magnetic dioxy
Change the mass spectrogram of Phosphorylated Peptide in titanium nano material enrichment β-casein enzymolysis liquid, figure C is double titanium functional magnetic nano materials
It is enriched with the mass spectrogram of Phosphorylated Peptide in β-casein enzymolysis liquid；
Fig. 6 is choosing of the double titanium functional magnetic nano materials of embodiment 4 to phosphorylation peptide gathering in β-casein enzymolysis product
Select mass spectra.Scheme the A mass spectrogram not enriched for the mixed enzymolysis product of β-casein and non-phosphorylating protein B SA, figure B is
Phosphorylated Peptide in β-casein enzymolysis product is enriched with using double titanium functional magnetic nano materials when the two mass ratio is 1:100
Mass spectrogram, figure C, which both is mass ratio, is enriched with β-casein enzymolysis products using double titanium functional magnetic nano materials when being 1:400
The mass spectrogram of middle Phosphorylated Peptide, figure D, which both is mass ratio, is enriched with β-using double titanium functional magnetic nano materials when being 1:800
The mass spectrogram of Phosphorylated Peptide in casein enzymolysis product；
Fig. 7 is that double titanium functional magnetic nano materials of embodiment 5 are enriched with the mass spectrogram of Phosphorylated Peptide in saliva.Scheme A be without
The mass spectrogram of phosphated peptide section in saliva is directly analyzed in enrichment, and figure B is that magnetic titanium dioxide nano material is enriched with phosphoric acid in saliva
Change the mass spectrogram of peptide, figure C is the mass spectrogram that double titanium functional magnetic nano materials are enriched with Phosphorylated Peptide in saliva；
Fig. 8 is that double titanium functional magnetic nano materials of embodiment 6 are enriched with the mass spectrogram of mono-/multi- Phosphorylated Peptide in milk.Scheming A is
The not enriched mass spectrogram for directly analyzing phosphated peptide section in milk, figure B are that magnetic titanium dioxide nano material is enriched in milk
The mass spectrogram of Phosphorylated Peptide, figure C are the mass spectrogram that double titanium functional magnetic nano materials are enriched with Phosphorylated Peptide in milk.
Specific embodiment
The present invention is realized using the interaction of double titanium functional magnetic nano materials and Phosphorylated Peptide in complex sample
The separation and concentration of Phosphorylated Peptide, specific embodiment introduced below.
Embodiment 1: the synthesis of hydrophilic magnetic mesoporous titanium dioxide material
(1) by 1.35 g FeCl3·6H2After magnetic agitation to solution clarification, 3.6 g are added in 75 mL ethylene glycol in O
NaAc, then be transferred in hydrothermal reaction kettle after ultrasound is sufficiently stirred, it heats 16 hours at 200 DEG C, after reaction kettle is cooling, uses
Deionized water and ethyl alcohol difference washed product three times, are dried in vacuo at 50 DEG C；
(2) 80 mg ultrasonic disperse of products therefrom in (1) is added dropwise 1.6 in the 200 ml ethyl alcohol containing 0.72 ml ammonium hydroxide dropwise
Gained mixed solution is reacted at 45 DEG C 24 h, is sufficiently washed with deionized water and dehydrated alcohol by ml tetrabutyl titanate；
(3) 2.5 g adenosine triphyosphate disodium salts are dissolved in 2 M sodium carbonate liquors, stir 10 at 0 DEG C
Min is added 0.8 ml γ-glycidyl ether oxygen propyl trimethoxy silicane, is stirred to react 12 hours at 65 DEG C；
(4) step (3) acquired solution is adjusted into pH value to 6 using concentrated hydrochloric acid, step (2) products therefrom is added, is stirred at 95 DEG C
It mixes 2 hours, is sufficiently washed with deionized water；
(5) step (4) products therefrom is dispersed in titanium sulfate solution, is reacted 2 hours at 25 DEG C, products therefrom spend from
Sub- water is dried in vacuo after sufficiently washing at 40 DEG C to get double titanium functional magnetic nano materials.
Double titanium functional magnetic nano materials obtained are detected with scanning electron microscope, testing conditions are: 15kV work
Make under voltage, dry material taken and is pasted on insulating tape on a small quantity, through metal spraying, vacuumize after, scanned under 8 microns of scale bars
Electron microscope observation, testing result are as shown in Figure 1.
Double titanium functional magnetic nano materials obtained are detected with transmission electron microscope, testing conditions are: 200kV work
Make under voltage, material dry on a small quantity is taken to be dispersed in dehydrated alcohol and infiltrated micro-grid net with mixed liquor, is inserted after dry
Enter instrument to vacuumize, projection electron microscope figure is observed under 100 nanometer-scale rulers, testing result is as shown in Figure 2.
Fig. 3 is the x-ray photoelectron spectroscopy of double titanium functional magnetic nano materials.
Embodiment 2: double titanium functional magnetic nano materials that embodiment 1 obtains are used for monophosphate as solid-phase adsorbent
Change the enrichment of mark peptide and dual phosphorylation mark peptide
(1) the mono-phosphorylated mark peptide of 1 mg or dual phosphorylation mark peptide the preparation of sample: are dissolved in 1 ml, 50 mM NH4HCO3It is molten
In liquid；
The bis- titanium functional magnetic nano material ultrasonic disperses of (2) 100 μ g 100 μ L contain 0.1 μ L step (1) acceptance of the bid peptide it is molten
In the sample solution of liquid, 37 DEG C of 20 min of incubation.Sample is rinsed three times with 100 μ L sample solutions.It is eluted with 10 μ L, 0.4 M ammonium hydroxide
30 min；
(3) mass spectral analysis: taking eluent and 1 μ L matrix solution point target in 1 μ L step (2), and mass spectrum point is carried out after natural drying
Analysis, mass spectrogram are as shown in Figure 4.
Analyze result: as seen from Figure 4, mono-phosphorylated mark peptide and dual phosphorylation mark the peptide signal peak after the enrichment of this material
Intensity is greatly improved.
Embodiment 3: double titanium functional magnetic nano materials that embodiment 1 obtains are used for phosphorylation as solid-phase adsorbent
The separation and concentration of Phosphorylated Peptide in albumen β-casein
(1) preparation of sample: 1 mg β-casein is in 50 mM NH4HCO337 DEG C of 16 h of enzymatic hydrolysis in solution；
The bis- titanium functional magnetic nano material ultrasonic disperses of (2) 100 μ g contain 100 fmol/ μ L step (1) β-in 100 μ L
In the sample solution of casein enzymolysis product, 37 DEG C of 20 min of incubation.Sample is rinsed three times with 100 μ L sample solutions.With 10 μ L
0.4 M ammonium hydroxide elutes 30 min；
(3) mass spectral analysis: taking eluent and 1 μ L matrix solution point target in 1 μ L step (2), and mass spectrum point is carried out after natural drying
Analysis, mass spectrogram are as shown in Figure 5.
Analyze result: as seen from Figure 5, the Phosphorylated Peptide from phosphorylated protein β-casein enzymolysis product is by this
Material is captured, and interference caused by non-phosphorylated peptide is substantially removed in stoste.
Embodiment 4: it is used to simulate using double titanium functional magnetic nano materials that embodiment 1 obtains as solid-phase adsorbent multiple
The separation and concentration of Phosphorylated Peptide in heterocycle border
(1) by mixed enzymolysis the product 1:100 in mass ratio, 1:400 and 1:800 of β-casein and non-phosphorylating protein B SA points
It is not configured to the simulation complex sample system of complexity rising；
The bis- titanium functional magnetic nano material ultrasonic disperses of (2) 100 μ g contain the loading that step (1) prepares solution in 100 μ L
In liquid, 37 DEG C of 30 min of incubation.Sample is rinsed three times with 200 μ L sample solutions.30 min are eluted with 8 μ L, 0.8 M ammonium hydroxide；
(3) mass spectral analysis: taking eluent and 1 μ L matrix solution point target in 1 μ L step (1), and mass spectrum point is carried out after natural drying
Analysis, mass spectrogram are as shown in Figure 6.
Analyze result: as seen from Figure 6, the impurity peaks from non-phosphorylating protein B SA are enriched with by this material
It is removed afterwards, and the Phosphorylated Peptide in phosphorylated protein β-casein enzymolysis product is captured by this material, signal peak strength
Substantially enhanced.
Embodiment 5: double titanium functional magnetic nano materials that embodiment 1 obtains are used in saliva as solid-phase adsorbent
The separation and concentration of Phosphorylated Peptide
The bis- titanium functional magnetic nano material ultrasonic disperses of (1) 100 μ g are in the sample solution that 100 μ L contain 5 μ L salivas, and 37
DEG C be incubated for 30 min.Sample is rinsed three times with 100 μ L sample solutions.30 min are eluted with 8 μ L, 0.4 M ammonium hydroxide；
(2) mass spectral analysis: taking eluent and 1 μ L matrix solution point target in 1 μ L step (1), and mass spectrum point is carried out after natural drying
Analysis, mass spectrogram are as shown in Figure 7.
Analyze result: as seen from Figure 7, this material can still be shown in the complex environment of saliva to mono-/multi- phosphorus
It is acidified the good concentration effect of peptide fragment.
Embodiment 6: double titanium functional magnetic nano materials that embodiment 1 obtains are used in milk as solid-phase adsorbent
The separation and concentration of Phosphorylated Peptide
The bis- titanium functional magnetic nano material ultrasonic disperses of (1) 100 μ g are in the sample solution that 100 μ L contain 5 μ L milk, and 37
DEG C be incubated for 30 min.Sample is rinsed three times with 100 μ L sample solutions.30 min are eluted with 8 μ L, 0.4 M ammonium hydroxide；
(2) mass spectral analysis: taking eluent and 1 μ L matrix solution point target in 1 μ L step (1), and mass spectrum point is carried out after natural drying
Analysis, mass spectrogram are as shown in Figure 8.
Analyze result: as seen from Figure 8, this material can still be shown in the complex environment of milk to mono-/multi- phosphorus
It is acidified the good concentration effect of peptide fragment.
Embodiment 7: the synthesis of double titanium functional magnetic nano materials
(1) by 1.35 g FeCl3·6H2After magnetic agitation to solution clarification, 3.6 g are added in 75 mL ethylene glycol in O
NaAc, then be transferred in hydrothermal reaction kettle after ultrasound is sufficiently stirred, it heats 12 hours at 180 DEG C, after reaction kettle is cooling, uses
Deionized water and ethyl alcohol difference washed product three times, are dried in vacuo at 40 DEG C；
(2) by 50 mg ultrasonic disperse of products therefrom in (1) in 500 ml ethyl alcohol, 1.5 ml metatitanic acid, four isopropyl is added dropwise dropwise
Gained mixed solution is reacted at 45 DEG C 20 h, is sufficiently washed with deionized water and dehydrated alcohol by ester；
(3) 2 g adenosine triphyosphate disodium salts are dissolved in sodium hydroxide solution, 30 min is stirred at 0 DEG C, are added
Enter 1 ml γ-glycidyl ether oxygen propyl trimethoxy silicane, is stirred to react 16 hours at 65 DEG C；
(4) step (3) acquired solution is adjusted into pH value to 5 using concentrated hydrochloric acid, step (2) products therefrom is added, stirs 3 at 90 DEG C
Hour, it is sufficiently washed with deionized water；
(5) step (4) products therefrom is dispersed in titanium sulfate solution, is reacted 2 hours at 45 DEG C, products therefrom spend from
Sub- water is dried in vacuo after sufficiently washing at 50 DEG C to get double titanium functional magnetic nano materials.
Embodiment 8: the synthesis of double titanium functional magnetic nano materials
(1) by 1.5 g FeCl3·6H2After magnetic agitation to solution clarification, 3.6 g are added in 80 mL ethylene glycol in O
NaAc, then be transferred in hydrothermal reaction kettle after ultrasound is sufficiently stirred, it is heated 16 hours at 200 DEG C, after reaction kettle is cooling,
Three times with deionized water and ethyl alcohol difference washed product, it is dried in vacuo at 40 DEG C；
(2) it by 50 mg ultrasonic disperse of products therefrom in (1) in the 200 ml methanol containing 0.05 mmol sodium hydroxide, is added
Acquired solution is reacted at 30 DEG C 20 h, is sufficiently washed with deionized water and dehydrated alcohol by 0.5 mg titanium sulfate；
(3) 5 g adenosine triphyosphate disodium salts are dissolved in 0.1M ammonium hydroxide, 30 min is stirred at 0 DEG C, be added 0.7
Ml γ-glycidyl ether oxygen propyl trimethoxy silicane is stirred to react 16 hours at 65 DEG C；
(4) step (2) products therefrom is added using 5M sulphur acid for adjusting pH value to 5 in step (3) acquired solution, is stirred at 90 DEG C
It 3 hours, is sufficiently washed with deionized water；
(5) step (4) products therefrom is dispersed in titanium tetrachloride solution, is reacted 2 hours at 45 DEG C, products therefrom is spent
Ionized water is dried in vacuo after sufficiently washing at 50 DEG C to get double titanium functional magnetic nano materials.
Embodiment 9: the synthesis of double titanium functional magnetic nano materials
(1) by 1 g FeCl3·6H2After magnetic agitation to solution clarification, 3.2 g NaAc are added in 60 mL ethylene glycol in O,
Be transferred in hydrothermal reaction kettle after ultrasound is sufficiently stirred, heated 12 hours at 200 DEG C again, after reaction kettle is cooling, spend from
Sub- water and ethyl alcohol difference washed product three times, are dried in vacuo at 40 DEG C；
(2) by 10 mg ultrasonic disperse of products therefrom in (1) in containing 0.5 g sodium carbonate ethanol/water mixed liquor (volume ratio 95:
5) it in, is added dropwise in 0.5 ml titanium tetrachloride solution dropwise, gained mixed solution is reacted into 30 h at 25 DEG C, uses deionized water
It is sufficiently washed with dehydrated alcohol；
(3) 2 g adenosine triphyosphate disodium salts are dissolved in sodium hydroxide solution, 30 min is stirred at 0 DEG C, are added
Enter 1 ml γ-glycidyl ether oxygen propyl trimethoxy silicane, is stirred to react 16 hours at 65 DEG C；
(4) step (2) products therefrom is added using 5M nitre acid for adjusting pH value to 5 in step (3) acquired solution, stirs 3 at 90 DEG C
Hour, it is sufficiently washed with deionized water；
(5) step (4) products therefrom is dispersed in nitric acid titanium solution, is reacted 2 hours at 20 DEG C, products therefrom spend from
Sub- water is dried in vacuo after sufficiently washing at 50 DEG C to get double titanium functional magnetic nano materials.
Claims (10)
1. a kind of method of double titanium functional magnetic nano material specific isolation enriching phosphated peptides, it is characterised in that specific step
It is rapid as follows, it disperses double titanium functional magnetic nano materials in Phosphorylated Peptide sample solution by ultrasonication, is incubated at 37 DEG C
It 10-60 minutes, is sufficiently washed with Phosphorylated Peptide sample solution, using volume ratio 0.5-20% ammonium hydroxide as eluent, is eluted at 37 DEG C
10-60 minutes, eluent point target is taken, is analyzed by mass spectrometry.
2. a kind of method of double titanium functional magnetic nano material specific isolation enriching phosphated peptides, which is characterized in that described
The synthetic method of double titanium functional magnetic nano materials, the specific steps are as follows:
(1) ferric trichloride is dissolved in ethylene glycol, after completely dissolution, anhydrous sodium acetate is added, turns after ultrasound is sufficiently stirred
It moves in hydrothermal reaction kettle, is heated 8-16 hours at 100-350 DEG C, after being cooled to room temperature, respectively with deionized water and anhydrous
Ethyl alcohol sufficiently washs products therefrom, is dried in vacuo at 50-100 DEG C；
(2) step (1) products therefrom is dispersed in solvent, TiO 2 precursor is added, gained mixed solution is existed
20-80 DEG C is stirred to react 12-48 hours, is sufficiently washed with deionized water and dehydrated alcohol respectively；
(3) adenosine triphyosphate disodium salt is dissolved in basic solvent, is stirred 5-30 minutes at 0-20 DEG C, is added
γ-glycidyl ether oxygen propyl trimethoxy silicane is stirred to react 6-18 hours at 10-80 DEG C；
(4) step (3) acquired solution is adjusted with acid pH value between 3-7, step (2) products therefrom is added, 50-95 DEG C it
Between stir 1-6 hours, sufficiently washed with deionized water and dehydrated alcohol；
(5) step (4) products therefrom is dispersed in the solvent containing titanium salt, is reacted 6-24 hours at 20-80 DEG C, gained
Product is dried in vacuo after sufficiently being washed with deionized water at 40-75 DEG C to get target product.
3. a kind of side of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that the solvent in step (2) is one or more of methanol, ethyl alcohol or ethyl alcohol/deionized water mixed solution.
4. a kind of side of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that TiO 2 precursor is tetraisopropyl titanate, tetrabutyl titanate, titanium sulfate or four chlorinations in step (2)
One or more of titanium.
5. a kind of side of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that one be additionally added before TiO 2 precursor is added in step (2) in sodium hydroxide, sodium carbonate or ammonium hydroxide
Kind is several.
6. a kind of side of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that the mass ratio of step (1) products therefrom and TiO 2 precursor is 30:1-15:1 in step (2).
7. a kind of side of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that step (3) neutral and alkali solvent is one of sodium hydrate aqueous solution, aqueous sodium carbonate or ammonium hydroxide or several
Kind.
8. a kind of side of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that γ-glycidyl ether oxygen propyl trimethoxy silicane and adenosine triphyosphate disodium salt in step (3)
Mass ratio be 1:5-1:1.
9. a kind of side of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that acid is one or more of concentrated hydrochloric acid, sulfuric acid or nitric acid in step (4).
10. a kind of double titanium functional magnetic nano material specific isolation enriching phosphated peptides according to claim 2
Method, it is characterised in that titanium salt is one or more of titanium sulfate, titanium tetrachloride or Titanium Nitrate in step (5).
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