CN107607640A - A kind of glycopeptide enrichment of nano composite material of boric acid modified and Mass Spectrometry detection method - Google Patents
A kind of glycopeptide enrichment of nano composite material of boric acid modified and Mass Spectrometry detection method Download PDFInfo
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Abstract
A kind of glycopeptide enrichment of the nano composite material of boric acid modified proposed by the present invention and Mass Spectrometry detection method, concretely comprise the following steps the nano composite material of boric acid modified being configured to material dispersion liquid by solvent of ultra-pure water；The material dispersion liquid and target glycopeptide solution are added in ammonium bicarbonate buffers, mixes and shakes enrichment；Nano composite material is centrifuged out, is fully washed with ammonium bicarbonate buffers, then with the elution of the trifluoroacetic acid of 50% acetonitrile/1%；Eluent mass spectral analysis；Wherein, the nano composite material of described boric acid modified is the metal organic framework nano composite material of boric acid modified.This method simple and fast, the nano composite material of the boric acid modified of synthesis have good biocompatibility, the selective enrichment available for glycopeptide in complex biological sample.
Description
Technical field
The invention belongs to advanced nano material and field of nanometer technology, and in particular to a kind of nano combined material of boric acid modified
The glycopeptide enrichment of material and Mass Spectrometry detection method, more particularly to a kind of metal organic framework of boric acid modified（MOF）Glycopeptide enrichment
With the method for MALDI-TOF MS and LC-MS/MS detection.
Background technology
The glycosylation of protein is posttranslational modification important and universal in life process, and they are passed with intercellular signal
Pass, cell division, propagation, many important complex biological processes such as differentiation and interaction it is closely bound up.Some researchs show,
The expression of glycopeptide is abnormal, can be as mankind's major disease such as the biological marker of many diseases, especially cancer.So to sugar
Early diagnosis of the research of peptide to disease has great significance.But the abundance of glycopeptide is often very low, and their mass spectrums
Response can be suppressed by the non-glycopeptide of high abundance and protein, and the salinity and surfactant in sample equally also can be to its mass spectrums
Behavior produces interference so that its ionizing efficiency is very low, and Mass Spectrometer Method is relatively difficult.Therefore analyzed using mass spectrometry method
Before glycopeptide in complex biological sample, it is very necessary to carry out selective enrichment 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. are found, tree species for bio-energy source develops rapidly.Due to mass-spectrometric technique（Mass
Spectrometry, MS）The characteristics of with high accuracy, high sensitivity and automation mechanized operation, and it being capable of accurate measurement 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, the identification of protein can be carried out rapidly, so as to provide reliable basis for the structure elucidation of protein.Therefore
Mass-spectrometric technique turns into platform indispensable in current proteomics research undisputedly, and the information quality of mass spectrometric data is straight
Connect the reliability for determining identification of proteins and identification quantity.
With deepening continuously for Recent study, many methods are all used to selective separation enrichment glycosylation albumen and more
Peptide, such as boric acid affinity chromatography, hydrazine chemistry, hydrophilic interaction chromatogram（HILIC）, red, orange, green, blue, yellow (ROGBY), volume exclusion method etc..Its
Mesoboric acid method causes extensive attention in terms of glycopeptide enrichment.
Metal-organic framework materials refer to that by organo-functional group be node centered on support, metal ion or metal cluster, lead to
Cross the network porous material for the three-dimensional periodic with regular nano pore being self-assembly of.With very big ratio table
The excellent properties such as area, stable nanoscale duct, regulatable pore passage structure, good heat endurance, and undersaturated match somebody with somebody
Coordination may occur with the analyte containing functional groups such as carboxyl, sulfonic groups for position metal, and MOFs materials turn 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.
Therefore, it is necessary to which the glycopeptide enrichment that is used for for designing a kind of nano composite material of new boric acid modified is examined with mass spectrum
Survey.
The content of the invention
Present invention aims at the glycopeptide enrichment for providing a kind of nano composite material of boric acid modified and Mass Spectrometry detection method.
A kind of glycopeptide enrichment of the nano composite material of boric acid modified provided by the invention and Mass Spectrometry detection method, specific step
It is rapid as follows：The nano composite material of boric acid modified is configured to 10 mg/mL material dispersion liquid using ultra-pure water as solvent；Should
Material dispersion liquid is added in 50 mM ammonium bicarbonate buffers with target glycopeptide solution, mixes and concussion is enriched with 45 points under 37 °C
Clock；Nano composite material is centrifuged out, is fully washed with 50 mM ammonium bicarbonate buffers, then with the trifluoro of 50% acetonitrile/1%
Acetic acid elutes；Eluent mass spectral analysis；
Wherein, the nano composite material of described boric acid modified is the metal organic framework nano composite material of boric acid modified.
In the present invention, mass spectral analysis is MALDI-TOF MS mass spectral analyses, is concretely comprised the following steps：1 μ L eluent points are taken to arrive
On MALDI-TOF MS sample introduction target plates, dry after put again plus 1 μ L concentration be 30 mg/mL DHB solution in
On the drop, matrix crystallization is formed, MALDI-TOF MS mass spectral analyses are carried out after drying.
In the present invention, mass spectral analysis is LC-MSMS mass spectral analyses, is concretely comprised the following steps：10 are dispersed in after eluent is freezed
In μ L A phases, 4 μ L dispersion liquids carried out LC-MSMS mass spectrums point in 110 minutes according to linear gradient from 2% B phases to 40% B phases
Analysis, wherein, A phases are the formic acid of water/0.1%, and B phases are the formic acid of acetonitrile/0.1%.
In the present invention, the specific preparation process of metal organic framework nano composite material of described boric acid modified is as follows：
（1）By FeCl3•6H2O, sodium citrate, ammonium acetate are dissolved in ethylene glycol, and magnetic agitation is transferred to anti-to clarifying after ultrasound
Kettle is answered, is reacted 16 hours at 200 DEG C, reactor is taken out, cool overnight, pours out magnetic ball, fully washed with deionized water and ethanol,
Vacuum drying obtains ferroso-ferric oxide magnetic ball at 50 DEG C；
（2）The aqueous solution of polyvinylpyrrolidone and polyethyleneimine is configured, by step（1）The ferroso-ferric oxide magnetic ball point of gained
Dissipate in the aqueous solution, at room temperature stirring reaction 6-20 hours, polyvinylpyrrolidone and the magnetic of polyethyleneimine cladding is made
Ball, magnet separation product, fully washed with dimethylformamide and water, be dried in vacuo at 50 DEG C；
（3）The dispersion steps in dimethylformamide and acetonitrile（2）Products therefrom, it is ultrasonic to fully dispersed；Add nine hydration nitre
Sour iron, to benzene hypoboric acid, terephthalic acid (TPA), stir, heating stirring 60 minutes at 120 DEG C；
（4）In step（3）Fe(NO3)39H2O is added in gained system, to benzene hypoboric acid, terephthalic acid (TPA), stirred,
Heating stirring 30 minutes at 120-150 DEG C, were repeated 3 times every 30 minutes, magnet separation product, with dimethylformamide, go from
Sub- water and absolute ethyl alcohol are fully washed, and the metal organic framework nanometer for the boric acid modified being dried in vacuo at 50 DEG C described in producing is answered
Condensation material.
In the present invention, step（1）Middle FeCl3•6H2O and the quality corresponding volume ratio of ethylene glycol are（0.8-1.5）g：（40-
80）ml.
In the present invention, step（1）Middle FeCl3•6H2O and the quality corresponding volume ratio of ethylene glycol are 1.15g：60ml.
In the present invention, step（1）Middle FeCl3•6H2O, sodium citrate, the mass ratio of ammonium acetate are：1.15：0.45：3.30.
In the present invention, step（2）The mass ratio of middle ferroso-ferric oxide magnetic ball, polyvinylpyrrolidone and polyethyleneimine is
1:10:4。
In the present invention, step（3）The magnetic ball and Fe(NO3)39H2O of middle polyvinylpyrrolidone and polyethyleneimine cladding,
Mass ratio to benzene hypoboric acid, terephthalic acid (TPA) is 1：16：8：8.
In the present invention, step（4）Middle Fe(NO3)39H2O, the mass ratio to benzene hypoboric acid, terephthalic acid (TPA) are 160 mg：
80 mg：80mg, reaction temperature are 120 DEG C, and the reaction time is 30 minutes, is repeated 3 times.
The beneficial effects of the present invention are：The synthetic method of the nano composite material of boric acid modified and its application, synthesis side
Method is simple and quick, and boric acid base group and glycopeptide can be combined closely by interaction, and MOF improves active surface and preferable body
Pore passage structure.The low-abundance glycopeptide that this nano composite material can be used in optionally enriched biological sample, and be used for
MALDI-TOF MS and LC-MS/MS are detected.Material provided by the present invention to glycopeptide test limit up to 500 amol/ μ L, to non-
The selectivity of glycopeptide is up to 1：100（Mass ratio）, nitrogen end glycopeptide can be detected directly from human serum and corresponds to different glycoprotein.
It is high by enrichment peptide fragment signal to noise ratio multiplication factor, there is preferable selectivity and high sensitivity, to the glycopeptide in complex biological sample
Detection have good effect.
The present invention has synthesized the nano composite material with reference to traditional MOF materials and boric acid material advantages first, and is applied to
The separation and concentration of glycopeptide.Due to the interaction between higher MOF surface area and boric acid base group and glycopeptide so that the nanometer is answered
Condensation material can carry out selective enrichment to the glycopeptide in complex biological sample, substantially increase its mass signal.Glycopeptide is examined
Limit is surveyed up to 500 amol/ μ L, to the selectivity of non-glycopeptide up to 1：100（Mass ratio）, nitrogen can be directly detected from human serum
End glycopeptide corresponds to different glycoprotein.
The synthetic method of the nano composite material of boric acid modified involved by this paper and its application, synthetic method are simply fast
Speed, reaction site is provided by boric acid base group, is combined closely with glycopeptide, reaches the effect of enrichment glycopeptide.And this material tool
It is magnetic, consistent excellent properties that are easy to operate, being easy to be made, possess MOF, boric acid base group and glycopeptide can be tight by interacting
Close combination, MOF improve active surface and preferable body pore passage structure.This nano composite material can be used for optionally being enriched with
Low-abundance glycopeptide in biological sample, and detected for MALDI-TOF MS and LC-MS/MS.
Brief description of the drawings
Fig. 1 is the electron micrograph of the nano composite material for the boric acid modified that embodiment 1 synthesizes, wherein：A), b) it is
Fe3O4@PVP/PEI@MOF (B) transmission electron microscope photo, c) it is Fe3O4@PVP/PEI@MOF (B) scanning electron shows
Micro mirror photo；
Fig. 2 is the energy dispersion X-ray spectrum and the content distribution of elements of the nano composite material for the boric acid modified that embodiment 1 synthesizes
Figure；
Fig. 3 is that the magnetic hysteresis of the nano composite material for the boric acid modified that embodiment 1 synthesizes returns curve map；
Fig. 4 is the IR Characterization spectrogram of the nano composite material for the boric acid modified that embodiment 1 synthesizes；
Fig. 5 is that the Raman of the nano composite material for the boric acid modified that embodiment 1 synthesizes characterizes spectrogram；
Fig. 6 is the X-ray diffraction pattern of the nano composite material for the boric acid modified that embodiment 1 synthesizes；
The HRP enzymolysis liquids that Fig. 7 is 200 fmol/ μ L in embodiment 2 are by former before the nano composite material a) enrichments of boric acid modified
The mass spectrogram of eluent after liquid b) enrichments；1 pmol/ μ L IgG enzymolysis liquids are rich by the nano composite material c) of boric acid modified
The mass spectrogram of eluent after stoste d) is enriched with before collection；
200 fmol/ μ L HRP enzymolysis liquids pass through the fresh obtained materials of nano composite material a) of boric acid modified in Fig. 8 embodiments 2
The mass spectrogram of eluent after gained is enriched with after the mass spectrogram b) material circulations of eluent use 5 times after material gained is enriched with；
200 fmol/ μ L HRP enzymolysis liquids pass through the fresh obtained materials of nano composite material a) of boric acid modified in Fig. 9 embodiments 2
The mass spectrogram of eluent after gained is enriched with after the mass spectrogram b) materials of eluent preserve 1 month at -20 DEG C after material gained is enriched with；
Figure 10 is HRP enzymolysis liquids mass spectrogram, HRP enzymolysis liquid after the nano composite material enrichment of boric acid modified in embodiment 2
Concentration is：a) 25 fmol/μL; b) 5 fmol/μL; c) 1 fmol/μL; d) 0.5 fmol/μL；
Figure 11 is that mass ratio is 1 in embodiment 3:Mass spectrogram before the mixed solution a) enrichments of 50 HRP and BSA enzymolysis liquids；b)
The mass spectrogram after the nano composite material enrichment of boric acid modified；Mass ratio is 1:The mixing of 100 HRP and BSA enzymolysis liquids is molten
Mass spectrogram before liquid c) enrichments；D) mass spectrogram after the nano composite material enrichment of boric acid modified.
Embodiment
The following examples are the further explanations to the present invention, rather than limit the scope of the present invention.
Embodiment 1：A kind of synthesis of the nano composite material of boric acid modified.
（1）Spent glycol synthesizes ferroso-ferric oxide magnetic ball as solvent, by 1.15g FeCl3•6H2O, 0.45g citric acids
Sodium, 3.30g ammonium acetates are dissolved in 60ml ethylene glycol, magnetic agitation（Gloves seal）1h is extremely clarified（Gloves seal）.After ultrasonic 5min,
It is transferred to reactor, 200 DEG C, 16h.Take out reactor, cool overnight.Magnetic ball is poured out, is washed 3 times（Each ultrasonic 5min）.With
Deionized water and ethanol fully wash magnetic ball, limpid pure to cleaning solution, are dried in vacuo at 50 DEG C；
（2）Configure polyvinylpyrrolidone（PVP）And polyethyleneimine（PEI）The aqueous solution, by step（1）Obtain four oxidations three
The mg of ferromagnetic ball 100 and 1g polyvinylpyrrolidones, 0.4g polyethyleneimines are soluble in water, and reaction 12h is stirred at room temperature.Magnet point
From product, fully washed with dimethylformamide and water, be dried in vacuo at 50 DEG C；
（3）The dispersion steps in 8 mL dimethylformamides and 8mL acetonitriles（2）The mg of products therefrom 20, ultrasound a period of time, fill
It is scattered；Add Fe(NO3)39H2O 320mg, to benzene hypoboric acid 160mg, terephthalic acid (TPA) 160mg, stir, 120 DEG C add
Thermal agitation 60 minutes；
（4）In step（3）Fe(NO3)39H2O 160mg is added in gained system, to benzene hypoboric acid 80mg, terephthalic acid (TPA) mg,
Stir, 150 DEG C of heating stirrings 30 minutes, be repeated 3 times, magnet separation product, with dimethylformamide, go every 30 minutes
Ionized water and absolute ethyl alcohol fully wash, and are dried in vacuo at 50 DEG C.
Fig. 1 is electron scanning micrograph and the transmission of the nano composite material for the boric acid modified that embodiment 1 synthesizes
Electron micrograph.Scanning electron microscope diagram can be seen that the polymer P DA layers of the layer of magnetic ball outer cladding, repairing
After adoring MOF, the crystallization shape on surface is different from the smooth layer of polymer, there is clearly crystal appearance；Transmission electron microscope figure
It can be seen that polymer P DA@MOF layers are about 30 nanometer thickness；ESEM model Philips XL30, by boric acid after purification
The synthetic method of the nano composite material of modification and its application are uniformly applied on conducting resinl, and SEM signs are carried out after metal spraying；Transmission
Electronic Speculum model JEM-2100F (J0EL), by the synthetic method of the nano composite material of boric acid modified after purification and its application
Alcohol dispersion liquid drop in and be covered with the copper mesh of carbon film, transmission electron microscope observation is carried out after drying and is taken pictures；
Fig. 2 is the content distribution of elements of the nano composite material for the boric acid modified that embodiment 1 synthesizes, wherein the quality of S elements point
Number accounts for 2.9%, is consistent with expection, table is as follows；
| The element number of nuclear charges | The symbol of element | Element term | Mass fraction |
| 5 | B K | Boron | 1.86 |
| 6 | C K | Carbon | 7.74 |
| 8 | O K | Oxygen | 26.92 |
| 26 | Fe K | Iron | 48.79 |
| 29 | Cu K | Copper | 14.69 |
Fig. 3 is the magnetic hysteresis loop of the nano composite material for the boric acid modified that embodiment 1 synthesizes；Although coated polymer and
After MOF layers, the magnetic response of material has declined, but still remains higher magnetic response intensity, about 43.7 emug-1；
Fig. 4 be embodiment 1 synthesize boric acid modified nano composite material IR Characterization spectrogram, the nano material occur compared with
More characteristic peaks, such as 3400cm-1Carboxyl characteristic peak, the 1500-1600cm at place-1Phenyl ring characteristic peak, the 560cm at place-1The Fe- at place
O-Fe vibration peaks, illustrate the successful synthesis of material；
Fig. 5 is that the Raman of the nano composite material for the boric acid modified that embodiment 1 synthesizes characterizes spectrogram, as IR Characterization spectrogram
Complementary data,<500cm-1There is the characteristic peak of magnetic ball, 1500-1600cm in place-1There is phenyl ring feature after being in coated polymer
Peak, illustrate the successful synthesis of material；
The X-ray diffraction pattern of the nano composite material for the boric acid modified that Fig. 6 synthesizes for embodiment 1,2 θ=8.2,13.0,
19.2,30.8 ° come from MOF, and 2 θ=33.3, and 34.2,36.0,57.2,62.6 ° come from magnetic ball kernel, this
Also the successful synthesis of magnetic MOF materials is just illustrated；X-ray diffractometer model Bruker D4 X-ray
diffractometer。
Embodiment 2：The nano composite material for the boric acid modified that embodiment 1 is synthesized is as SPME adsorbing separation
Medium is used for the enrichment of low concentration HRP enzymolysis liquids and MALDI-TOF MS are detected.
（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, is boiled ten minutes.It is 1 according to mass ratio:50 pancreas
The ratio of protease and standard protein, trypsase (trypsin) is added, 37 °C are incubated 16 hours, can obtain 2 mg/mL's
HRP tryptose enzymolysis liquids；4 mg IgG standard proteins accurately are weighed, it is 4 mg/ to be made into concentration with 25 mM ammonium bicarbonate solns
ML standard protein solution, pH are about 8.3, are 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 aqueous solution of the nano composite material of 10 mg/mL boric acid modifieds is prepared with ultra-pure water.Take 20 μ L material
Solution removes supernatant, addition cushioning liquid is dilute in 0.6 mL centrifuge tube after washing 2 times with 50 mM ammonium bicarbonate buffers
Release the HRP enzymolysis liquids of rear various concentrations（Cumulative volume is 100 μ L）, mix, concussion enrichment 45 minutes under 37 °C；Centrifuge material
Material, sucks supernatant, with 50 mM ammonium bicarbonate buffers detergent three times, then adds the trifluoro of acetonitriles of 10 μ L 50%/1%
Acetic acid, 37 DEG C of concussions elute 30 minutes, centrifuge material, suction out the standby rear use of eluent.
（3）Point target：Take 1 μ L steps（2）Described eluent point is on MALDI-TOF MS sample introduction target plates, after drying again
Point plus the 2,5- dihydroxy-benzoic acids that 1 μ L concentration is 30 mg/mL（DHB）Solution is dried on the drop, forming matrix crystallization
Carry out mass spectral analysis again afterwards.
（4）Mass spectral analysis is inhaled using the synthetic method of the nano composite material of boric acid modified and its application as SPME
Attached separating medium is enriched with obtained glycopeptide and compared with the stoste mass spectrogram before enrichment.
Concentration be 200 fmol/ μ L HRP enzymolysis liquids by boric acid modified nano composite material synthetic method and its
Occur 19 glycopeptide for belonging to HRP peaks 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 1 pmol/ μ L IgG enzymolysis liquids pass through the synthetic method of the nano composite material of boric acid modified and its answered
Occurs the glycopeptide peak that twenty-one provision belongs to IgG after enrichment, 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）.
Embodiment 3：The nano composite material for the boric acid modified that embodiment 1 is synthesized is situated between as SPME adsorbing separation
Matter is used for HRP enzymolysis liquids and bovine serum albumin(BSA)（BSA）The enrichment of the mixed solution of enzymolysis liquid detects with MALDI-TOF MS.
（1）The preparation of standard protein enzymolysis liquid：2 mg standard proteins HRP and 5 mg standard protein BSA accurately are weighed, are used
25 mM ammonium bicarbonate solns are made into the standard protein solution that concentration is 2 mg/mL and 5 mg/mL, and pH is about 8.3, boils 10
Minute.It is 1 according to mass ratio:50 trypsase and the ratio of standard protein, trypsase (trypsin) is added, 37 °C incubate
Educate 16 hours, can obtain 2 mg/mL HRP tryptoses enzymolysis liquid and 5 mg/mL BSA enzymolysis liquids.
（2）The enrichment of sample：
Glycopeptide is enriched with：With ultra-pure water prepare 10 mg/mL boric acid modifieds nano composite material synthetic method and its application it is molten
Liquid.20 μ L material solution is taken to remove supernatant after washing 2 times with 50 mM ammonium bicarbonate buffers in 0.6 mL centrifuge tube,
1 μ L 2 mg/mL HRP tryptose enzymolysis liquids are first added, are respectively 1 according to HRP and BSA mass ratio:50、1:100 add
BSA enzymolysis liquids, then adding 50 mM ammonium bicarbonate buffers of respective volume makes system be made into the body that cumulative volume is 100 μ L
System, mix, concussion enrichment 45 minutes under 37 °C；Material is centrifuged, supernatant is sucked, with 50 mM ammonium bicarbonate buffers
Detergent three times, then adds the trifluoroacetic acid solution of 10 μ L 50% acetonitrile/1%, 37 DEG C of concussion elutions 30 minutes, centrifugation point
From material, the standby rear use of eluent is suctioned out.
（3）Point target：Take 1 μ L steps（2）Described eluent point is on MALDI-TOF MS sample introduction target plates, after drying again
Point plus the 2,5- dihydroxy-benzoic acids that 1 μ L concentration is 30 mg/mL（DHB）Solution is dried on the drop, forming matrix crystallization
Carry out mass spectral analysis again afterwards.
（4）Mass spectral analysis is inhaled using the synthetic method of the nano composite material of boric acid modified and its application as SPME
Attached separating medium is enriched with obtained glycopeptide and compared with the stoste mass spectrogram before enrichment.
Mass ratio is 1:Synthesis side of 50 HRP and BSA the enzymolysis mixed liquor by the nano composite material of boric acid modified
After method and its application enrichment, it will be clear that 13 glycopeptide peaks for deriving from HRP from mass spectrogram（m/z=2591.4,
m/z=2611.4, m/z=3087.7, m/z=3222.9, 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=4838.7, m/z=4984.7）.
Embodiment 4：The nano composite material for the boric acid modified that embodiment 1 is synthesized is situated between as SPME adsorbing separation
Matter is used for glycopeptide enrichment in Healthy Human Serum sample and detected with MALDI-TOF MS and LC-MS/MS.
（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 at 37 DEG C add 20 mM in the dark
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 aqueous solution of the nano composite material of 10 mg/mL boric acid modifieds is prepared with ultra-pure water.Take 100 μ L material
Expect that solution in 0.6 mL centrifuge tube, removes supernatant, adds 100 μ L with 50 after washing 2 times with 50 mM ammonium bicarbonate buffers
The sero-enzyme solution of mM ammonium bicarbonate buffers dilution freezes liquid, mixes, concussion enrichment 45 minutes under 37 °C；Centrifuge material
Material, sucks supernatant, with 50 mM ammonium bicarbonate buffers solution detergent three times, then adds 10 μ L 50% acetonitrile/1%
Trifluoroacetic acid solution, 37 DEG C of concussions elute 30 minutes, centrifuge material, suction out standby after eluent freezes.（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.It 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%.
After synthetic method and its application enrichment of the Healthy Human Serum by the nano composite material of boric acid modified, it can recognize
209 nitrogen end glycopeptide corresponds to 89 kinds of different glycoprotein.
Claims (10)
1. a kind of glycopeptide enrichment of nano composite material of boric acid modified and Mass Spectrometry detection method, it is characterised in that specific steps are such as
Under：The nano composite material of boric acid modified is configured to 10 mg/mL material dispersion liquid using ultra-pure water as solvent；By the material
Dispersion liquid is added in 50 mM ammonium bicarbonate buffers with target glycopeptide solution, mixes and concussion is enriched with 45 minutes under 37 °C；From
The heart isolates nano composite material, is fully washed with 50 mM ammonium bicarbonate buffers, is then washed with the trifluoroacetic acid of 50% acetonitrile/1%
It is de-；Eluent mass spectral analysis；
Wherein, the nano composite material of described boric acid modified is the metal organic framework nano composite material of boric acid modified.
2. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 1 and Mass Spectrometry detection method, it is special
Sign is that the mass spectral analysis is MALDI-TOF MS mass spectral analyses, concretely comprises the following steps：1 μ L eluents point is taken to MALDI-TOF
On MS sample introduction target plates, put again after drying plus DHB solution that 1 μ L concentration is 30 mg/mL be on the drop,
Matrix crystallization is formed, MALDI-TOF MS mass spectral analyses are carried out after drying.
3. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 1 and Mass Spectrometry detection method, it is special
Sign is that the mass spectral analysis is LC-MSMS mass spectral analyses, concretely comprises the following steps：10 μ L A phases are dispersed in after eluent is freezed
In, 4 μ L dispersion liquids carried out LC-MSMS mass spectral analyses in 110 minutes according to linear gradient from 2% B phases to 40% B phases, its
In, A phases are the formic acid of water/0.1%, and B phases are the formic acid of acetonitrile/0.1%.
4. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 1 and Mass Spectrometry detection method, it is special
Sign is that the preparation process of the metal organic framework nano composite material of described boric acid modified is as follows：
By FeCl3•6H2O, sodium citrate, ammonium acetate are dissolved in ethylene glycol, and magnetic agitation is transferred to reaction to clarifying after ultrasound
Kettle, react 16 hours at 200 DEG C, take out reactor, cool overnight, pour out magnetic ball, fully washed with deionized water and ethanol,
Vacuum drying obtains ferroso-ferric oxide magnetic ball at 50 DEG C；
The aqueous solution of polyvinylpyrrolidone and polyethyleneimine is configured, by step（1）The ferroso-ferric oxide magnetic ball of gained disperses
In the aqueous solution, at room temperature stirring reaction 6-20 hours, polyvinylpyrrolidone and the magnetic ball of polyethyleneimine cladding is made,
Magnet separation product, fully washed with dimethylformamide and water, be dried in vacuo at 50 DEG C；
The dispersion steps in dimethylformamide and acetonitrile（2）Products therefrom, it is ultrasonic to fully dispersed；Add nine nitric hydrates
Iron, to benzene hypoboric acid, terephthalic acid (TPA), stir, heating stirring 60 minutes at 120 DEG C；
In step（3）Fe(NO3)39H2O is added in gained system, to benzene hypoboric acid, terephthalic acid (TPA), stirred, 120-
Heating stirring 30 minutes at 150 DEG C, were repeated 3 times every 30 minutes, magnet separation product, with dimethylformamide, deionized water
Fully washed with absolute ethyl alcohol, the nano combined material of metal organic framework for producing described boric acid modified is dried in vacuo at 50 DEG C
Material.
5. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 4 and Mass Spectrometry detection method, it is special
Sign is step（1）Middle FeCl3•6H2O and the quality corresponding volume ratio of ethylene glycol are（0.8-1.5）g：（40-80）ml.
6. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 5 and Mass Spectrometry detection method, it is special
Sign is step（1）Middle FeCl3•6H2O and the quality corresponding volume ratio of ethylene glycol are 1.15g：60ml.
7. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 4 and Mass Spectrometry detection method, it is special
Sign is step（1）Middle FeCl3•6H2O, sodium citrate, the mass ratio of ammonium acetate are：1.15：0.45：3.30.
8. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 4 and Mass Spectrometry detection method, it is special
Sign is step（2）The mass ratio of middle ferroso-ferric oxide magnetic ball, polyvinylpyrrolidone and polyethyleneimine is 1:10:4.
9. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 4 and Mass Spectrometry detection method, it is special
Sign is step（3）The magnetic ball and Fe(NO3)39H2O of middle polyvinylpyrrolidone and polyethyleneimine cladding, to benzene hypoboric acid,
The mass ratio of terephthalic acid (TPA) is 1：16：8：8.
10. the glycopeptide enrichment of the nano composite material of boric acid modified according to claim 4 and Mass Spectrometry detection method, it is special
Sign is step（4）Middle Fe(NO3)39H2O, the mass ratio to benzene hypoboric acid, terephthalic acid (TPA) are 160 mg：80 mg：80mg,
Reaction temperature is 120 DEG C, and the reaction time is 30 minutes, is repeated 3 times.
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