CN109433158A - Magnetic nanometer composite material and the preparation method and application thereof for the enrichment of multi-mode peptide fragment - Google Patents
Magnetic nanometer composite material and the preparation method and application thereof for the enrichment of multi-mode peptide fragment Download PDFInfo
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- 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
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- 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
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/223—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material containing metals, e.g. organo-metallic compounds, coordination complexes
- B01J20/226—Coordination polymers, e.g. metal-organic frameworks [MOF], zeolitic imidazolate frameworks [ZIF]
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- 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
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
- B01J20/28007—Sorbent size or size distribution, e.g. particle size with size in the range 1-100 nanometers, e.g. nanosized particles, nanofibers, nanotubes, nanowires or the like
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- 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
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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- 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
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/46—Materials comprising a mixture of inorganic and organic materials
Abstract
The invention discloses a kind of magnetic nanometer composite materials and the preparation method and application thereof for the enrichment of multi-mode peptide fragment, and the magnetic nanometer composite material is by Fe3O4Magnetic ball is coated on Fe3O4The SiO of magnetic ball surface2Layer is grown on SiO2Metal-organic framework layer on layer is constituted；The metal-organic framework layer is by metal ion Zr4+It is formed with two kinds of organic ligands by coordination, two kinds of organic ligands are for terephthalic acid (TPA) and to benzene hypoboric acid.The magnetic nanometer composite material is with Fe3O4Magnetic ball has good magnetic responsiveness energy as kernel；With Zr4+Affine site as phosphorylated polypeptide, affine site of the boric acid base group as glycopeptide on terephthalic acid (TPA), to both be able to achieve the enrichment for phosphorylated polypeptide, it is able to achieve the enrichment to glycopeptide again, and there is very high bioaccumulation efficiency, it is had a very important significance in studying physiological behavior protein phosphorylation and glycosylation process, and application prospect is good.
Description
Technical field
The invention belongs to technical field of biological materials, the magnetic Nano for relating to Sync enrichment various modes peptide fragment is compound
Material and the preparation method and application thereof.
Background technique
Magnetic metal-organic backbone nanocomposite is by magnetic nano-balls and to be wrapped in the gold of magnetic Nano ball surface
Category-organic backbone (Metal-Organic Frameworks, MOFs) is constituted, simultaneous while with good magnetic responsiveness energy
Have the peculiar properties such as the porosity height, good mechanical performance, aromatic ligand of MOFs are abundant and surface property is easy to adjust, because
This, magnetic metal-organic backbone nanosphere is concerned in recent years, has been widely used in field of biomedicine, especially
Albumen or peptide separation, drug delivery, magnetic resonance imaging etc..
Magnetic metal-organic backbone nanocomposite is all single for the enrichment application of phosphorylated polypeptide and glycopeptide at present
Mode, that is, it is only used for a kind of enrichment of peptide fragment (phosphorylated polypeptide or glycopeptide).Weibing Zhang etc. discloses one kind
Magnetic metal-organic backbone nanosphere preparation method, and it is more in phosphorylation to give the magnetic metal-organic backbone nanosphere
The application of peptide enrichment aspect, the magnetic metal-organic backbone nanosphere is with Fe3+As metal ion, trimethylbenzene is as organic
Ligand uses methyl methacrylate (Methyl methacrylate, MMA) coated magnetic nanosphere, then by MMA packet first
The magnetic nano-balls addition wrapped up in is added sequentially to containing Fe3+Solution and trimethylbenzene solution in be stirred to react, complete once from
Assembling so repeats 31 times, i.e. acquisition magnetic metal-organic backbone nanosphere, and the magnetic metal-organic backbone nanosphere can only
For enrichment (the Facile preparation of core-shell magnetic metal- to phosphorylated polypeptide
Organic framework nanoparticles for the selective capture of phosphopeptides,
ACS Appl.Mater.Interfaces., 2015,7,16338-16347, Weibing Zhang etc.).Pengyuan Yang
Etc. disclosing a kind of preparation method of magnetic metal-organic backbone nanocomposite, and give the organic bone of the magnetic metal-
Application of the frame nanocomposite in terms of glycopeptide enrichment, is compounded to form magnetic graphite for graphene and magnetic nano-balls first
Alkene, then with Zn2+As metal ion, 2-methylimidazole is organic ligand, and ultrasound obtains magnetic metal-organic backbone under room temperature
Composite material, the magnetic metal-organic framework composite material have preferable enrichment to apply (Development of only for glycopeptide
versatile metal-organic framework functionalized magnetic graphene core-shell
biocomposite for highly specific recognition of glycopeptides,ACS
Appl.Mater.Interfaces., 2016,8,27482-27489, Pengyuan Yang etc.).
However, research has shown that abnormal protein phosphorylation and protein glycosylation all have with cancer it is certain contact, therefore
The magnetic adsorptive material for developing energy enriching phosphated peptide section and glycopeptide section, will have the diagnoses and treatment of Cancerous disease great
Meaning.
Summary of the invention
The purpose of the present invention is intended to provide a kind of rich for multi-mode peptide fragment for above-mentioned problems of the prior art
Magnetic nanometer composite material of collection and preparation method thereof, to be able to achieve the enrichment of two kinds of peptide fragments of phosphorylated polypeptide and glycopeptide.
Another object of the present invention is intended to provide the above-mentioned magnetic nanometer composite material for the enrichment of multi-mode peptide fragment and is being enriched with
Application in terms of phosphorylated polypeptide and glycopeptide.
Magnetic nanometer composite material of the present invention for the enrichment of multi-mode peptide fragment, by Fe3O4Magnetic ball is coated on Fe3O4
The SiO of magnetic ball surface2Layer is grown on SiO2Metal-organic framework layer on layer is constituted；The metal-organic framework layer is by gold
Belong to ion Zr4+It is formed with two kinds of organic ligands by coordination, two kinds of organic ligands are for terephthalic acid (TPA) and to benzene hypoboric acid.
Magnetic nanometer composite material of the present invention for the enrichment of multi-mode peptide fragment, is presented complete spherical shape, and partial size is equal
Even and narrow distribution, average grain diameter are 350~450nm or so, and the nanoparticle that this shape is regular, of uniform size is relatively suitble to
Enrichment for polypeptide with separate application.The magnetic nanometer composite material is with superparamagnetic iron oxide (Fe3O4Magnetic ball)
As kernel, there is high magnetic saturation intensity, to have good magnetic responsiveness energy to externally-applied magnetic field；What the present invention used
Fe3O4Magnetic ball accounts for 60% or so of magnetic nanometer composite material quality, to make the saturation magnetization of magnetic nanometer composite material
Reach 36emu g-1Left and right.It is coated on Fe3O4The SiO of magnetic ball surface2Layer is used as middle layer, can chelate Zr4+Metal ion from
And be conducive to the growth in situ of metal-organic framework.Fe3O4The SiO of magnetic ball surface cladding2With good dispersibility, can solve
Certainly it is easy to lead using poly-dopamine (PDA), polyvinylpyrrolidone (PVP) as middle layer in conventional metals-organic framework materials
The problem of causing material to reunite.The raw material that the present invention synthesizes metal-organic framework selects Zr4+As metal ion, on the one hand it is
Synthesize one of the component of metal-organic framework, while a kind of affine site as enriched phosphorus acidification polypeptide.The present invention is to benzene
Dioctyl phthalate and to benzene hypoboric acid as organic double ligands, on the one hand it be one of the component of synthesis metal-organic framework, another party
Boric acid base group on benzene hypoboric acid can be used as a kind of affine site of enrichment glycopeptide.
The preparation method of magnetic nanometer composite material of the present invention for the enrichment of multi-mode peptide fragment, mainly by outer
Prolong the mechanism of growth to realize, first using sol-gal process in Fe3O4Magnetic ball surface wraps up one layer of SiO2, then with zirconium chloride
(ZrCl4), terephthalic acid (TPA) and to benzene hypoboric acid be raw material using one kettle way in SiO2Layer surface generates metal-organic framework layer.
The preparation method of magnetic nanometer composite material of the present invention for the enrichment of multi-mode peptide fragment, steps are as follows:
(1) Fe is prepared3O4/SiO2Nanoparticle
Under ultrasound condition, there is Fe to evenly dispersed3O4Ammonium hydroxide and silane coupling agent are sequentially added in the suspension of magnetic ball,
It is stirred to react 2~6h after ultrasonic disperse is uniform, Magneto separate is carried out to gained reaction solution later and collects the solid isolated producing
Object, obtained solid product are successively washed with ethyl alcohol, deionized water, dimethylformamide and obtain Fe3O4/SiO2Nanoparticle is (i.e.
SiO2Layer cladding Fe3O4The nanoparticle of magnetic ball)；Described evenly dispersed have Fe3O4The suspension of magnetic ball is by Fe3O4Magnetic ball is uniform
It is scattered in the mixed liquor being made of ethyl alcohol and deionized water and obtains；The Fe3O4The quality of magnetic ball, ammonium hydroxide and silane coupling agent
Volume ratio is 100:(0.5~2): (0.5~2), the quality of mass volume ratio is in terms of mg, and volume is in terms of mL；
(2) Fe is prepared3O4/SiO2/ MOF magnetic nanometer composite material
Under agitation, there is Fe for evenly dispersed3O4/SiO2The suspension and ZrCl of nanoparticle4Solution, to benzene two
Formic acid solution is added sequentially to benzene hypoboric acid solution to form reaction system in 110~140 DEG C of dimethylformamide, continues
It is stirred to react 2~6h；Magneto separate is carried out to gained reaction solution later and collects the solid product isolated, obtained solid product according to
It is secondary to be washed with dimethylformamide, ethyl alcohol and deionized water and obtain Fe after drying3O4/SiO2/ MOF magnetic Nano is compound
Material is (i.e. in Fe3O4/SiO2Nanoparticle surface grows the magnetic nanometer composite material for having metal-organic framework layer)；It is described anti-
Answer Fe in system3O4/SiO2Nanoparticle and ZrCl4Mass ratio be 1:(1~3)；The Zr4+With terephthalic acid (TPA) and to benzene
The molar ratio of hypoboric acid is 1:1；The terephthalic acid (TPA) is (2~1): (1~2) with the molar ratio to benzene hypoboric acid.
The preparation method of the above-mentioned magnetic nanometer composite material for the enrichment of multi-mode peptide fragment, the Fe3O4Magnetic ball is mainly
Using iron chloride, ammonium acetate, sodium citrate as raw material, using ethylene glycol as solvent hydro-thermal method synthesize partial size probably 200nm~
The superparamagnetic iron oxide nanosphere of 300nm；Furthermore by adjusting the controllable magnetic ball particle diameter distribution of the hydro-thermal reaction time.System
Standby Fe3O4The specific implementation of magnetic ball can be obtained with reference to the customary preparation methods disclosed in the prior art, referring to The
design and synthesis of a hydrophilic core–shell–shell structured magnetic
metal-organic framework as a novel immobilized metal ion affinity platform
For phosphoproteome research Chem.Commun., 2014,50,6228-6231, Chunhui Deng etc. with
And Ti4+-immobilized multilayer polysaccharide coated magnetic nanoparticles for
highly selective enrichment of phosphopeptides J.Mater.Chem.B 2014,2,4473-
4480, Hanfa Zou etc..
The preparation method of the above-mentioned magnetic nanometer composite material for the enrichment of multi-mode peptide fragment, it is used in step (1)
Fe3O4Magnetic ball is pre-processed, to Fe3O4While magnetic ball cleans, the groups such as magnetic ball surface carboxyl and hydroxyl are activated.
Preprocessing process are as follows: first by Fe3O4Magnetic ball, which is put into hydrochloric acid (concentration of hydrochloric acid 0.1M), is ultrasonically treated 10~60min, right later
Ultrasonic products therefrom carries out Magneto separate and collects the solid product isolated, and obtained solid product is washed through deionized water.
The preparation method of the above-mentioned magnetic nanometer composite material for the enrichment of multi-mode peptide fragment, in step (1), is used to prepare
Fe3O4The volume ratio of ethyl alcohol and deionized water is 4:1 in the mixed liquor of magnetic ball suspension.
The preparation method of the above-mentioned magnetic nanometer composite material for the enrichment of multi-mode peptide fragment, in step (2), it is described uniformly
It is dispersed with Fe3O4/SiO2The suspension of nanoparticle is by Fe3O4/SiO2Nanoparticle is dispersed in dimethylformamide
It obtains；ZrCl4Solution, terephthalic acid solution are by zirconium chloride, terephthalic acid (TPA), to benzene hypoboric acid to benzene hypoboric acid solution
It is dissolved separately in dimethylformamide and obtains.By adjusting Zr4+, terephthalic acid (TPA), to the ratio and reaction of benzene hypoboric acid when
Between, the regulation to metal-organic framework materials structure, above-mentioned Zr may be implemented4+It is formed with terephthalic acid (TPA) and to benzene hypoboric acid
Organic ligand molar ratio be 1:1 when, be conducive to the formation of metal-organic framework；Regulate and control terephthalic acid (TPA) and to two boron of benzene
When the ratio of acid is 1:1, be conducive to introduce more boric acid base groups.
The preparation method of the above-mentioned magnetic nanometer composite material for the enrichment of multi-mode peptide fragment, step (1) and step (2) are washed
The purpose washed, which is for removing, is adsorbed on the unreacted material in solid product surface, and general every kind of washing lotion washs 3~5 times i.e.
It can.It uses in the step (1) and is washed with step (2) as the identical dimethylformamide of reaction environment, in order to avoid introducing
Other miscellaneous solvents, are unfavorable for subsequent reactions.
The preparation method of the above-mentioned magnetic nanometer composite material for the enrichment of multi-mode peptide fragment, the stirring operation used is normal
The mechanical stirring of rule.In step (1), sol-gal process prepares Fe3O4/SiO2During nanoparticle stirring rate be 500~
600rpm.In step (2), one kettle way prepares Fe3O4/SiO2During/MOF magnetic nanometer composite material stirring rate be 700~
800rpm。
Application of the magnetic nanometer composite material of the present invention in phosphorylated polypeptide and glycopeptide enrichment, for biological sample
Phosphorylated polypeptide and glycopeptide in this equally have good concentration effect, in studying physiological behavior protein phosphorylation and glycosylation
Tool has very important significance in the process.
The operation of the acidification of magnetic nanometer composite material enriched phosphorus polypeptide and glycopeptide of the present invention are as follows:
(1) enriched phosphorus is acidified polypeptide: first by phosphorylated protein beta-casein trypsin digestion at phosphorylated polypeptide,
And be diluted with buffer, magnetic nanometer composite material of the present invention is then added and is mixed, at room temperature using shaking
Bed, which shakes 10~60min, makes phosphorylated polypeptide all be enriched in magnetic nanometer composite material surface, the then effect of externally-applied magnetic field again
It is lower to be separated the magnetic nanometer composite material of adsorption phosphorylated polypeptide from solution using Magneto separate, then by surface
Adsorbed phosphorylated polypeptide magnetic nanometer composite material be added to desorption attached liquid in phosphorylated polypeptide is compound from magnetic Nano
Desorption is got off on material.Wherein adsorb phosphorylated polypeptide is the metal-organic framework on magnetic nanometer composite material surface,
Middle metal ion Zr4+The enrichment to phosphorylated polypeptide is realized using fixing metal ions affinity chromatography method (IMAC) principle.
PH by increasing buffer solution, which is realized, is adsorbed phosphorylated polypeptide from the disengaging of magnetic nanometer composite material surface, to realize
The enrichment and separation of phosphorylated polypeptide.
(2) be enriched with glycopeptide: first will glycosylation protein immunoglobulin G trypsin digestion at glycopeptide, and with buffer
Liquid is diluted, and magnetic nanometer composite material of the present invention is then added and is mixed, shakes 10 using shaking table at room temperature
~60min makes glycopeptide be enriched in magnetic nanometer composite material surface, then utilizes Magneto separate will under the action of externally-applied magnetic field again
The magnetic nanometer composite material of adsorption glycopeptide is separated from solution, then by the magnetic Nano of adsorption glycopeptide
Composite material be added to desorption attached liquid in by glycopeptide desorption is got off from magnetic nanometer composite material.Wherein absorption glycopeptide is
The metal-organic framework on magnetic nanometer composite material surface, wherein organic ligand utilizes benzene to the boric acid base group on benzene hypoboric acid
The affinity chromatography method principle of boric acid realizes the enrichment to glycopeptide.Component by changing buffer, which realizes, is adsorbed phosphoric acid
Change polypeptide to be detached from from magnetic nanometer composite material surface, to realize the enrichment and separation of glycopeptide.
Compared with prior art, the invention has the following advantages:
1, provided by the present invention for the magnetic nanometer composite material of multi-mode peptide fragment enrichment, with Fe3O4In magnetic ball is used as
Core has good magnetic responsiveness energy；In Fe3O4Magnetic ball surface is introduced by Zr4+And terephthalic acid (TPA), to benzene hypoboric acid composition
Metal-organic framework, with Zr4+As the affine site of phosphorylated polypeptide, terephthalic acid (TPA) is conducive to the structure of metal-organic framework
It builds, the building of metal-organic framework on the one hand can be participated in benzene hypoboric acid, on the other hand boric acid base group thereon is as glycopeptide
Affine site, to not only be able to achieve the enrichment for phosphorylated polypeptide, but also be able to achieve the enrichment to glycopeptide, and have very Gao Fu
Collect efficiency, is had a very important significance in studying physiological behavior protein phosphorylation and glycosylation process, and application prospect is good
It is good.
2, provided by the present invention for the magnetic nanometer composite material of multi-mode peptide fragment enrichment, in Fe3O4Magnetic ball surface introduces
Metal-organic framework, the network structure of organic inorganic hybridization make nanosphere have fabulous stability, are conducive to nanosphere
Do not allow degradable or collapse, its pattern can be kept, for a long time convenient for the popularization and application of magnetic nanometer composite material.
3, it provided by the present invention for the preparation method of the magnetic nanometer composite material of multi-mode peptide fragment enrichment, uses first
Sol-gal process is in Fe3O4Magnetic ball surface superscribes SiO2Then layer generates one layer of gold by epitaxial growth regime using one kettle way
Category-organic backbone, whole process is easy to operate, reaction condition is mild, and it is multiple that magnetic Nano can be prepared in a short time
Condensation material, therefore be easy to promote in biomedicine field.
Detailed description of the invention
Fig. 1 is structural characterization and its preparation of the magnetic nanometer composite material of the present invention for the enrichment of multi-mode peptide fragment
Method flow diagram.
Fig. 2 is the magnetic Nano material morphology characterization figure of preparation of the embodiment of the present invention, and wherein A is prepared by embodiment 3
Fe3O4/SiO2Scanning electron microscope (SEM) figure of nanoparticle, B are Fe prepared by embodiment 163O4/SiO2/ MOF magnetic Nano is compound
Scanning electron microscope (SEM) figure of material, C are Fe prepared by embodiment 33O4/SiO2Transmission electron microscope (TEM) figure of nanoparticle, D are
Fe prepared by embodiment 163O4/SiO2Transmission electron microscope (TEM) figure of/MOF magnetic nanometer composite material.
Fig. 3 is Fe prepared by the embodiment of the present invention 163O4/SiO2The element of/MOF magnetic nanometer composite material forms characterization
Figure.
Fig. 4 is the X ray diffracting spectrum of the magnetic Nano material of preparation of the embodiment of the present invention, and wherein a corresponding embodiment 1 is made
Standby Fe3O4Magnetic ball, Fe prepared by b corresponding embodiment 33O4/SiO2Nanoparticle, Fe prepared by c corresponding embodiment 163O4/
SiO2/ MOF magnetic nanometer composite material.
Fig. 5 is Fe prepared by the embodiment of the present invention 163O4/SiO2Nitrogen adsorption-desorption of/MOF magnetic nanometer composite material
Attached curve.
Fig. 6 is that the magnetic Nano material of preparation of the embodiment of the present invention is returned in -18000Oe to the magnetic hysteresis within the scope of 18000Oe
Line chart spectrum, the Fe that wherein prepared by a corresponding embodiment 13O4Magnetic ball, Fe prepared by b corresponding embodiment 33O4/SiO2Nanoparticle, c pairs
The Fe for answering embodiment 16 to prepare3O4/SiO2/ MOF magnetic nanometer composite material.
Fig. 7 is that magnetic nanometer composite material of the present invention illustrates the enrichment and separation process of phosphorylated polypeptide and glycopeptide
Figure.
Fig. 8 is the MS map of beta-casein digestive juice in application examples 1 of the present invention, wherein (a) is the β-of not enriched processing
The MS map of casein digest liquid, (b) Fe to be prepared through embodiment 163O4/SiO2At/MOF magnetic nanometer composite material enrichment
The MS map of the beta-casein digestive juice of reason.
Fig. 9 is the MS map of immunoglobulin G digestive juice in application examples 2 of the present invention, wherein (a) is not enriched processing
The MS map of immunoglobulin G digestive juice, (b) Fe to be prepared through embodiment 163O4/SiO2/ MOF magnetic nanometer composite material
It is enriched with the MS figure of the immunoglobulin G digestive juice of processing.
Specific embodiment
Clear, complete description is carried out below with reference to technical solution of the attached drawing to various embodiments of the present invention, it is clear that is retouched
Stating embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, originally
Field those of ordinary skill obtained all other embodiment without making creative work, belongs to this hair
Bright protected range.
Magnetic nanometer composite material structure proposed by the present invention for the enrichment of multi-mode peptide fragment, as shown in Figure 1 C, the magnetic
Property nanocomposite is by Fe3O4Magnetic ball, is coated on Fe3O4The SiO of magnetic ball surface2Middle layer, growth in situ are in SiO2Middle layer
On metal-organic framework constitute.The present invention is that the process flow provided based on Fig. 1 prepares the magnetic nanometer composite material,
Fe is prepared first with hydro-thermal method3O4Nanoscale magnetic bead (SPIOs), as shown in Figure 1A；Again according to sol-gal process obtained
Fe3O4Magnetic ball surface coats one layer of SiO2Middle layer obtains SiO2Middle layer coats Fe3O4Magnetic nano-particle (SPIOs@
SiO2), as shown in Figure 1B；Finally by metal ion Zr4+, organic ligand (being formed by terephthalic acid (TPA) and to benzene hypoboric acid) with
Fe3O4/SiO2Magnetic nano-particle passes through one kettle way together and Fe is prepared3O4/SiO2/ MOF magnetic nanometer composite material
(SPIOs@SiO2/MOF)。
To keep technical solution provided by the invention clearer, is provided below with reference to embodiment and more detailed illustrate to conciliate
It releases.
1 hydro-thermal method of embodiment prepares superparamagnetism Fe3O4Nanoscale magnetic bead
The Fe used in following embodiment3O4The specific preparation process of magnetic ball are as follows: by raw material 1.157gFeCl3·6H2O、
3.303gNH4Ac (ammonium acetate) and 0.4gNa3CT (sodium citrate) is added in the reaction kettle for filling 60mL ethylene glycol, and magnetic force stirs
Mixing 1h is uniformly mixed above-mentioned raw materials；Then stirrer is removed, reaction kettle is warming up to 200 DEG C, is reacted 16 hours；It again will reaction
Kettle is cooled to room temperature, and is carried out Magneto separate to reaction solution and is collected the solid product isolated；Then ethyl alcohol, deionized water are successively used
(every kind of washing lotion is washed five times) is washed to solid product to get Fe is arrived3O4Magnetic ball.
The Fe obtained by this method3O4Magnetic ball can it is evenly dispersed in water, form stable superparamagnetic nano particle
Suspension.To obtained Fe3O4Magnetic ball carry out DLS (Dynamic Light Scattering, dynamic light scattering) analysis shows that,
Resulting Fe3O4Magnetic spherolite diameter is in 300nm or so.
By adjusting 12~16h of the hydro-thermal reaction time, adjustable obtained Fe3O4Magnetic spherolite diameter 200~300nm it
Between.
Embodiment 2- embodiment 10Fe3O4Magnetic ball wraps up silica (SiO2) layer (Fe of selection 300nm3O4Magnetic ball)
Raw material is weighed according to table 1, and the technological parameter provided in table 1 is combined to prepare Fe according to following methods3O4/SiO2It receives
Rice corpuscles:
(1) HCl that 20mL concentration is 0.1M is added to and fills 100mg Fe3O4In bis- mouthfuls of bottles of 100mL of magnetic ball, ultrasound
Magneto separate is carried out after processing 30min, and collects the Fe isolated3O4Magnetic ball, the Fe isolated3O4Magnetic ball is cleaned with deionized water
I.e. three times completions are to Fe3O4The pretreatment of magnetic ball.
(2) by pretreated Fe3O4Magnetic ball is added to by ethyl alcohol and the deionized water mixed liquor that 4:1 is formed by volume
In, obtain Fe3O4Magnetic ball suspension, later to Fe under ultrasound condition3O40.5~2mL ammonium hydroxide is sequentially added in magnetic ball suspension
With 0.5~2mL silane coupling agent (Tetraethylorthosilane, TEOS, Sigma), ultrasonic disperse 30min is dispersed
Uniform mixed liquor, gained mixed liquor under mechanical stirring (500~600rpm) react 2~6h, later to gained reaction solution into
Row Magneto separate simultaneously collects the solid product isolated, and obtained solid product successively uses ethyl alcohol, deionized water, dimethylformamide
(DMF) (every kind of washing lotion washing is three times) is washed to get Fe is arrived3O4/SiO2Nanoparticle.To obtained Fe3O4/SiO2Nanoparticle
Carry out DLS (Dynamic Light Scattering, dynamic light scattering) analysis shows that, resulting Fe3O4/SiO2Nanoparticle
Seed diameter is shown in Table 1 in 320~420nm or so.
Table 1 prepares Fe3O4/SiO2The raw material and its proportion and technological parameter of nanoparticle
From table 1 it follows that by dosage and the reaction time of regulation ammonium hydroxide and silane coupling agent, adjustable package
In Fe3O4The SiO of magnetic ball surface2The thickness of middle layer.From SiO2Middle layer coats fastness, size and saving drug etc.
Factor considers, now selects the Fe of 350nm thickness3O4/SiO2Nanoparticle is used for following example F e3O4/SiO2/ MOFs magnetic Nano
The preparation of composite material.
Embodiment 11- embodiment 18 prepares Fe3O4/SiO2/ MOF magnetic nanometer composite material
Following embodiment is using one kettle way, and the reaction unit used is oil bath heating, churned mechanically two mouthfuls of bottles are anti-
Answer container.
By 116mg Fe3O4/SiO2Nanoparticle evenly spreads in 2mL DMF and obtains Fe3O4/SiO2Nanoparticle suspends
116~348mg zirconium chloride is dissolved in 4mLDMF and obtains chlorination zirconium solution, terephthalic acid (TPA) is dissolved in 4mLDMF by liquid
Terephthalic acid solution is obtained, benzene hypoboric acid will be dissolved in 4mLDMF and be obtained to benzene hypoboric acid solution.In chlorination zirconium solution
Zr4+Substance amount and terephthalic acid solution and to the terephthalic acid (TPA) in benzene hypoboric acid solution and to benzene hypoboric acid substance
Measuring the ratio between summation is 1:1；Terephthalic acid (TPA) (PTA) is (2~1): (1~2) with the molar ratio to benzene hypoboric acid (PBA).
Fe is prepared according to following methods in conjunction with the technological parameter provided in table 23O4/SiO2/ MOF magnetic nanometer composite material:
Under mechanical agitation, 30mL DMF is added into reaction vessel, is warming up to 110~140 DEG C, then sequentially adds Fe3O4/
SiO2Nanoparticle suspension, chlorination zirconium solution, terephthalic acid solution, to benzene hypoboric acid solution, later at 110~140 DEG C
Continue to be stirred to react 2~6h；Magneto separate is carried out to gained reaction solution after reaction and collects the solid product isolated, gained
Solid product successively washs (every kind of washing lotion is washed three times) with DMF, ethyl alcohol and deionized water, and washing products therefrom is dry through vacuum
It is dry to get arrive Fe3O4/SiO2/ MOF magnetic nanometer composite material.To obtained Fe3O4/SiO2/ MOF magnetic nanometer composite material
Carry out DLS (Dynamic Light Scattering, dynamic light scattering) analysis shows that, resulting Fe3O4/SiO2/ MOF magnetic
Property nanocomposite partial size is shown in Table 1 in 350~780nm or so.
Table 2 prepares Fe3O4/SiO2The raw material and its proportion and technological parameter of/MOFs magnetic nanometer composite material
From Table 2, it can be seen that passing through regulation zirconium chloride, terephthalic acid (TPA), to benzene hypoboric acid and reaction time, can adjust
It is whole to be grown in Fe3O4/SiO2Nanoparticle SiO2Metal organic frame thickness in middle layer.And it can be seen that by analysis
In the case that PTA amount is less, it is unfavorable for the composition of metal-organic framework, obtained nano-particles size is basically unchanged, with
PTA amount increases, and metal-organic framework largely synthesizes, it is contemplated that will affect the enrichment to glycopeptide when PBA content is very little, because
This comprehensively considers, and the preferred molar ratio of PTA and PBA are 1:1.In addition, (such as above-mentioned 6h) too long between when reacted, obtained nanometer
Particle diameter reaches 780nm, and metal-organic framework prepared by surface is connected with each other, and causes nanoparticle reunion not disperse, partial size
Become larger, is extremely unfavorable for the enrichment application to albumen or peptide fragment.
Structural characterization
In order to probe into SiO2Whether middle layer and metal-organic framework are successfully combined to Fe3O4On nanoscale magnetic bead, to reality
Apply the Fe of the preparation of example 13O4Fe prepared by magnetic ball, embodiment 33O4/SiO2Fe prepared by nanoparticle, embodiment 163O4/SiO2/
The appearance and size and microstructure of MOF magnetic nanometer composite material are characterized, as shown in Figures 2 to 4.
Fe prepared by embodiment 33O4/SiO2Fe prepared by nanoparticle and embodiment 163O4/SiO2/ MOF magnetic Nano
Composite material carries out morphology analysis using scanning electron microscope (SEM) and transmission electron microscope (TEM), as a result as shown in Figure 2；From figure
2 as can be seen that the Fe prepared3O4/SiO2Nanoparticle and Fe3O4/SiO2/ MOFs magnetic nanometer composite material is equal at size
One, the spherical shape of regular appearance；In addition, finding out that nanoparticle surface is very smooth from Fig. 2 C, and there is one layer to swoon, shows SiO2In
Interbed is successfully wrapped in Fe3O4Magnetic ball surface, and in Fig. 2 D, coarse network structure is presented in nanoparticle surface, this proof
Metal-organic framework has successfully been synthesized to Fe3O4/SiO2Nanoparticle surface.
Fe prepared by embodiment 163O4/SiO2/ MOF magnetic nanometer composite material carries out X-ray energy spectrum analysis (EDX),
It is as shown in Figure 3 to analyze result.It can be with Fe from Fig. 33O4/SiO2In/MOF magnetic nanometer composite material, C element atomic percent
For 34.04%, N element atomic percent be 4.02%, O Elements Atom percentage is 44.02%, B element atomic percent is
2.89%, Si Elements Atom percentage are that 4.64%, Fe atomic percent is 5.79%, Zr Elements Atom percentage 4.6%.From
Above-mentioned testing result can be seen that Fe3O4There are Si, Zr, B element in/Polymer/MOFs composite nano materials, further demonstrate,prove
Bright SiO2(metal ion in MOFs layers is Zr for middle layer and MOFs layers4+, organic ligand PBA contains B element) succeed
Fe3O4Magnetic ball surface is formed, the Zr of high-content4+Be conducive to the enrichment application for phosphorylated polypeptide, the B of high-content is conducive to pair
In the enrichment application of glycopeptide.
Fe prepared by embodiment 13O4Fe prepared by magnetic ball, embodiment 33O4/SiO2It is prepared by nanoparticle and embodiment 16
Fe3O4/SiO2/ MOF magnetic nanometer composite material carries out X-ray diffraction analysis, and analysis result is as shown in Figure 4.It can from Fig. 4
To find out, three kinds of materials are all had and standard Fe3O4The consistent characteristic peak of diffraction maximum, illustrates Fe3O4/SiO2/ MOF magnetic Nano is multiple
Condensation material remains Fe3O4The crystal structure of magnetic ball；Fe simultaneously3O4/SiO2/ MOF magnetic nanometer composite material (7.4 °, 8.4 °,
25.8 ° of and) there are three new diffraction maximums, which is the characteristic diffraction peak of metal-organic framework, illustrates metal-
Organic backbone successfully synthesizes on magnetic nano-particle surface.
Fe prepared by embodiment 163O4/SiO2/ MOF magnetic nanometer composite material carries out N2The attached test of adsorption/desorption is surveyed
Test result is as shown in Figure 5.From figure 5 it can be seen that gained N2The attached isothermal curve of adsorption/desorption shows that this magnetic metal-is organic
Skeleton nanocomposite has biggish specific surface area and porosity, illustrates that metal-organic framework is successfully synthesized in magnetic ball
Surface is conducive to the enrichment application for peptide fragment.
In conclusion Fe3O4/SiO2The shell of/MOF magnetic nanometer composite material has metal-organic framework, and
The structure does not have a significant effect the magnetic crystal structures of composite nano materials, and this unique MOF shell is beneficial in phosphoric acid
Change the application in polypeptide and glycoprotein capture and separation.
Magnetism testing
Fe prepared by embodiment 13O4Fe prepared by magnetic ball, embodiment 33O4/SiO2It is prepared by nanoparticle, embodiment 16
Fe3O4/SiO2/ MOF magnetic nanometer composite material is using Model BHV-525 type vibrating specimen magnetometer (VSM) in -18000Oe
Magnetic Test is carried out within the scope of to 18000Oe, the hysteresis loop tested is as shown in Figure 6.From fig. 6 it can be seen that all
The hysteresis loop of sample passes through origin, no remanent magnetism and coercivity, illustrates Fe3O4Magnetic ball, Fe3O4/SiO2Nanoparticle, Fe3O4/
SiO2/ MOF magnetic nanometer composite material all has superparamagnetism, wherein Fe3O4/SiO2/ MOF magnetic nanometer composite material is satisfied
Reach 36emu g with the intensity of magnetization-1Left and right.
Application examples
Invention further provides above-mentioned Fe3O4/SiO2/ MOF magnetic nanometer composite material enriched phosphorus acidification polypeptide and
Application in terms of glycopeptide, Fe3O4/SiO2/ MOF magnetic nanometer composite material for phosphorylated polypeptide and glycopeptide enrichment with separate
Process, as shown in fig. 7, first by Fe3O4/SiO2/ MOF magnetic nanometer composite material is added in sample to be processed, is then existed
It is enriched in shaking table, how much the time can be adjusted according to sample, will be consolidated after enrichment process by Magneto separate mode
Body product is separated, and has the magnetic ball of phosphorylated polypeptide or glycopeptide to solve adsorption with the buffer of desorption again later
Absorption, to obtain the buffer containing phosphorylated polypeptide or glycopeptide.It can be to obtaining containing phosphorylated polypeptide or glycopeptide
Buffer carries out MS (Mass Spectrometry, mass spectrum) analysis, to further determine that Fe3O4/SiO2/ MOF magnetic Nano is multiple
Concentration effect of the condensation material to phosphorylated polypeptide or glycopeptide.
The enrichment of phosphorylated polypeptide in 1 beta-casein digestive juice of application examples
1mg beta-casein is taken to be dissolved in the NH that 1ml 50Mm pH is 8.24HCO3In buffer, the pancreas egg of 25 μ g is added
White enzyme digests 16h under the conditions of 37 DEG C；Then with the first buffer (acetonitrile solution that volumetric concentration is 50%, wherein containing
0.25% trifluoroacetic acid, i.e. 50%ACN-H2O, 0.25%TFA) it is diluted to 10-6The concentration of M obtains beta-casein digestive juice.
The Fe for taking 1mg embodiment 16 to prepare3O4/SiO2/ MOF magnetic nanometer composite material is added to 200 μ l beta-casein digestive juice samples
In, then 45min is enriched in room temperature under the conditions of shaking table 150-200rpm；Later with 3 times (400 μ every time of the first buffer solution for cleaning
L) polypeptide of non-specific adsorption is removed from magnetic nanometer composite material surface；The magnetism of phosphorylated polypeptide will finally have been adsorbed
Nanocomposite is added in 10 the second buffers of μ l (ammonia spirit that volumetric concentration is 10%), in shaking table 800-
1200rpm is aggressively shaken desorption 30min under state, isolates magnetic nanometer composite material using Magneto separate, obtains desorption
Liquid.Then the beta-casein digestive juice of 1 μ l desorption attached liquid and the not enriched processing of 1 μ l is taken to be analyzed by mass spectrometry, analysis result is such as
Shown in Fig. 8.
From figure 8, it is seen that the beta-casein digestive juice of not enriched processing, has to a phosphorylation with Mass Spectrometer Method
The signal of polypeptide, and signal is very low [seeing Fig. 8 (a)]；Use Fe3O4/SiO2After the enrichment of/MOF magnetic nanometer composite material, entire matter
It is all that the signal peak of phosphorylated polypeptide [is shown in Fig. 8 (b), β on spectrogram1s、β2sFor the phosphorus in the mono-phosphorylated site of two different locations
It is acidified the characteristic peak of polypeptide, β3mFor the characteristic peak of the phosphorylated polypeptide in polyphosphoric acid site].Above-mentioned analysis result finds out, can be with
Detect all phosphorylated polypeptide, and intensity is high and does not have miscellaneous peptide, shows Fe of the present invention3O4/SiO2/ MOF is magnetic
Nanocomposite enriched phosphorus can be acidified polypeptide well, while have selectivity and very high efficiency well.
Application examples 2 glycosylates the enrichment of glycopeptide in protein immunoglobulin G digestive juice
2mg immunoglobulin G is taken to be dissolved in the NH that 1ml 50Mm pH is 8.24HCO3In buffer, the pancreas of 50 μ g is added
Protease digests 16h under the conditions of 37 DEG C；Then with the first buffer (100mM NH4HCO3Aqueous solution) it is diluted to 10-7M's is dense
Degree obtains immunoglobulin G digestive juice.The Fe for taking 1mg embodiment 16 to prepare3O4/SiO2/ MOFs magnetic nanometer composite material is added
Into 100 μ l immunoglobulin G digestive juice samples, 45min then is enriched in room temperature under the conditions of shaking table 150-200rpm；Later
The polypeptide of non-specific adsorption is removed from magnetic nanometer composite material surface with the first buffer solution for cleaning 3 times (400 μ l every time)；
The magnetic nanometer composite material for having adsorbed glycopeptide is finally added to 10 the second buffers of μ l (acetonitrile water that volumetric concentration is 50%
Solution, wherein the trifluoroacetic acid for being 1% containing volumetric concentration, i.e. 50%ACN-H2O includes 1%TFA) in, in shaking table 800-
1200rpm is aggressively shaken desorption 30min under state, isolates magnetic nanometer composite material using Magneto separate, obtains desorption
Liquid.Then it takes the immunoglobulin G digestive juice of 1 μ l desorption attached liquid and the not enriched processing of 1 μ l to be analyzed by mass spectrometry, analyzes result
As shown in Figure 9.
From fig. 9, it can be seen that the immunoglobulin G digestive juice of not enriched processing, has to three glycopeptides with mass spectrum inspection
Signal, and signal is very low, and entire MS figure is substantially the signal [seeing Fig. 9 (a)] of miscellaneous peptide fragment；Use Fe3O4/SiO2/ MOF magnetic
Property nanocomposite enrichment after, can detecte the signal to 25 glycopeptides on entire mass spectrogram, and entire MS figure is all sugar
The signal peak [seeing Fig. 9 (b), I label is all the characteristic peak of glycopeptide] of peptide.Above-mentioned analysis is the result shows that of the present invention
Fe3O4/SiO2/ MOF magnetic nanometer composite material can be enriched with glycopeptide well, while have selectivity and very high effect well
Rate.
In conclusion Fe of the present invention3O4/SiO2Multi-mode peptide fragment may be implemented in/MOF magnetic nanometer composite material
Enrichment.
Claims (6)
1. the magnetic nanometer composite material for the enrichment of multi-mode peptide fragment, it is characterised in that the magnetic nanometer composite material is by Fe3O4
Magnetic ball is coated on Fe3O4The SiO of magnetic ball surface2Layer is grown on SiO2Metal-organic framework layer on layer is constituted；The metal-
Organic frame layer is by metal ion Zr4+It is formed with two kinds of organic ligands by coordination, two kinds of organic ligands are to benzene two
Formic acid and to benzene hypoboric acid.
2. the preparation method described in a kind of claim 1 for the magnetic nanometer composite material of multi-mode peptide fragment enrichment, feature
It is that steps are as follows:
(1) Fe is prepared3O4/SiO2Nanoparticle
Under ultrasound condition, there is Fe to evenly dispersed3O4Ammonium hydroxide and silane coupling agent are sequentially added in the suspension of magnetic ball, through super
Sound is stirred to react 2~6h after being uniformly dispersed, carry out Magneto separate to gained reaction solution later and collect the solid product isolated, institute
Solid product is successively washed with ethyl alcohol, deionized water, dimethylformamide and obtains Fe3O4/SiO2Nanoparticle；It is described equal
It is even to be dispersed with Fe3O4The suspension of magnetic ball is by Fe3O4Magnetic ball is dispersed in the mixed liquor being made of ethyl alcohol and deionized water
It obtains；The Fe3O4The mass volume ratio of magnetic ball, ammonium hydroxide and silane coupling agent is 100:(0.5~2): (0.5~2), mass body
The quality of product ratio is in terms of mg, and volume is in terms of mL；
(2) Fe is prepared3O4/SiO2/ MOF magnetic nanometer composite material
Under agitation, there is Fe for evenly dispersed3O4/SiO2The suspension and ZrCl of nanoparticle4Solution, terephthalic acid (TPA) are molten
Liquid is added sequentially to benzene hypoboric acid solution to form reaction system in 110~140 DEG C of dimethylformamide, and it is anti-to continue stirring
Answer 2~6h；Magneto separate is carried out to gained reaction solution later and collects the solid product isolated, obtained solid product successively uses two
Methylformamide, ethyl alcohol and deionized water wash and obtain Fe after drying3O4/SiO2/ MOFs magnetic nanometer composite material；
Fe in the reaction system3O4/SiO2Nanoparticle and ZrCl4Mass ratio be 1:(1~3)；The Zr4+With terephthalic acid (TPA)
It is 1:1 with the molar ratio to benzene hypoboric acid；The terephthalic acid (TPA) is (2~1): (1~2) with the molar ratio to benzene hypoboric acid.
3. the preparation method for the magnetic nanometer composite material of multi-mode peptide fragment enrichment according to claim 2, feature
It is Fe employed in step (1)3O4Magnetic ball is pre-processed, preprocessing process are as follows: first by Fe3O4Magnetic ball is put into
It is ultrasonically treated 10~60min in hydrochloric acid, Magneto separate is carried out to ultrasonic products therefrom later and collects the solid product isolated, institute
Solid product is obtained to wash through deionized water.
4. the preparation method according to Claims 2 or 3 for the magnetic nanometer composite material of multi-mode peptide fragment enrichment, special
Sign is in the mixed liquor that the volume ratio of ethyl alcohol and deionized water is 4:1.
5. the preparation method according to Claims 2 or 3 for the magnetic nanometer composite material of multi-mode peptide fragment enrichment, special
Sign is described evenly dispersed there is Fe3O4/SiO2The suspension of nanoparticle is by Fe3O4/SiO2Nanoparticle is dispersed in
It is obtained in dimethylformamide；ZrCl4Solution, terephthalic acid solution are by zirconium chloride, terephthaldehyde to benzene hypoboric acid solution
Acid is dissolved separately in dimethylformamide benzene hypoboric acid and obtains.
6. application of the magnetic nanometer composite material described in claim 1 in phosphorylated polypeptide and glycopeptide enrichment.
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