CN106732409A - The synthetic method of the metal organic framework nano composite material of sulfonic group modification and its application - Google Patents
The synthetic method of the metal organic framework nano composite material of sulfonic group modification and its application Download PDFInfo
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Abstract
The present invention proposes synthetic method and its application of a kind of metal organic framework nano composite material of sulfonic group modification.First, hydro-thermal method synthesis magnetic ball, auto polymerization in dopamine hydrochloride weakly alkaline solution is dispersed in by magnetic ball；The magnetic ball that poly-dopamine is coated successively is dispersed in the dimethyl formamide solution of zirconium chloride and 2 sulfonic group terephthalic acid (TPA)s, obtains the metal organic framework nano composite material of sulfonic group modification.The metal organic framework nano composite material of sulfonic group modification can occur hydrophilic interaction by hydrophilic interaction with glycopeptide, and specific enrichment glycopeptide reaches the effect of material efficiently concentrating.The synthetic method simple and fast, the material of synthesis has preferable hydrophily and biocompatibility, can be used for the selective enrichment of glycopeptide in complex biological sample.
Description
Technical field
The invention belongs to advanced nano material and field of nanometer technology, and in particular to the metal organic framework of sulfonic group modification
The synthetic method of nano composite material and its application, more particularly to a kind of magnetic ball surface cladding poly-dopamine and sulfonic group modification
The metal organic framework of metal ion centered on zirconium（MOF）The synthetic method of nano composite material and its for glycopeptide enrichment with
The application of MALDI-TOF MS and LC-MS/MS detection.
Background technology
The glycosylation of protein is important and universal posttranslational modification in life process, and they are passed with intercellular signal
Pass, cell division, propagation, differentiation and interact etc. many important complex biological processes it is closely bound up.Some researchs show,
The expression exception of glycopeptide or Phosphorylated Peptide, can be used as the great disease of the mankind such as the biological marker of many diseases, especially cancer
Disease.So the early diagnosis to the research of glycopeptide to disease has great significance.But the abundance of glycopeptide is often very low, and
And the response of their mass spectrums can be suppressed by the non-glycopeptide of high abundance and protein, the salinity and surfactant in sample are equally
Interference can be produced to its mass spectrum behavior so that its ionizing efficiency is very low, and Mass Spectrometer Method is relatively difficult.Therefore matter is being used
Before glycopeptide in Spectral Analysis complex biological sample, it is very necessary that selective enrichment is carried out to the peptide fragment in sample.
Since the eighties in 19th century, a series of new Soft ionization techniques such as fast atom bombardment ionization, ground substance assistant laser
After desorption ionization, electron spray ionisation etc. find, tree species for bio-energy source is developed rapidly.Due to mass-spectrometric technique（Mass
Spectrometry, MS）The characteristics of with high accuracy, high sensitivity and automation mechanized operation, and it can accurately measure peptide
Relative molecular weight and sulfonic acid sequence of section and protein etc., quickly and accurately obtain multiple proteins property parameters, with reference to
Bioinformatics tools, can rapidly carry out the identification of protein, so that for the structure elucidation of protein provides reliable basis.Therefore
Mass-spectrometric technique is undisputedly as platform indispensable in current proteomics research, and the information quality of mass spectrometric data is straight
Connect the reliability and identification quantity for determining identification of proteins.
With deepening continuously for Recent study, many methods are all used to selective separation enrichment glycosylation albumen and many
Peptide, such as boric acid affinity chromatography, hydrazine chemistry, hydrophilic interaction chromatogram（HILIC）, red, orange, green, blue, yellow (ROGBY), volume exclusion method etc..Its
Middle HILIC methods are most widely used, and effect is also more preferable compared with other method, in HILIC methods, such as metal organic framework material
Material, nano material etc., all cause extensive attention in terms of glycopeptide enrichment.
Metal-organic framework materials refer to by organo-functional group be support, metal ion or node centered on metal cluster, lead to
Cross the network porous material of the three-dimensional periodic with regular nano pore being self-assembly of.With very big ratio table
The excellent properties such as nanoscale duct, regulatable pore passage structure, good heat endurance of area, stabilization, and undersaturated match somebody with somebody
Position metal may occur coordination with the analyte containing functional groups such as carboxyl, sulfonic groups, and MOFs materials have turned into be had
Chemical machine, inorganic chemistry, the study hotspot in physical chemistry field.MOFs is nowadays in gas separation, the selection of Benzene and Homologues
Property absorption, medicament research and development etc. have played its no small effect.In recent years, MOFs materials Preliminary Applications in proteins/peptides point
From enrichment, and MOFs materials are shown in proteome research, such as low abundance peptide enrichment there are good potentiality.
The content of the invention
With MOF technologies be combined HILIC by the present invention, devises a kind of magnetic ball surface cladding poly-dopamine and sulfonic group is repaiied
The metal organic framework of metal ion centered on zirconium of decorations（MOF）Nano composite material, the aqueous favoring provided by sulfonic group
Interaction is combined closely with glycopeptide, reaches the effect of enrichment glycopeptide.
Present invention aim at the synthetic method for providing the metal organic framework nano composite material that a kind of sulfonic group is modified,
The metal organic framework of metal ion centered on zirconium that especially a kind of magnetic ball surface cladding poly-dopamine and sulfonic group are modified
（MOF）The synthetic method of nano composite material.
Metal organic framework nano composite material another object of the present invention is to provide sulfonic group modification is rich in glycopeptide
Application in collection and MALDI-TOF MS and LC-MS/MS detection.
The synthetic method of the metal organic framework nano composite material of the sulfonic group modification that the present invention is provided, specific steps are such as
Under：
（1）By FeCl3•6H2O is dissolved in ethylene glycol, and ultrasound after magnetic agitation to clarification, plus sodium acetate stirring is transferred to reactor,
Reacted 16 hours at 200 DEG C, take out reactor, cooled down 12 hours, fully the generated ferroso-ferric oxide magnetic ball of washing, at 50 DEG C
Lower vacuum drying；
（2）By step（1）The ferroso-ferric oxide magnetic ball for being obtained is dissolved in trihydroxy methyl sulfonic acid methylmethane buffering with dopamine hydrochloride
In liquid, be stirred at room temperature reaction 16 hours, with magnet separation product after, fully washed with deionized water and absolute ethyl alcohol, at 50 DEG C
Lower vacuum drying, obtains the magnetic ball of poly-dopamine cladding；
（3）The dispersion steps in dimethylformamide（2）The magnetic ball of the poly-dopamine cladding of gained, ultrasound to fully dispersed, plus
Enter zirconium chloride to stir, heating stirring 60-360 minutes at 150 DEG C；
（4）In step（3）Part 2- sulfonic group terephthalic acid (TPA)s are added in gained system, is stirred, heating stirring at 150 DEG C
30 minutes, magnet separation product is used, fully washed with dimethylformamide, deionized water and absolute ethyl alcohol, the vacuum at 50 DEG C
Dry, obtain final product the metal organic framework nano composite material of gained sulfonic group modification.
In the present invention, step（1）Middle FeCl3•6H2The quality corresponding volume ratio of O and ethylene glycol is（0.9-1.8）g：（50-
100）ml.
In the present invention, step（1）Middle FeCl3•6H2The quality corresponding volume ratio of O and ethylene glycol is 1.35g：75ml.
In the present invention, step（2）Middle trihydroxy methyl sulfonic group aminomethane buffer with solvent as deionized water and ethanol, volume
Than being 1：1, pH=8.5.
In the present invention, step（2）The mass ratio of middle ferroso-ferric oxide magnetic ball and dopamine hydrochloride is 2.4：1.
In the present invention, step（3）The magnetic ball of middle poly-dopamine cladding and the mass ratio of zirconium chloride are 100 mg：150 mg,
Reaction temperature is 150 DEG C, and the reaction time is 60 minutes.
In the present invention, step（2）The magnetic ball and step of middle products therefrom poly-dopamine cladding（4）Middle 2- sulfonic groups are to benzene two
The mass ratio of formic acid is（75-125）mg：（280-360）Mg, reaction temperature is 120-180 DEG C, and the reaction time is 15-45 minutes.
In the present invention, step（2）The magnetic ball and step of middle products therefrom poly-dopamine cladding（4）Middle 2- sulfonic groups are to benzene two
The mass ratio of formic acid is 100 mg：320 mg, reaction temperature is 150 DEG C, and the reaction time is 30 minutes.
The present invention also proposes that the synthetic method of the metal organic framework nano composite material of sulfonic group modification is enriched with glycopeptide
With the application in Mass Spectrometric Identification：The metal organic framework nano composite material that sulfonic group is modified is configured to by solvent of ultra-pure water
It is material dispersion liquid, by the material dispersion liquid and target glycopeptide solution 90% acetonitrile/0.1% trifluoroacetic acid buffer solution of addition, mixes
Merge and be incubated in instrument is digested, nano composite material is gone out by centrifugation, with 90% acetonitrile/0.1% trifluoroacetic acid and 80% second
Nitrile/1% phosphate buffer washs nano composite material, is then eluted with 30% acetonitrile/0.1% formic acid, takes 1 μ L eluents and directly exists
Target is put on MALDI-TOF MS sample introduction target plates, is put again after drying plus 1 μ L concentration is the DHB solution of 30mg/mL
In matrix crystallization on the drop, is formed, mass spectral analysis is carried out.
The present invention has synthesized the nano composite material with reference to tradition MOF materials and HILIC material advantages first, and is applied to
The separation and concentration of glycopeptide.Due to the hydrophilic interaction between surface area and sulfonic group and glycopeptide MOF higher so that the nanometer
Composite can carry out selective enrichment to the glycopeptide in complex biological sample, substantially increase its mass signal.To glycopeptide
Test limit up to 100 amol/ μ L, to the selectivity of non-glycopeptide up to 1：100（Mass ratio）, directly can be detected from human serum
Nitrogen end glycopeptide corresponds to different glycoprotein.
Magnetic ball surface cladding poly-dopamine of the invention and the metal of metal ion centered on zirconium of sulfonic group modification have
Machine skeleton（MOF）Nano composite material, synthetic method is simple and quick, and sulfonic group and glycopeptide can closely be tied by hydrophilic interaction
Close, MOF improves active surface and preferable body pore passage structure.This nano composite material can be used for optionally enriched biological
Low-abundance glycopeptide in sample, and for MALDI-TOF MS and LC-MS/MS detection.
The beneficial effects of the present invention are：The metal organic framework nano composite material synthetic method of sulfonic group modification is simple
Quickly, sulfonic group and glycopeptide can be combined closely by hydrophilic interaction, and MOF improves active surface and preferable body opening road
Structure.The low-abundance glycopeptide that this nano composite material can be used in optionally enriched biological sample, and for MALDI-TOF
MS and LC-MS/MS is detected.Material provided by the present invention is to glycopeptide test limit up to 100 amol/ μ L, the selection to non-glycopeptide
Property is up to 1：100（Mass ratio）, directly can detect that nitrogen end glycopeptide corresponds to different glycoprotein from human serum.By enrichment peptide fragment
Signal to noise ratio multiplication factor is high, and with preferable selectivity and high sensitivity, the detection to the glycopeptide in complex biological sample has very
Good effect.
Brief description of the drawings
Fig. 1 shines for the SEM of the metal organic framework nano composite material that the sulfonic group of embodiment 1 is modified
Piece and transmission electron microscope photo；Wherein：SEM:A) Fe3O4@PDA, b) Fe3O4@PDA@Zr-SO3H, TEM: c)
Fe3O4@PDA@Zr-SO3H, d) Fe3O4@PDA@Zr-SO3H（Enlarged drawing）；
The energy dispersion X-ray spectrum of the metal organic framework nano composite material that Fig. 2 is modified for the sulfonic group of embodiment 1 and unit
Cellulose content is distributed；
The N2 adsorption curve of the metal organic framework nano composite material that Fig. 3 is modified for the sulfonic group of embodiment 1；Accompanying drawing：Aperture
Distribution curve；
The magnetic hysteresis loop of the metal organic framework nano composite material that Fig. 4 is modified for the sulfonic group of embodiment 1；
The hydrophily test chart of the metal organic framework nano composite material that Fig. 5 is modified for the sulfonic group of embodiment 1；Wherein（a）
After for 15 minutes,（b）After for 30 minutes,（c）After for 45 minutes,（d）After Magnetic Isolation 3 seconds；
The IR Characterization spectrogram of the metal organic framework nano composite material that Fig. 6 is modified for the sulfonic group of embodiment 1；
Fig. 7 characterizes spectrogram for the Raman of the metal organic framework nano composite material that the sulfonic group of embodiment 1 is modified；
The X-ray diffraction pattern of the metal organic framework nano composite material that Fig. 8 is modified for the sulfonic group of embodiment 1；
Fig. 9 is that the HRP enzymolysis liquids of 250 fmol/ μ L in embodiment 2 are nano combined by metal organic framework that sulfonic group is modified
The mass spectrogram of eluent after stoste b) is enriched with before material a) enrichments；What the IgG enzymolysis liquids of 1 pmol/ μ L were modified by sulfonic group
The mass spectrogram of eluent after stoste d) is enriched with before metal organic framework nano composite material c) enrichments；
The HRP enzymolysis liquids of 250 fmol/ μ L are nano combined by metal organic framework that sulfonic group is modified in Figure 10 embodiments 2
Mass spectrogram b) the material circulations of eluent use eluent after gained enrichment after 5 times after the fresh prepared material gained enrichments of material a)
Mass spectrogram；
The HRP enzymolysis liquids of 250 fmol/ μ L are nano combined by metal organic framework that sulfonic group is modified in Figure 11 embodiments 2
After the mass spectrogram b) materials of eluent are preserved 1 month at -20 DEG C after the fresh prepared material gained enrichments of material a) after gained enrichment
The mass spectrogram of eluent；
Figure 12 is HRP enzymolysis liquids matter after the metal organic framework nano composite material that sulfonic group is modified is enriched with embodiment 2
Spectrogram, HRP hydrolyzate contents are：a) 25 fmol/μL; b) 5 fmol/μL; c) 1 fmol/μL; d) 0.1 fmol/μ
L；
Figure 13 be embodiment 3 in mass ratio be 1:Mass spectrogram before the mixed solution a) enrichments of 50 HRP and BSA enzymolysis liquids；b)
By mass spectrogram after the metal organic framework nano composite material enrichment of sulfonic group modification；Mass ratio is 1:100 HRP and BSA
Mass spectrogram before the mixed solution c) enrichments of enzymolysis liquid；D) the metal organic framework nano composite material modified by sulfonic group
Mass spectrogram after enrichment.
Specific embodiment
Following embodiment is further illustrated to of the invention, rather than limitation the scope of the present invention.
Embodiment 1：The synthesis of the metal organic framework nano composite material of sulfonic group modification.
（1）Spent glycol synthesizes ferroso-ferric oxide magnetic ball as solvent, by 1.35g FeCl3•6H2O is dissolved in 75ml second two
Alcohol, magnetic agitation（Gloves are sealed）To clarify, afterwards plus 3.6g crushing sodium acetate stir to dissolve and continue stir 0.5h（Gloves are sealed
Mouthful）.After ultrasonic 5min, reactor, 200 DEG C, 16h are transferred to.Take out reactor, cool overnight.Magnetic ball is poured out, is washed 5 times（Often
Secondary ultrasonic 5min）.Magnetic ball is fully washed with deionized water and ethanol, it is limpid pure to cleaning solution, it is vacuum dried at 50 DEG C；
（2）Configuration trihydroxy methyl sulfonic acid methylmethane（Tris）Buffer solution（Solvent is deionized water and ethanol, volume ratio 1:1, pH=
8.5）, by step（1）The obtained mg of ferroso-ferric oxide magnetic ball 120 is dissolved in 80mLTris buffer solutions with 0.32g dopamine hydrochlorides
（Include 0.05g Tris, 40mL water, 40mL ethanol）, reaction 16h is stirred at room temperature.Magnet separation product, with deionized water and nothing
Water-ethanol is fully washed, and is vacuum dried at 50 DEG C；
（3）The dispersion steps in 75 mL dimethylformamides（2）The mg of products therefrom 100, it is ultrasound a period of time, fully dispersed；
Zirconium chloride 150mg is added, is stirred, 150 DEG C of heating stirrings 360 minutes；
（4）In step（3）Part 2- sulfonic group terephthalic acid (TPA) 320mg are added in gained system, is stirred, 150 DEG C of heating
Stirring 30 minutes, magnet separation product is fully washed with dimethylformamide, deionized water and absolute ethyl alcohol, true at 50 DEG C
Sky is dried.
Fig. 1 is the electron scanning micrograph and transmission electron microscope photo of embodiment 1.SEM
Figure it can be seen that polymer P DA the layer of layer of magnetic ball outer cladding, after modification MOF, the crystallization shape on surface be polymerized
The smooth layer of thing is different, has clearly crystal to occur；Transmission electron microscope figure can be seen that MOF layers of polymer P DA@about 50
Nanometer thickness；ESEM model Philips XL30, the metal organic framework of sulfonic group modification after purification is nano combined
Applying material carries out SEM signs on conducting resinl after metal spraying；Transmission electron microscope model JEM-2100F (J0EL), by after purification
The alcohol dispersion liquid of the metal organic framework nano composite material of sulfonic group modification is dropped in and is covered with the copper mesh of carbon film, and drying is laggard
Row transmission electron microscope observation is simultaneously taken pictures；
Fig. 2 is the content distribution of elements of embodiment 1, and the mass fraction of wherein S elements accounts for 2.9%, is consistent with expection, and table is such as
Under；
| The element number of nuclear charges | The symbol of element | Element term | Mass fraction | Deviation |
| 40 | Zr | Zirconium | 41.6 | 1.2 |
| 8 | O | Oxygen | 20.5 | 0.2 |
| 6 | C | Carbon | 16.2 | 3.4 |
| 16 | S | Sulphur | 2.9 | 1.0 |
| 7 | N | Nitrogen | 12.5 | 1.5 |
| 26 | Fe | Iron | 6.2 | 1.3 |
Fig. 3 is the N2 adsorption curve and pore size distribution curve of embodiment 1；As can be seen from Figure, the ratio of the nano composite material
Surface area is larger, the enrichment application beneficial to after, and the size in hole is 3.20 rans；
Fig. 4 is the magnetic hysteresis loop of embodiment 1；Although after polymer and MOF layers has been coated, under the magnetic response of material
Drop, but still remain magnetic response intensity higher, about 26.4 emug-1；
Fig. 5 is the hydrophily test chart of embodiment 1；Material is disperseed to form the aqueous solution of stable uniform in aqueous, and
15 minutes, 30 minutes, still keep dispersion homogeneous after 45 minutes；And with attraction after, then become at once in 3 seconds settled solution with
Material is separated；
Fig. 6 is the IR Characterization spectrogram of embodiment 1, and the nano material occurs in that more characteristic peak, such as 3400cm-1The carboxylic at place
Base characteristic peak, 1500-1600cm-1The phenyl ring characteristic peak at place, 560cm-1The Fe-O-Fe vibration peaks at place, illustrate the successful conjunction of material
Into；
Fig. 7 characterizes spectrogram for the Raman of embodiment 1, as the complementary data of IR Characterization spectrogram,<500cm-1There is magnetic ball in place
Characteristic peak, 1500-1600cm-1There is phenyl ring characteristic peak after being in cladding DOPA amine layer, illustrate the successful synthesis of material；
Fig. 8 is the X-ray diffraction pattern of embodiment 1,2 θ=8.2, and 13.0,19.2,30.8 ° come from MOF, and 2 θ=
33.3,34.2,36.0,57.2,62.6 ° come from magnetic ball kernel, and this also just illustrates the successful conjunction of magnetic MOF materials
Into；X-ray diffractometer model Bruker D4 X-ray diffractometer.
Embodiment 2：The metal organic framework nano composite material of the sulfonic group modification that embodiment 1 is obtained is micro- as solid phase
Extraction adsorbing separation medium is used for low concentration HRP enzymolysis liquids and the enrichment of β-Casein enzymolysis liquids is examined with MALDI-TOF MS
Survey.
（1）The preparation of standard protein enzymolysis liquid：2 mg HRP standard proteins accurately are weighed, with 25 mM ammonium bicarbonate solns
The standard protein solution that concentration is 2 mg/mL is made into, pH is about 8.3, boiled ten minutes.It is 1 according to mass ratio:50 pancreas
The ratio of protease and standard protein, adds trypsase (trypsin), and 37 °C are incubated 16 hours, can obtain 2 mg/mL's
HRP tryptose enzymolysis liquids；4 mg IgG standard proteins accurately are weighed, concentration is made into for 4 mg/ with 25 mM ammonium bicarbonate solns
The standard protein solution of mL, pH is about 8.3, is boiled ten minutes.It is 1 according to mass ratio:50 trypsase and standard protein
Ratio, add trypsase (trypsin), 37 °C be incubated 16 hours, can obtain 4 mg/mL IgG tryptose enzymolysis liquids.
（2）The enrichment of sample：
Glycopeptide is enriched with：The solution of the metal organic framework nano composite material that 10 mg/mL sulfonic groups are modified is prepared with ultra-pure water.
The material solution of 20 μ L is taken in the centrifuge tube of 0.6 mL, 2 are washed with the cushioning liquid that volume fraction is 90% acetonitrile and 0.1%TFA
It is secondary after removal supernatant, addition with cushioning liquid dilute after various concentrations HRP enzymolysis liquids（Cumulative volume is 100 μ L）, mix,
Enrichment 30 minutes is shaken under 37 °C；Centrifugation material, sucks supernatant, with 90% acetonitrile 0.1%TFA solution detergent one
Time, then with twice of 80% acetonitrile/1% phosphoric acid solution detergent, it is subsequently adding 30% acetonitrile/0.1% formic acid solution of 10 μ L, 37
DEG C concussion wash-out 20 minutes, centrifugation material suctions out the standby rear use of eluent.
（3）Point target：Take 1 μ L steps（2）Described eluent point on MALDI-TOF MS sample introduction target plates, after drying again
Point plus 1 μ L concentration are the 2,5- dihydroxy-benzoic acids of 30 mg/mL（DHB）Solution is dried in matrix crystallization on the drop, is formed
Carry out mass spectral analysis again afterwards.
（4）Mass spectral analysis is divided using the metal organic framework nano composite material that sulfonic group is modified as SPME absorption
Compared from the medium glycopeptide that obtains of enrichment and with the stoste mass spectrogram before enrichment.
Concentration is the metal organic framework nano composite material modified by sulfonic group of HRP enzymolysis liquids of 250 fmol/ μ L
19 glycopeptide peaks for belonging to HRP are occurred in that after enrichment, in mass spectrogram（m/z=1843.0, m/z=2541.4, m/z=
2591.4, m/z=2611.4, m/z=3074.5, m/z=3087.7, m/z=3222.9, m/z=3321.8, m/z=
3353.7, m/z=3369.7, m/z=3605.0, m/z=3672.1, m/z=3894.1, m/z=4056.2, m/z=
4222.4, m/z=4719.6, m/z=4821.7, m/z=4838.7, m/z=4984.7）.
Concentration is that the metal organic framework nano composite material modified by sulfonic group of IgG enzymolysis liquids of 1 pmol/ μ L is rich
Occur in that twenty-one provision belongs to the glycopeptide peak of IgG after collection, in mass spectrogram（M/z=2399.3, m/z=2431.3, m/z=
2457.3, m/z=2488.3, m/z=2561.4, m/z=2602.4, m/z=2618.4, m/z=2634.4, m/z=2650.4, m/z=
2764.5, m/z=2781.5, m/z=2796.5, m/z=2805.5, m/z=2837.5, m/z=2853.5, m/z=2926.6, m/z=
2958.6, m/z=2967.6, m/z=3000.0, m/z=3130.0, m/z=3161.7）
Fig. 9 is that the HRP enzymolysis liquids of 250 fmol/ μ L in embodiment 2 are nano combined by metal organic framework that sulfonic group is modified
The mass spectrogram of eluent after stoste b) is enriched with before material a) enrichments；What the IgG enzymolysis liquids of 1 pmol/ μ L were modified by sulfonic group
The mass spectrogram of eluent after stoste d) is enriched with before metal organic framework nano composite material c) enrichments；
The HRP enzymolysis liquids of 250 fmol/ μ L are nano combined by metal organic framework that sulfonic group is modified in Figure 10 embodiments 2
Mass spectrogram b) the material circulations of eluent use eluent after gained enrichment after 5 times after the fresh prepared material gained enrichments of material a)
Mass spectrogram；
The HRP enzymolysis liquids of 250 fmol/ μ L are nano combined by metal organic framework that sulfonic group is modified in Figure 11 embodiments 2
After the mass spectrogram b) materials of eluent are preserved 1 month at -20 DEG C after the fresh prepared material gained enrichments of material a) after gained enrichment
The mass spectrogram of eluent；
Figure 12 is HRP enzymolysis liquids matter after the metal organic framework nano composite material that sulfonic group is modified is enriched with embodiment 2
Spectrogram, HRP hydrolyzate contents are：a) 25 fmol/μL; b) 5 fmol/μL; c) 1 fmol/μL; d) 0.1 fmol/μ
L；
The metal organic framework nano composite material that mass spectral analysis is modified using sulfonic group is used as SPME adsorbing separation medium
The glycopeptide that obtains of enrichment is simultaneously compared with the stoste mass spectrogram before enrichment, it can be seen that the material is excellent in terms of glycopeptide is enriched with
Performance.
Embodiment 3：The metal organic framework nano composite material of the sulfonic group modification that embodiment 1 is obtained is micro- as solid phase
Extraction adsorbing separation medium is used for HRP enzymolysis liquids and bovine serum albumin(BSA)（BSA）The enrichment of the mixed solution of enzymolysis liquid with
MALDI-TOF MS are detected.
（1）The preparation of standard protein enzymolysis liquid：2 mg standard proteins HRP and 5 mg standard protein BSA accurately are weighed, is used
It is 2 mg/mL and the standard protein solution of 5 mg/mL that 25 mM ammonium bicarbonate solns are made into concentration, and pH is about 8.3, boils 10
Minute.It is 1 according to mass ratio:50 trypsase and the ratio of standard protein, add trypsase (trypsin), and 37 °C incubate
Educate 16 hours, can obtain the HRP tryptoses enzymolysis liquid of 2 mg/mL and the BSA enzymolysis liquids of 5 mg/mL.
（2）The enrichment of sample：
Glycopeptide is enriched with：The solution of the metal organic framework nano composite material that 10 mg/mL sulfonic groups are modified is prepared with ultra-pure water.
The material solution of 20 μ L is taken in the centrifuge tube of 0.6 mL, 2 are washed with the cushioning liquid that volume fraction is 90% acetonitrile and 0.1%TFA
Removal supernatant, first adds the HRP tryptose enzymolysis liquids of 2 mg/mL of 1 μ L after secondary, is according to the mass ratio of HRP and BSA respectively
1:50、1:100 add BSA enzymolysis liquids, and the volume fraction for being subsequently added respective volume is that the aqueous solution of 90% acetonitrile/0.1%TFA makes
System is made into the system that cumulative volume is 100 μ L, mixes, and enrichment 30 minutes is shaken under 37 °C；Centrifugation material, sucks
Clear liquid, with 90% acetonitrile 1%TFA solution detergent one time, then with twice of 80% acetonitrile/1% phosphoric acid solution detergent, Ran Houjia
Enter 30% acetonitrile/0.1% formic acid solution of 10 μ L, 37 DEG C of concussions are eluted 20 minutes, centrifugation material, after sucking-off eluent is standby
With.
（3）Point target：Take 1 μ L steps（2）Described eluent point on MALDI-TOF MS sample introduction target plates, after drying again
Point plus 1 μ L concentration are the 2,5- dihydroxy-benzoic acids of 30 mg/mL（DHB）Solution is dried in matrix crystallization on the drop, is formed
Carry out mass spectral analysis again afterwards.
（4）Mass spectral analysis is divided using the metal organic framework nano composite material that sulfonic group is modified as SPME absorption
Compared from the medium glycopeptide that obtains of enrichment and with the stoste mass spectrogram before enrichment.
Mass ratio is 1:The enzymolysis mixed liquor of 50 HRP and BSA is multiple by the metal organic framework nanometer that sulfonic group is modified
After condensation material enrichment, it will be clear that 12 glycopeptide peaks from HRP from mass spectrogram（m/z=2541.4, m/z=
2591.4, m/z=2611.4, m/z=3222.9, m/z=3321.8, m/z=3353.7, m/z=3369.7, m/z=
3672.1, m/z=4056.2, m/z=4222.4, m/z=4838.7, m/z=4984.7）
Embodiment 4：The metal organic framework nano composite material of the sulfonic group modification that embodiment 1 is obtained is used as SPME
Adsorbing separation medium is used for glycopeptide enrichment and MALDI-TOF MS and LC-MS/MS detection in Healthy Human Serum sample.
（1）Preparation of samples：
Glycopeptide enrichment prepares：2 μ L human serums are scattered in the mM ammonium bicarbonate solns of 198 μ L 25, boil 10 minutes and are deformed.
Afterwards 10 mM dithiothreitol (DTT)s are added at 60 DEG C（DTT）Carry out 30 minutes reduction reactions, after add 20 mM in the dark at 37 DEG C
Indole-3-acetic acid（IAA）Carry out 1 hour alkylated reaction.It is afterwards 1 according to mass ratio:50 trypsase and protein concentration
Ratio, add trypsase (trypsin), 37 °C be incubated 16 hours, freeze it is stand-by.
（2）The enrichment of sample：
Glycopeptide is enriched with：The solution of the metal organic framework nano composite material that 10 mg/mL sulfonic groups are modified is prepared with ultra-pure water.
The material solution of 40 μ L is taken in the centrifuge tube of 0.6 mL, 2 are washed with the cushioning liquid that volume fraction is 90% acetonitrile and 0.1%TFA
Removal supernatant after secondary, the lyophilized liquid of sero-enzyme solution for adding the cushioning liquid of 100 μ L 90% acetonitrile and 1%TFA to dilute, mixes,
Enrichment 30 minutes is shaken under 37 °C；Centrifugation material, sucks supernatant, with 90% acetonitrile 0.1%TFA solution detergent one
Time, then with twice of 80% acetonitrile/1% phosphoric acid solution detergent, it is subsequently adding 30% acetonitrile/0.1% formic acid solution of 10 μ L, 37
DEG C concussion wash-out 20 minutes, centrifugation material, suction out eluent it is lyophilized after it is standby.（LC-MS/MS）
（3）Glycopeptide mass spectral analysis：
LC-MSMS：By step（2）The lyophilized liquid of gained is dispersed in 10 μ L A phases（H2O/0.1%FA）In.The instrument is EASY-
The system of nLC 1000 are simultaneously connected with Orbitrap Fusion mass spectrometer.4 μ L dispersion liquids are according to linear gradient
From 2% B phases in 110 minutes（Acetonitrile/0.1%FA）Enter analytical column to 40% B phase sample introductions（C18, 75 μm x 50 cm）.Color
Spectrum post stabilizes 10 minutes in initial conditions, and column flow rate is 200 nL/min.Laser voltage is 2.0 kV.Orbitrap mass spectrums are soft
Part automatically switches between MS and MS/MS patterns.Can reach the resolution ratio of m/z=200.The data obtained by mass spectrum are based on 2015 3
The Uniprot-SwissProt databases of the issue of the moon 11 carry out searching storehouse, and the fragment ion masses number tolerance degree of deviation is 0.050
Da, error rate（FDR）Less than 1%.
Healthy Human Serum is by that after the metal organic framework nano composite material enrichment that sulfonic group is modified, can recognize 177
Bar nitrogen end glycopeptide corresponds to 85 kinds of different glycoprotein.
Claims (9)
1. sulfonic group modification metal organic framework nano composite material synthetic method, it is characterised in that comprise the following steps that：
By FeCl3•6H2O is dissolved in ethylene glycol, and ultrasound after magnetic agitation to clarification, plus sodium acetate stirring is transferred to reactor, 200
Reacted 16 hours at DEG C, take out reactor, cooled down 12 hours, fully washing, be vacuum dried at 50 DEG C, obtain ferroso-ferric oxide
Magnetic ball；
By step（1）The ferroso-ferric oxide magnetic ball for being obtained is dissolved in trihydroxy methyl sulfonic group aminomethane buffer with dopamine hydrochloride
In, be stirred at room temperature reaction 16 hours, with magnet separation product after, fully washed with deionized water and absolute ethyl alcohol, at 50 DEG C
Vacuum drying, obtains the magnetic ball of poly-dopamine cladding；
The dispersion steps in dimethylformamide（2）The magnetic ball of the poly-dopamine cladding of gained, ultrasound adds chlorine to fully dispersed
Change zirconium to stir, heating stirring 60-360 minutes at 150 DEG C；
（4）In step（3）Part 2- sulfonic group terephthalic acid (TPA)s are added in gained system, is stirred, heating stirring at 150 DEG C
30 minutes, magnet separation product is used, fully washed with dimethylformamide, deionized water and absolute ethyl alcohol, the vacuum at 50 DEG C
Dry, obtain final product the metal organic framework nano composite material of gained sulfonic group modification.
2. the synthetic method of the metal organic framework nano composite material of sulfonic group according to claim 1 modification, it is special
It is step to levy（1）Middle FeCl3•6H2The quality corresponding volume ratio of O and ethylene glycol is（0.9-1.8）g：（50-100）ml.
3. the synthetic method of the metal organic framework nano composite material of sulfonic group according to claim 2 modification, it is special
It is step to levy（1）Middle FeCl3•6H2The quality corresponding volume ratio of O and ethylene glycol is 1.35g：75ml.
4. the synthetic method of the metal organic framework nano composite material of sulfonic group according to claim 1 modification, it is special
It is step to levy（2）Middle trihydroxy methyl sulfonic group aminomethane buffer is with solvent as deionized water and ethanol, deionized water and ethanol
Volume ratio is 1：1, pH=8.5.
5. the synthetic method of the metal organic framework nano composite material of sulfonic group according to claim 1 modification, it is special
It is step to levy（2）The mass ratio of middle ferroso-ferric oxide magnetic ball and dopamine hydrochloride is 2.4：1.
6. the synthetic method of the metal organic framework nano composite material of sulfonic group according to claim 1 modification, it is special
It is step to levy（3）The magnetic ball of middle poly-dopamine cladding and the mass ratio of zirconium chloride are 100 mg：150 mg, reaction temperature is
150 DEG C, the reaction time is 60 minutes.
7. the synthetic method of the metal organic framework nano composite material of sulfonic group according to claim 1 modification, it is special
It is step to levy（2）The magnetic ball and step of middle products therefrom poly-dopamine cladding（4）The quality of middle 2- sulfonic groups terephthalic acid (TPA)
Than for（75-125）mg：（280-360）Mg, reaction temperature is 120-180 DEG C, and the reaction time is 15-45 minutes.
8. the synthetic method of the metal organic framework nano composite material of sulfonic group according to claim 7 modification, it is special
It is step to levy（2）The magnetic ball and step of middle products therefrom poly-dopamine cladding（4）The quality of middle 2- sulfonic groups terephthalic acid (TPA)
Than being 100 mg：320 mg, reaction temperature is 150 DEG C, and the reaction time is 30 minutes.
9. a kind of metal organic framework nano composite material of the sulfonic group modification that synthetic method as claimed in claim 1 is obtained exists
Glycopeptide is enriched with and the application in Mass Spectrometric Identification, it is characterised in that：The metal organic framework nano composite material that sulfonic group is modified
Material dispersion liquid is configured as by solvent of ultra-pure water, the material dispersion liquid and target glycopeptide solution are added into 90% acetonitrile/0.1%
In trifluoroacetic acid buffer solution, mix simultaneously digest instrument in be incubated, nano composite material is gone out by centrifugation, with 90% acetonitrile/
0.1% trifluoroacetic acid and 80% acetonitrile/1% phosphate buffer washing nano composite material, are then washed with 30% acetonitrile/0.1% formic acid
It is de-, take 1 μ L eluents and target is directly put on MALDI-TOF MS sample introduction target plates, put again after drying plus 1 μ L concentration is 30mg/mL's
DHB solution carries out mass spectral analysis in matrix crystallization on the drop, is formed.
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| CN109675441B (en) * | 2019-01-11 | 2020-08-11 | 浙江大学 | Heparin sodium modified metal organic framework film and preparation method and application thereof |
| CN113150294A (en) * | 2021-03-05 | 2021-07-23 | 中国科学院兰州化学物理研究所 | Porous material with polycarboxylic acid ultrahigh hydrophilicity |
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