CN114011376B - Metal oxidation affinity chromatography magnetic mesoporous nano material, preparation method and application - Google Patents
Metal oxidation affinity chromatography magnetic mesoporous nano material, preparation method and application Download PDFInfo
- Publication number
- CN114011376B CN114011376B CN202111282363.5A CN202111282363A CN114011376B CN 114011376 B CN114011376 B CN 114011376B CN 202111282363 A CN202111282363 A CN 202111282363A CN 114011376 B CN114011376 B CN 114011376B
- Authority
- CN
- China
- Prior art keywords
- affinity chromatography
- product
- phosphorylated
- magnetic mesoporous
- metal oxidation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 50
- 238000001042 affinity chromatography Methods 0.000 title claims abstract description 25
- 229910052751 metal Inorganic materials 0.000 title claims abstract description 25
- 239000002184 metal Substances 0.000 title claims abstract description 25
- 230000003647 oxidation Effects 0.000 title claims abstract description 25
- 238000007254 oxidation reaction Methods 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 30
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 108010026552 Proteome Proteins 0.000 claims abstract description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 12
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 8
- 229910010413 TiO 2 Inorganic materials 0.000 claims abstract description 7
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 54
- 239000008367 deionised water Substances 0.000 claims description 35
- 229910021641 deionized water Inorganic materials 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 30
- 238000005406 washing Methods 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 21
- 238000000926 separation method Methods 0.000 claims description 19
- 238000003756 stirring Methods 0.000 claims description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 17
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 15
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 14
- 238000004503 metal oxide affinity chromatography Methods 0.000 claims description 13
- 238000001291 vacuum drying Methods 0.000 claims description 13
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 12
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
- 239000004408 titanium dioxide Substances 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 10
- 238000004949 mass spectrometry Methods 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 102000004142 Trypsin Human genes 0.000 claims description 9
- 108090000631 Trypsin Proteins 0.000 claims description 9
- 239000012588 trypsin Substances 0.000 claims description 9
- 239000006185 dispersion Substances 0.000 claims description 8
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 239000002243 precursor Substances 0.000 claims description 8
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 7
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 7
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 7
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 7
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- VXUYXOFXAQZZMF-UHFFFAOYSA-N titanium(IV) isopropoxide Chemical compound CC(C)O[Ti](OC(C)C)(OC(C)C)OC(C)C VXUYXOFXAQZZMF-UHFFFAOYSA-N 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 4
- 239000005695 Ammonium acetate Substances 0.000 claims description 4
- 229940043376 ammonium acetate Drugs 0.000 claims description 4
- 235000019257 ammonium acetate Nutrition 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- HQFCOGRKGVGYBB-UHFFFAOYSA-N ethanol;nitric acid Chemical compound CCO.O[N+]([O-])=O HQFCOGRKGVGYBB-UHFFFAOYSA-N 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 3
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 3
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 3
- 239000001099 ammonium carbonate Substances 0.000 claims description 3
- 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 3
- 239000012460 protein solution Substances 0.000 claims description 3
- 230000017854 proteolysis Effects 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 229910000348 titanium sulfate Inorganic materials 0.000 claims description 3
- 230000005389 magnetism Effects 0.000 claims description 2
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 2
- XJDNKRIXUMDJCW-UHFFFAOYSA-J titanium tetrachloride Chemical compound Cl[Ti](Cl)(Cl)Cl XJDNKRIXUMDJCW-UHFFFAOYSA-J 0.000 claims description 2
- 238000001819 mass spectrum Methods 0.000 abstract description 15
- 108091005981 phosphorylated proteins Proteins 0.000 abstract description 9
- 238000002372 labelling Methods 0.000 abstract description 3
- 239000002105 nanoparticle Substances 0.000 abstract description 3
- 238000003980 solgel method Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 39
- 102000011632 Caseins Human genes 0.000 description 34
- 235000021247 β-casein Nutrition 0.000 description 32
- 108010076119 Caseins Proteins 0.000 description 29
- 239000000463 material Substances 0.000 description 18
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 16
- 229940098773 bovine serum albumin Drugs 0.000 description 16
- 230000002255 enzymatic effect Effects 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 12
- 239000000413 hydrolysate Substances 0.000 description 10
- 239000010410 layer Substances 0.000 description 8
- 108010033276 Peptide Fragments Proteins 0.000 description 6
- 102000007079 Peptide Fragments Human genes 0.000 description 6
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 6
- 101000741065 Bos taurus Beta-casein Proteins 0.000 description 5
- 108010001441 Phosphopeptides Proteins 0.000 description 4
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 3
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 229910017604 nitric acid Inorganic materials 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000009210 therapy by ultrasound Methods 0.000 description 3
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- VHJLVAABSRFDPM-IMJSIDKUSA-N L-1,4-dithiothreitol Chemical compound SC[C@H](O)[C@@H](O)CS VHJLVAABSRFDPM-IMJSIDKUSA-N 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 229910002808 Si–O–Si Inorganic materials 0.000 description 1
- 229910003077 Ti−O Inorganic materials 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 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 description 1
- 239000005018 casein Substances 0.000 description 1
- 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 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011247 coating layer Substances 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000002336 sorption--desorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28019—Spherical, ellipsoidal or cylindrical
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
- B01J20/28057—Surface area, e.g. B.E.T specific surface area
- B01J20/28061—Surface area, e.g. B.E.T specific surface area being in the range 100-500 m2/g
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
- B01J20/28078—Pore diameter
- B01J20/28083—Pore diameter being in the range 2-50 nm, i.e. mesopores
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Abstract
The invention discloses a metal oxidation affinity chromatography magnetic mesoporous nano material, a preparation method and application thereof, wherein the nano material is Fe 3 O 4 @mSiO 2 @TiO 2 The method is characterized in that a mesoporous silica layer with a proper aperture is introduced on the surface of the magnetic ferroferric oxide nanoparticle by a sol-gel method, and then the surface of the magnetic mesoporous nanomaterial is coated with the titania layer, so that the whole process of enzymolysis of protein and enrichment of phosphorylated peptide in the same system is realized, and a quantitative analysis method of a non-labeled phosphorylated proteome is established by combining mass spectrum, and can be used for quantifying phosphorylated protein in a complex sample. The metal oxidation affinity chromatography magnetic mesoporous nanomaterial has the characteristics of large surface area, low detection limit and high specificity for capturing phosphorylated peptides, can be combined with a non-labeling quantitative method, and can be used for quantitatively researching phosphorylated proteins, so that the metal oxidation affinity chromatography magnetic mesoporous nanomaterial has great potential in phosphoproteome research.
Description
Technical Field
The invention belongs to a method for separating, enriching and quantifying phosphorylated peptides, and particularly relates to a metal oxidation affinity chromatography magnetic mesoporous nanomaterial and a preparation method thereof as well as a method for quantifying phosphorylated proteome by combining mass spectrum.
Background
Protein phosphorylation involves a number of biological processes including signaling, cell differentiation, and regulation of gene expression. Phosphorylated proteomics allows qualitative and quantitative studies of phosphorylated proteins by means of biological mass spectrometry techniques, so that phosphorylated proteins are reliably identified and quantified. However, the low stoichiometric ratio of phosphorylated proteins and the low ionization efficiency make direct mass spectrometry studies of phosphorylated proteomes a great challenge. Quantitative proteomics techniques based on mass spectrometry include both label-based and unlabeled methods. The unlabeled quantification has good application prospects because no expensive labeling reagent is needed, applicable sample types are large, and applicable sample amounts are large. However, in the research of phosphorylated proteome, the method is influenced by factors such as a plurality of phosphorylated peptide enrichment steps, and the like, and is easy to introduce operation errors.
Therefore, it is necessary to develop a metal oxide affinity chromatography magnetic mesoporous nanomaterial capable of integrating proteolysis and phosphopeptide enrichment, and to establish a non-labeled quantitative analysis method of a phosphorylated proteome with application value on the basis of the metal oxide affinity chromatography magnetic mesoporous nanomaterial.
Disclosure of Invention
The invention aims to: the first object of the invention is to provide a metal oxidation affinity chromatography magnetic mesoporous nanomaterial which simultaneously realizes protein enzymolysis and peptide enrichment; a second object of the present invention is to provide a method for preparing the above nanomaterial; a third object of the present invention is to provide the use of the above-mentioned nanomaterials in non-labeled quantitative analysis of phosphorylated proteomes.
The technical scheme is as follows: the invention relates to a metal oxidation affinity chromatography magnetic mesoporous nanomaterial, which is Fe 3 O 4 @mSiO 2 @TiO 2 Has a spherical structure; the core of the spherical structure is ferroferric oxide for providing magnetism, the outer layer of the ferroferric oxide is coated with a mesoporous silica layer for proteolysis, and the outer part of the mesoporous silica layer is coated with a titanium dioxide layer for enrichment of phosphorylated peptides.
The invention also provides a preparation method of the metal oxidation affinity chromatography magnetic mesoporous nanomaterial, which comprises the following steps:
fully dissolving ferric trichloride, ammonium acetate and sodium citrate in ethylene glycol, stirring at 100-170 ℃, transferring the formed black reaction liquid to a high-pressure reaction kettle, placing the reaction kettle in a baking oven at 100-200 ℃ for reaction for 8-16 hours, cooling to room temperature, hysteresis separation, collecting a product, washing with absolute ethyl alcohol and deionized water, and then vacuum drying at 50-100 ℃;
uniformly dispersing the product obtained in the step (1) in absolute ethyl alcohol by ultrasonic, adding deionized water and ammonia water, uniformly mixing the solution by ultrasonic, adding tetraethyl orthosilicate, stirring for 5-20 hours, hysteresis separation, collecting the product, washing the product with absolute ethyl alcohol and deionized water, and then drying the product in vacuum at 50-100 ℃;
uniformly dispersing the product obtained in the step (2), cetyltrimethylammonium chloride and triethanolamine in deionized water by ultrasonic, stirring for 1-5 hours at 50-100 ℃, adding a cyclohexane mixed solution of tetraethyl orthosilicate and isopropanol, stirring for 5-20 hours, hysteresis separating and collecting the product, washing with absolute ethyl alcohol and deionized water, refluxing in a nitric acid ethanol solution at 50-100 ℃, washing with absolute ethyl alcohol and deionized water again, and vacuum drying at 50-100 ℃;
step (4), uniformly dispersing the product obtained in the step (3) in a solvent by ultrasonic, adding a titanium dioxide precursor, stirring at 20-80 ℃ for reaction for 5-10 hours, performing hysteresis separation, collecting the product, washing with deionized water and ethanol, and then drying in vacuum; thus obtaining the nano material.
Further, in the step (1), the mass ratio of the ferric trichloride, the ammonium acetate and the sodium citrate is as follows: (2-4): (3-10): (1-1.5).
Further, in the step (2), the mass-volume ratio of the product obtained in the step (1) to the absolute ethyl alcohol is 1:1000; the volume ratio of the tetraethoxysilane to the dispersion liquid is 1: (300-400).
Further, in the step (3), the mass ratio of the product obtained in the step (2), cetyltrimethylammonium chloride and triethanolamine is 1:30: (1-2); the cyclohexane mixed solution comprises 5 weight percent of tetraethyl orthosilicate and 2.5 weight percent of isopropanol; and the reflux times of the product in the nitric acid ethanol solution are 3-5 times.
Further, in the step (4), the mass ratio of the product obtained in the step (3) to the titanium dioxide precursor is (10-20): 1, a step of; wherein the titanium dioxide precursor is one or a mixture of more of tetraisopropyl titanate, n-butyl titanate, titanium sulfate or titanium tetrachloride; before adding the titanium dioxide precursor, one or more of sodium hydroxide, sodium carbonate or ammonia water are also added.
The invention also protects the application of the metal oxidation affinity chromatography magnetic mesoporous nanomaterial in quantitative analysis of unlabeled phosphorylated proteome, and the specific steps of quantitative analysis are as follows:
step one, placing a metal oxidation affinity chromatography magnetic mesoporous nano material in a solvent to prepare a dispersion liquid;
step two, adding a protein solution into the dispersion, mixing, adding trypsin, and incubating at 37 ℃; finally adding trifluoroacetic acid and acetonitrile, and carrying out oscillation incubation at room temperature;
and thirdly, separating the nano material from the solution obtained in the second step through an external magnetic field, washing with a buffer solution to remove the non-phosphorylated peptide, adding ammonia water to perform oscillation elution at 37 ℃, taking the eluent to spot a target, and naturally drying to perform mass spectrometry analysis.
Further, in the first step, the concentration of the dispersion liquid is 10-30 mg/mL; the solvent was 25 mM sodium bicarbonate solution.
Further, in the second step, the mass ratio of trypsin to protein is 1: 50-1: 200; the incubation time at 37 ℃ is 1-16 hours; the incubation time is 10-30 minutes under the condition of shaking at room temperature.
Further, in step three, the buffer contains 15mM ammonium bicarbonate, 20-50wt% acetonitrile and 0.5-5wt% trifluoroacetic acid.
The invention synthesizes a metal oxidation affinity chromatography magnetic mesoporous nano material, which takes magnetic microspheres as a matrix and takes titanium oxide bonds and ordered mesopores as main functional groups. The nanoparticle has high affinity, proper porous structure and excellent magnetic property. The mesoporous structure of the surface not only provides a high specific surface area, but also serves as a temporary enzyme reactor, and effectively accelerates the digestion of trypsin. Titanium dioxide can be used for enrichment of phosphorylated peptides due to its amphiphilic properties and good physicochemical stability. The monolithic material exhibits high selectivity for phosphorylated peptide enrichment, excellent sensitivity and good reusability.
The preparation principle of the invention is as follows: the method is characterized in that a mesoporous silicon dioxide layer with a proper aperture is introduced to the surface of magnetic ferroferric oxide nanoparticles through a sol-gel method, and then the titanium dioxide layer is coated on the surface of a magnetic mesoporous nano material, so that the whole process of enzymolysis of proteins and enrichment of phosphorylated peptides in the same system is realized, and a quantitative analysis method of a non-labeled phosphorylated proteome is established by combining mass spectrum, and can be used for quantifying phosphorylated proteins in complex samples.
The beneficial effects are that: compared with the prior art, the invention has the remarkable advantages that: the metal oxidation affinity chromatography magnetic mesoporous nanomaterial prepared by the invention has the characteristics of large surface area, low detection limit and high specificity for capturing phosphorylated peptide; the metal oxidation affinity chromatography magnetic mesoporous nano material prepared by the invention has a proper mesoporous structure, and can reduce enzymolysis digestion time to 1 hour; the method for quantifying the phosphorylated proteome can carry out trypsin digestion and phosphopeptide enrichment in the same system, eliminates the operation errors caused by multiple steps of pretreatment of the phosphorylated proteome, can be combined with a non-labeling quantification method, and carries out quantitative research on the phosphorylated protein, thus showing great potential in the research of the phosphorylated proteome.
Drawings
FIG. 1 is a transmission electron microscope and a scanning electron microscope photograph of the metal oxide affinity chromatography magnetic mesoporous nanomaterial obtained in example 1;
FIG. 2 is an infrared spectrum of the magnetic mesoporous nanomaterial of metal oxide affinity chromatography obtained in example 1;
FIG. 3 is a hysteresis curve of the magnetic mesoporous nanomaterial of metal oxide affinity chromatography obtained in example 1;
FIG. 4 is a graph showing the nitrogen-adsorption desorption curve of the metal oxide affinity chromatography magnetic mesoporous nanomaterial obtained in example 1;
FIG. 5 is a graph showing the effect of the metal oxide affinity chromatography magnetic mesoporous nanomaterial obtained in example 4 on bovine serum albumin enzymolysis rate;
FIG. 6 is a mass spectrum of the magnetic mesoporous nanomaterial of example 5 for separating and enriching phosphorylated peptides in standard beta-casein enzymatic hydrolysate;
FIG. 7 is a mass spectrum of the magnetic mesoporous nanomaterial of example 5 for separation and enrichment of standard phosphorylated protein beta-casein enzymatic hydrolysate and bovine serum albumin casein mixed solution;
FIG. 8 is a mass spectrum of the repeatability of phosphorylated peptides in the magnetic mesoporous nanomaterial-enriched beta-casein mixed enzymatic hydrolysate of the metal oxide affinity chromatography of example 6;
FIG. 9 is a linear regression curve of the magnetic mesoporous nanomaterial of example 7 on the phosphopeptide (m/z 2061.77) of beta-casein concentration and beta-casein in a series of mixtures of beta-casein concentration and bovine serum albumin.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and examples.
Example 1
Preparation of metal oxidation affinity chromatography magnetic mesoporous nano material
(1) Dissolving 4.05g FeCl3. 6H2O,11.56g NH4Ac,1.12g Na3C6H5O7.2H2O in 210 mL glycol, magnetically stirring at 170 ℃ for 1 hour, transferring the black reaction liquid formed by heating to a 100 mL high-pressure reaction kettle, placing the reaction kettle in a 200 ℃ oven for reaction for 16 hours, naturally cooling to room temperature after the reaction is finished, taking out the reaction kettle, hysteresis separation and collection of products, alternately washing with absolute ethyl alcohol and deionized water, and vacuum drying at 60 ℃ to obtain Fe 3 O 4 。
(2) Fe obtained in the step (1) 3 O 4 0.10 And g, mixing the mixture uniformly by using 100 mL absolute ethyl alcohol in an ultrasonic way for 30 minutes, adding 25 mL deionized water, ammonia water 3.125 mL, performing ultrasonic treatment for 15 minutes, adding 400 mu L of tetraethyl orthosilicate, stirring for 12 hours, performing hysteresis separation, collecting a product, washing the product with absolute ethyl alcohol and deionized water, and performing vacuum drying at 60 ℃.
(3) 0.10g of the product obtained in the step (2), 3.0g of cetyltrimethylammonium chloride and 0.10g of triethanolamine were sonicated for 1 hour to uniformly disperse in 60 mL deionized water, stirred at 60℃and 200 rpm for 1 hour, a cyclohexane mixture of 20 mL containing 5wt% tetraethyl orthosilicate and 2.5wt% isopropyl alcohol was added, stirred for 12 hours, the product was collected by hysteresis separation, washed with absolute ethanol and deionized water, refluxed in a nitric acid/ethanol solution for 4 times at 85℃and the product was collected by hysteresis separation again, washed with absolute ethanol and deionized water, and vacuum-dried at 60℃for 12 hours.
(4) Ultrasonically treating 0.15 g of the product obtained in the step (3) with 200 mL of absolute ethyl alcohol for 30 hours, adding 0.75 mL ammonia water solution, ultrasonically treating for 15 minutes, adding 1.50 g tetrabutyl titanate, stirring at 45 ℃ for 6 hours, hysteresis separating and collecting the product, washing with deionized water and ethanol, and vacuum drying at 60 ℃ for 12 hours to obtain Fe 3 O 4 @mSiO 2 @TiO 2 。
Referring to fig. 1, (a) is a scanning electron micrograph of the material prepared in this example; (b) As can be seen from the transmission electron microscope photograph of the material prepared in the embodiment, the whole appearance is spherical, the particle size is about 500 nm, and the clear ordered mesoporous silicon shell layer and the outermost TiO layer are shown 2 The coating layer illustrates that the preparation material of the embodiment has a mesoporous structure and a core-shell structure.
Referring to FIG. 2, the infrared test results demonstrate that 800 cm of the materials prepared in this example -1 The symmetrical telescopic vibration of Si-O-Si is weakened by 500-700 cm -1 The Ti-O characteristic absorption peak appears at the position, compared with unmodified ferroferric oxide, the preparation method can prepare the material containing TiO by a sol-gel method 2 A material of functional groups.
Referring to FIG. 3, it can be seen that Fe 3 O 4 @nSiO 2 @mSiO 2 @TiO 2 Is of the magnetic saturation strength of (2)20 emu/g, which indicates that the material prepared by the embodiment has superparamagnetism;
referring to FIG. 4, it can be seen from the graph that the adsorption branch in the nitrogen adsorption and desorption curve is calculated, so that the BET specific surface area of the material is 264 m m/g, the pore diameter is 6.38 nm, and the material has a large specific surface area and a mesoporous structure.
Example 2
(1) Dissolving 2.67g FeCl3. 6H2O,6.94g NH4Ac,1.12g Na3C6H5O7.2H2O in 210 mL glycol, magnetically stirring at 100deg.C for 1 hr, transferring the black reaction liquid to 100 mL high-pressure reaction kettle, placing the reaction kettle in 100 deg.C oven for reaction for 12 hr, naturally cooling to room temperature, taking out the reaction kettle, hysteresis separating to collect product, alternately washing with absolute ethanol and deionized water, and vacuum drying at 100deg.C to obtain Fe 3 O 4 。
(2) Fe obtained in the step (1) 3 O 4 0.10 And g, mixing the mixture uniformly by using 100 mL absolute ethyl alcohol in an ultrasonic way for 30 minutes, adding 25 mL deionized water, ammonia water 3.125 mL, performing ultrasonic treatment for 15 minutes, adding 320 mu L of tetraethyl orthosilicate, stirring for 12 hours, performing hysteresis separation, collecting a product, washing the product with the absolute ethyl alcohol and the deionized water, and performing vacuum drying at 100 ℃.
(3) 0.10g of the product obtained in the step (2), 3.0g of cetyltrimethylammonium chloride and 0.20g of triethanolamine were sonicated for 1 hour to uniformly disperse in 60 mL deionized water, stirred at 100 ℃ and 200 rpm for 5 hours, a cyclohexane mixture of 20 mL containing 5wt% tetraethyl orthosilicate and 2.5wt% isopropyl alcohol was added, stirred for 12 hours, the product was collected by hysteresis separation, washed with absolute ethanol and deionized water, refluxed in a nitric acid/ethanol solution for 3 times at 60 ℃, the product was collected by hysteresis separation again, washed with absolute ethanol and deionized water, and vacuum dried at 80 ℃ for 12 hours.
(4) Ultrasonic treating the product of step (3) with 0.15 g of absolute ethyl alcohol (200 mL) for 30 hours, adding 0.75. 0.75 mL of 0.1wt% sodium hydroxide solution, ultrasonic treating for 15 minutes, adding 0.30g of tetraisopropyl titanate, stirring at 45 ℃ for 6 hours, hysteresis separating and collecting the product, washing with deionized water and ethanol, and vacuum drying at 60 ℃ for 12 hours to obtain Fe 3 O 4 @mSiO 2 @TiO 2 。
Example 3
(1) Dissolving 5.34g FeCl3. 6H2O,23.12g NH4Ac,1.68g Na3C6H5O7.2H2O in 210 mL glycol, magnetically stirring at 150deg.C for 1 hr, transferring the black reaction liquid to a 100 mL high-pressure reaction kettle, placing the reaction kettle in a 150 deg.C oven for reaction for 12 hr, naturally cooling to room temperature, taking out the reaction kettle, hysteresis separating to collect product, alternately washing with absolute ethanol and deionized water, and vacuum drying at 100deg.C to obtain Fe 3 O 4 。
(2) Fe obtained in the step (1) 3 O 4 0.10 And g, mixing the mixture uniformly by using 100 mL absolute ethyl alcohol in an ultrasonic way for 30 minutes, adding 25 mL deionized water, ammonia water 3.125 mL, performing ultrasonic treatment for 15 minutes, adding 427 mu L of tetraethyl orthosilicate, stirring for 12 hours, performing hysteresis separation, collecting a product, washing the product with absolute ethyl alcohol and deionized water, and performing vacuum drying at 80 ℃.
(3) The product from step (2) was sonicated for 1 hour with 0.10g, 3.0g cetyl trimethylammonium chloride, 0.10g triethanolamine to uniformly disperse in 60 mL deionized water, stirred at 80 ℃ at 200 rpm for 5 hours, added with 20 mL cyclohexane mixture containing 5wt% tetraethyl orthosilicate and 2.5wt% isopropyl alcohol, stirred for 12 hours, collected by hysteresis separation, washed with absolute ethanol and deionized water, refluxed 5 times in nitric acid/ethanol solution at 80 ℃, collected by hysteresis separation again, washed with absolute ethanol and deionized water, and vacuum dried at 100 ℃ for 12 hours.
(4) Ultrasonically treating the product obtained in the step (3) with 0.15 g of 200 mL of absolute ethyl alcohol for 30 hours, adding 0.75. 0.75 mL of 0.3 mol/L sodium carbonate solution, ultrasonically treating for 15 minutes, adding 2.0g of titanium sulfate, stirring at 45 ℃ for 6 hours, hysteresis separating and collecting the product, washing with deionized water and ethanol, and vacuum drying at 60 ℃ for 12 hours to obtain Fe 3 O 4 @mSiO 2 @TiO 2 。
Example 4
The metal oxidation affinity chromatography magnetic mesoporous nanomaterial obtained in example 1 is used for enzymolysis of bovine serum albumin.
(1) Sample preparation: the standard protein 1 mg is weighed into an EP tube, 25 mM sodium bicarbonate solution is added in an appropriate volume to make the protein concentration 1 mg/mL, the protein is boiled at 100 ℃ for 10 minutes, 1 mol/L dithiothreitol is added after the protein is cooled to room temperature to make the final concentration 10 mM, and the protein is incubated for 30 minutes by shaking at 56 ℃. The EP tube was removed and cooled to room temperature, and 500 mM of iodoacetamide was added to give a final iodoacetamide concentration of 30 mM and incubated at room temperature for 50 minutes in the absence of light.
(2) Bovine serum albumin standard protein solution (1.5X10) -7 M) is an enzymolysis object, the mass ratio of enzyme to protein is 1:2000, the mass ratio of protein to material is 1:50, the enzymolysis system is 200 mu L, and incubation is carried out at 37 ℃ for different times, namely 10 minutes, 1 hour, 2 hours and 16 hours respectively.
(3) Taking 1 mu L of the solution in the step (2) to spot the target, and naturally drying to perform mass spectrometry analysis. As shown in FIG. 5, the number of the detected enzymatic peptide fragments in the bovine serum albumin is counted, and as shown in FIG. 5, when the mesoporous size of the obtained metal oxide affinity chromatography magnetic mesoporous nanomaterial is 6.38 and nm, the detected enzymatic peptide fragments are 6, 8, 10 and 14 respectively in 10 minutes, 1 hour, 2 hours and 16 hours, and compared with the material without the pore diameter and with the pore diameter of 12.51 and 1.82 nm, the number of the detected peptide fragments is more, which indicates that the mesoporous structure of the material prepared by the embodiment can accelerate the enzymolysis.
Example 5
The metal oxide affinity chromatography magnetic mesoporous nanomaterial obtained in example 1 is used for separating and enriching phosphorylated peptides in a mixture of phosphorylated protein beta-casein enzymatic hydrolysate, bovine serum albumin and beta-casein enzymatic hydrolysate.
(1) Preparation of the sample: 1 mg beta-casein, bovine serum albumin and beta-casein mixture were weighed into an EP tube, added with a proper volume of 25 mM sodium bicarbonate solution to make the protein concentration 1 mg/mL, boiled at 100 ℃ for 10 minutes, cooled to room temperature, added with 1M dithiothreitol to make the final concentration 10 mM, and incubated at 56 ℃ for 30 minutes with shaking. The EP tube was removed and cooled to room temperature, and 500 mM iodoacetamide was added to give a final iodoacetamide concentration of 30 mM and incubated at room temperature for 50 minutes in the dark. The mixture of beta-casein, bovine serum albumin and beta-casein is sucked and trypsin liquid is added. And (3) performing enzymolysis at 37 ℃ for 16 hours, and stopping the enzymolysis by trifluoroacetic acid.
(2) And adding a loading buffer solution, wherein the buffer solution comprises 15mM ammonium bicarbonate, 20-50wt% acetonitrile and 0.5-5wt% trifluoroacetic acid, and vibrating and incubating for 30 minutes. Hysteresis separation. 200. Mu.L of wash buffer was added to wash 3 times to remove non-phosphorylated peptide fragments. 10. Mu.L of 10% NH was added 3 ·H 2 And (3) the O eluent is incubated for 15 minutes by shaking, and the eluted product is collected.
(3) Taking 1 mu L of the eluting solution in the step (2) to spot the target, and naturally drying to perform mass spectrometry analysis.
The mass spectrum is shown in FIG. 6, wherein (a) shows the mass spectrum of the phosphorylated peptide in the non-enriched beta-casein enzymatic hydrolysate, and (b) shows the mass spectrum of the phosphorylated peptide in the beta-casein enzymatic hydrolysate enriched in the preparation material of example 1. As can be seen from fig. 6, the non-phosphorylated peptides were detected before enrichment with metal oxide affinity chromatography magnetic mesoporous nanomaterial. And after enrichment of the metal oxidation affinity chromatography magnetic mesoporous nanomaterial, the phosphorylated peptide is identified.
Referring to fig. 7, where (a) is a mass spectrum of the non-enriched beta-casein enzymatic hydrolysate and bovine serum albumin in a mass ratio of 1:2000, and (b) is a mass spectrum of the beta-casein enzymatic hydrolysate and bovine serum albumin in a mass ratio of 1:2000 after enrichment, fig. 7 shows that only non-phosphorylated peptides are detected before enrichment, and phosphorylated peptides in beta-casein are clearly detected after enrichment when the mass ratio of beta-casein enzymatic hydrolysate and bovine serum albumin is 1:2000.
Example 6
The metal oxidation affinity chromatography magnetic mesoporous nanomaterial obtained in example 1 is repeatedly used for 5 times to enrich the phosphorylated peptide in the beta-casein mixed enzymolysis product.
(1) Preparation of the sample: 1 mg beta-casein, bovine serum albumin and beta-casein mixture were weighed into an EP tube, added with a proper volume of 25 mM sodium bicarbonate solution to make the protein concentration 1 mg/mL, boiled at 100 ℃ for 10 minutes, cooled to room temperature, added with 1M dithiothreitol to make the final concentration 10 mM, and incubated at 56 ℃ for 30 minutes with shaking. The EP tube was removed and cooled to room temperature, and 500 mM iodoacetamide was added to give a final iodoacetamide concentration of 30 mM and incubated at room temperature for 50 minutes in the dark. The mixture of beta-casein, bovine serum albumin and beta-casein is sucked and trypsin liquid is added. And (3) performing enzymolysis at 37 ℃ for 16 hours, and stopping the enzymolysis by trifluoroacetic acid.
(2) Adding a loading buffer solution, shaking and incubating for 30 minutes. Hysteresis separation. 200. Mu.L of wash buffer was added to wash 3 times to remove non-phosphorylated peptide fragments. 10. Mu.L of 10% ammonia eluent was added, incubated with shaking for 15 minutes, and the eluted product was collected.
(3) Taking 1 mu L of the elution solution point target in the step (2), and naturally drying to perform mass spectrometry analysis, wherein the mass spectrum is shown in FIG. 8.
(4) Washing the enrichment material 3 times with elution buffer, loading buffer, and repeating steps (2) and (3) for a total of 5 times with regenerated enrichment material.
(5) Taking 1 mu L of the elution solution point target in the steps (3) and (4), and naturally drying for mass spectrometry analysis.
The mass spectrum is shown in fig. 8, wherein (a) is a mass spectrum of the phosphorylated peptide after the first enrichment, and (b) is a mass spectrum of the phosphorylated peptide after the fifth enrichment, and as seen in fig. 8, the phosphorylated peptide in the beta-casein enzymatic hydrolysate can be effectively enriched after the enrichment material is reused for 5 times by comparing the phosphorylated peptide spectrum in the beta-casein enzymatic hydrolysate enriched by the metal oxidation affinity chromatography magnetic mesoporous nanomaterial for the 1 st and 5 th enrichment.
Example 7
The magnetic mesoporous nanomaterial of metal oxide affinity chromatography obtained in example 1 was used to quantify β -casein relative to the intensity of phosphorylated peptide (m/z 2061.77) in a range of β -casein concentrations and bovine serum albumin mixtures.
(1) Preparation of the sample: a proper amount of standard protein beta-casein stock solution and bovine serum albumin with the final concentration of 10 mmol/L are sucked, and diluted into solutions with different concentrations (10 fmol/mu L, 50 fmol/mu L,100 fmol/mu L, 250 fmol/mu L, 500 fmol/mu L and 1000 fmol/mu L) by using 25 mM sodium bicarbonate solution, and vortex mixing is uniform. 200 mu L of beta-casein solution with different concentrations are respectively taken and mixed with eggs according to the materialsThe stock solutions of materials were added simultaneously in a white mass ratio of 50:1, respectively. Trypsin solution was added. And (3) performing enzymolysis at 37 ℃, stopping enzymolysis by trifluoroacetic acid, adding acetonitrile, and performing shake incubation for 30 minutes. Hysteresis separation. 200. Mu.L of wash buffer was added to wash 3 times to remove non-phosphorylated peptide fragments. 10. Mu.L of 10% NH was added 3 ·H 2 And (3) the O eluent is incubated for 15 minutes by shaking, and the eluted product is collected. Each set of samples was run in duplicate 3 times.
(2) Taking 1 mu L of the eluting solution in the step (1) to spot the target, and naturally drying to perform mass spectrometry analysis.
(3) Characteristic phosphopeptide peak intensities were recorded and linear regression analysis was performed with peak intensity on the ordinate (Y) and beta-casein concentration on the abscissa (X).
As can be seen from fig. 9, the characteristic peptide peak intensity (Y) of β -casein is linearly dependent on the β -casein concentration (X), regression equation: y=5.77x+168.87, linear correlation coefficient: r=0.9987, rsd values of less than 15.94% each, indicating that relative quantification of β -casein can be performed depending on the strength of the phosphorylated peptide.
Claims (9)
1. The preparation method of the metal oxidation affinity chromatography magnetic mesoporous nanomaterial is characterized by comprising the following steps of:
fully dissolving ferric trichloride, ammonium acetate and sodium citrate in ethylene glycol, stirring at 100-170 ℃, transferring the formed black reaction liquid to a high-pressure reaction kettle, placing the reaction kettle in a 100-200 ℃ oven for reaction for 8-16 hours, cooling to room temperature, hysteresis separating and collecting a product, washing with absolute ethyl alcohol and deionized water, and then vacuum drying at 50-100 ℃;
uniformly dispersing the product obtained in the step (1) in absolute ethyl alcohol by ultrasonic, adding deionized water and ammonia water, uniformly mixing the solution by ultrasonic, adding tetraethyl orthosilicate, stirring for 5-20 hours, hysteresis separation, collecting the product, washing the product with absolute ethyl alcohol and deionized water, and then drying the product in vacuum at 50-100 ℃;
uniformly dispersing the product obtained in the step (2), cetyltrimethylammonium chloride and triethanolamine in deionized water by ultrasonic, stirring for 1-5 hours at 50-100 ℃, adding a cyclohexane mixed solution of tetraethyl orthosilicate and isopropanol, stirring for 5-20 hours, hysteresis separating and collecting the product, washing with absolute ethyl alcohol and deionized water, refluxing in a nitric acid ethanol solution at 50-100 ℃, washing with absolute ethyl alcohol and deionized water again, and vacuum drying at 50-100 ℃;
step (4), uniformly dispersing the product obtained in the step (3) in a solvent by ultrasonic, adding a titanium dioxide precursor, stirring at 20-80 ℃ for reaction for 5-10 hours, performing hysteresis separation, collecting the product, washing with deionized water and ethanol, and then drying in vacuum; obtaining the nano material which is Fe 3 O 4 @mSiO 2 @TiO 2 Has a spherical structure; the core of the spherical structure is ferroferric oxide for providing magnetism, the outer layer of the ferroferric oxide is coated with a mesoporous silica layer for proteolysis, and the outer part of the mesoporous silica layer is coated with a titanium dioxide layer for enrichment of phosphorylated peptides.
2. The method for preparing the metal oxidation affinity chromatography magnetic mesoporous nanomaterial according to claim 1, wherein the method comprises the following steps of: in the step (1), the mass ratio of the ferric trichloride to the ammonium acetate to the sodium citrate is as follows: (2-4): (3-10): (1-1.5).
3. The method for preparing the metal oxidation affinity chromatography magnetic mesoporous nanomaterial according to claim 1, wherein the method comprises the following steps of: in the step (2), the mass-volume ratio of the product obtained in the step (1) to the absolute ethyl alcohol is 1:1000; the volume ratio of the tetraethoxysilane to the dispersion liquid is 1: (300-400).
4. The method for preparing the metal oxidation affinity chromatography magnetic mesoporous nanomaterial according to claim 1, wherein the method comprises the following steps of: in the step (3), the mass ratio of the product obtained in the step (2), cetyltrimethylammonium chloride and triethanolamine is 1:30: (1-2); the cyclohexane mixed solution comprises 5 weight percent of tetraethyl orthosilicate and 2.5 weight percent of isopropanol; and the reflux times of the product in the nitric acid ethanol solution are 3-5 times.
5. The method for preparing the metal oxidation affinity chromatography magnetic mesoporous nanomaterial according to claim 1, wherein the method comprises the following steps of: in the step (4), the mass ratio of the product obtained in the step (3) to the titanium dioxide precursor is (10-20): 1, a step of; wherein the titanium dioxide precursor is one or a mixture of more of tetraisopropyl titanate, n-butyl titanate, titanium sulfate or titanium tetrachloride; before adding the titanium dioxide precursor, one or more of sodium hydroxide, sodium carbonate or ammonia water are also added.
6. Use of the metal oxide affinity chromatography magnetic mesoporous nanomaterial prepared by the preparation method of claim 1 in quantitative analysis of unlabeled phosphorylated proteomes, characterized by the following specific steps of:
step one, placing a metal oxidation affinity chromatography magnetic mesoporous nano material in a solvent to prepare a dispersion liquid;
step two, adding a protein solution into the dispersion, mixing, adding trypsin, and incubating at 37 ℃; finally adding trifluoroacetic acid and acetonitrile, and carrying out oscillation incubation at room temperature;
and thirdly, separating the nano material from the solution obtained in the second step through an external magnetic field, washing with a buffer solution to remove the non-phosphorylated peptide, adding ammonia water to perform oscillation elution at 37 ℃, taking the eluent to spot a target, and naturally drying to perform mass spectrometry analysis.
7. The use according to claim 6, characterized in that: in the first step, the concentration of the dispersion liquid is 10-30 mg/mL; the solvent was 25 mM sodium bicarbonate solution.
8. The use according to claim 6, characterized in that: in the second step, the mass ratio of trypsin to protein is 1: 50-1: 200; the incubation time at 37 ℃ is 1-16 hours; the incubation time is 10-30 minutes under the condition of shaking at room temperature.
9. The use according to claim 6, characterized in that: in the third step, the buffer solution comprises 15mM ammonium bicarbonate, 20-50wt% of acetonitrile and 0.5-5wt% of trifluoroacetic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111282363.5A CN114011376B (en) | 2021-11-01 | 2021-11-01 | Metal oxidation affinity chromatography magnetic mesoporous nano material, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111282363.5A CN114011376B (en) | 2021-11-01 | 2021-11-01 | Metal oxidation affinity chromatography magnetic mesoporous nano material, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114011376A CN114011376A (en) | 2022-02-08 |
| CN114011376B true CN114011376B (en) | 2024-02-27 |
Family
ID=80059415
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111282363.5A Active CN114011376B (en) | 2021-11-01 | 2021-11-01 | Metal oxidation affinity chromatography magnetic mesoporous nano material, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114011376B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114755337B (en) * | 2022-04-18 | 2023-01-31 | 河南大学 | Disulfide bond mediated photo-crosslinking magnetic silica affinity probe and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103157430A (en) * | 2011-12-09 | 2013-06-19 | 中国科学院大连化学物理研究所 | Sea-urchin-shaped core-shell type Fe3O4@TiO2 magnetic microspheres, and preparation and application thereof |
| CN103920473A (en) * | 2014-04-28 | 2014-07-16 | 扬州大学 | Method for preparing carbon-modified titanium dioxide composite magnetic nano adsorbent |
| CN104445438A (en) * | 2014-12-20 | 2015-03-25 | 张仁超 | Preparation method of superparamagnetic ferroferric oxide composite magnetic mesoporous material |
| CN104857933A (en) * | 2015-05-18 | 2015-08-26 | 苏州汇通色谱分离纯化有限公司 | Preparation and application of core-shell type magnetic metal organic framework nano-particles |
| CN105363426A (en) * | 2015-12-08 | 2016-03-02 | 复旦大学 | Peptide identification method by using mesoporous silica composite combined with mass spectrum |
| CN106140163A (en) * | 2016-06-24 | 2016-11-23 | 许昌学院 | A kind of preparation method of mesoporous TiO 2 magnetic Nano material |
-
2021
- 2021-11-01 CN CN202111282363.5A patent/CN114011376B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103157430A (en) * | 2011-12-09 | 2013-06-19 | 中国科学院大连化学物理研究所 | Sea-urchin-shaped core-shell type Fe3O4@TiO2 magnetic microspheres, and preparation and application thereof |
| CN103920473A (en) * | 2014-04-28 | 2014-07-16 | 扬州大学 | Method for preparing carbon-modified titanium dioxide composite magnetic nano adsorbent |
| CN104445438A (en) * | 2014-12-20 | 2015-03-25 | 张仁超 | Preparation method of superparamagnetic ferroferric oxide composite magnetic mesoporous material |
| CN104857933A (en) * | 2015-05-18 | 2015-08-26 | 苏州汇通色谱分离纯化有限公司 | Preparation and application of core-shell type magnetic metal organic framework nano-particles |
| CN105363426A (en) * | 2015-12-08 | 2016-03-02 | 复旦大学 | Peptide identification method by using mesoporous silica composite combined with mass spectrum |
| CN106140163A (en) * | 2016-06-24 | 2016-11-23 | 许昌学院 | A kind of preparation method of mesoporous TiO 2 magnetic Nano material |
Non-Patent Citations (3)
| Title |
|---|
| Facile synthesis of novel Fe3O4@SiO2@mSiO2@TiO2core-shell microspheres with mesoporous structure and their photocatalytic performance;Jing Ma等;《Journal of Alloys and Compounds》;第456-463页 * |
| 徐尚元.磁性介孔氧化硅复合材料的功能化制备及应用.《中国优秀硕士学位论文全文数据库 工程科技I辑》.2018,第B015-191页. * |
| 磁性介孔氧化硅复合材料的功能化制备及应用;徐尚元;《中国优秀硕士学位论文全文数据库 工程科技I辑》;第B015-191页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114011376A (en) | 2022-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Facile synthesis of zwitterionic polymer-coated core–shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides | |
| Li et al. | Highly selective and rapid enrichment of phosphorylated peptides using gallium oxide‐coated magnetic microspheres for MALDI‐TOF‐MS and nano‐LC‐ESI‐MS/MS/MS analysis | |
| Li et al. | Preparation of titanium-grafted magnetic mesoporous silica for the enrichment of endogenous serum phosphopeptides | |
| CN103143331A (en) | Synthetic method for magnetic metal organic framework composite material coated by [Cu3(btc)2] on surfaces of ferroferric oxide microspheres and application of composite material | |
| Huan et al. | A magnetic nanofiber-based zwitterionic hydrophilic material for the selective capture and identification of glycopeptides | |
| Ma et al. | Ligand-free strategy for ultrafast and highly selective enrichment of glycopeptides using Ag-coated magnetic nanoarchitectures | |
| CN106552603A (en) | PH response type magnetic metal organic frame composite nano materials and preparation method and application | |
| CN109433158A (en) | Magnetic nanometer composite material and the preparation method and application thereof for the enrichment of multi-mode peptide fragment | |
| Chen et al. | Preparation of C60‐functionalized magnetic silica microspheres for the enrichment of low‐concentration peptides and proteins for MALDI‐TOF MS analysis | |
| CN114011376B (en) | Metal oxidation affinity chromatography magnetic mesoporous nano material, preparation method and application | |
| CN109942667A (en) | The methods and applications of two-dimensional metallic organic backbone nanometer sheet enriching phosphated peptide section | |
| CN110665465A (en) | Magnetic covalent organic framework material for glycopeptide enrichment and preparation method and application thereof | |
| Hu et al. | Dual metal cations coated magnetic mesoporous silica probe for highly selective capture of endogenous phosphopeptides in biological samples | |
| Zhu et al. | Nanostructure stable hydrophilic hierarchical porous metal-organic frameworks for highly efficient enrichment of glycopeptides | |
| CN113457630B (en) | Preparation method of magnetic amphiphilic metal organic framework material for enriching glycopeptides | |
| CN111617746A (en) | Polyion liquid modified nano material, preparation method thereof and application thereof in enrichment of phosphorylated peptide | |
| Yang et al. | Boronic acid-functionalized mesoporous magnetic particles with a hydrophilic surface for the multimodal enrichment of glycopeptides for glycoproteomics | |
| CN111458429A (en) | Preparation and application of chitosan modified magnetic nano material | |
| CN113828284A (en) | Preparation and application method of multi-metal multifunctional magnetic nano material | |
| He et al. | Removal of adsorption sites on the external surface of mesoporous adsorbent for eliminating the interference of proteins in enrichment of phosphopeptides/nucleotides | |
| CN117563553A (en) | Preparation and application of angiotensin converting enzyme ACE functionalized magnetic nano-microsphere | |
| Li et al. | Organic molecule-assisted synthesis of Fe3O4/graphene oxide nanocomposites for selective capture of low-abundance peptides and phosphopeptides | |
| CN110130099B (en) | Magnetic nanofiber-based zwitterionic hydrophilic materials for selective capture and recognition of glycopeptides | |
| CN112675821B (en) | Magnetic covalent organic framework material for glycopeptide enrichment based on amphiphilic site and preparation method and application thereof | |
| CN105717288B (en) | A kind of magnetic nanoparticle of surface modification and the preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |