CN103877955B - Based on acid amide type integral post and the Synthesis and applications of hydrophilic interaction mechanism enrichment glycopeptide - Google Patents
Based on acid amide type integral post and the Synthesis and applications of hydrophilic interaction mechanism enrichment glycopeptide Download PDFInfo
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Abstract
The present invention relates to the Synthesis and applications of the special acid amide type integral post of a kind of glycopeptide enrichment.Described integral post adopts monomer with acid amides functional group and crosslinking agent, adds pore-foaming agent and the initator of proper proportion, the integral post that the method for being gathered by fast light is prepared in UV transparent quartz capillary.Such integral post has the advantages such as making is simple and quick, Stability Analysis of Structures is homogeneous, hydrophily is better, acid amides functional group is many, and integral post of the present invention can realize the on-column enrichment based on hydrophilic interaction mechanism to glycopeptide in micro-biological sample.In the structure of the pretreatment of biological sample glycoprotein and online glycoprotein assay platform, there is good practical value and application prospect.
Description
Technical field
The present invention relates to glycopeptide enriching apparatus, i.e. a kind of acid amide type integral post based on hydrophilic interaction mechanism enrichment glycopeptide and preparation thereof and the application in biological sample glycoprotein preconditioning technique.
Background technology
In the research field of proteomics, the Nomenclature Composition and Structure of Complexes of protein receives the concern of people always on the impact of its function, especially for the albumen of posttranslational modification, different decorating sites and modification group produce tremendous influence to the function of albumen, but the study hotspot become in proteomics because of the feature of itself low abundance and high complexity and difficult point.
Glycoprotein is the albumen that a class has important biomolecule meaning, has indispensable effect, and be biomarker and the clinical treatment target of some diseases in the bioprocess such as cell transport, intracellular signaling, immunological regulation, biological irritability.As an important technology in glycoprotein research, mass spectrum is adopted to make the 26S Proteasome Structure and Function exploration of people to glycoprotein have further understanding to the glycosylation site information of glycoprotein and the parsing of sugar chain structure.But glycoprotein abundance is lower, the ratio that the glycopeptide produced after enzymolysis accounts for total peptide section is little, in Mass Spectrometric Identification process glycopeptide information easily cover by other peptide segment informations, therefore in sample pretreatment process, enrichment effectively must be carried out to glycoprotein and glycopeptide.
Glycopeptide enrichment method conventional at present includes hydrazides oxidizing process, the material enrichment of boric acid functionalization and hydrophilic Interaction Chromatography.The wherein hydrophilic Interaction Chromatography glycopeptide that mainly utilizes sugar chain portion to bring and the difference of non-glycopeptide in hydrophily, thus realize the selective enrichment of glycopeptide, the feature of the method is can on the basis not destroying this body structure of glycopeptide, the various glycopeptide with dissimilar sugar chain is carried out to the enrichment of universality, simultaneously operating condition is gentle, and with following liquid-phase-mass spectrometry system compatible.In recent years, a lot of seminar has all developed the dissimilar material based on hydrophilic Interaction Chromatography, comprises commercialization agarose gel particle, hydrophilic polymers microballoon and surface hydrophilic and modifies (Selman, the M.H. such as magnetic ball, McDonnell, L.A., Palmblad, M., Ruhaak, L.R., Deelder, A.M., Wuhrer, M., Anal.Chem.2010,82,1073 – 1081.Yu, L.Li, X.Guo, Z.Zhang, X.Liang, X.Chem.Eur.J.2009,15,12618-12626; Yeh, C.H.Chen, S.H.Li, D.T.Lin, H.P.Huang, H.J.Chang, C.I.Shih, W.L.Chern, C.L.Shi, F.K.Hsu, J.L.J.Chromatogr.A2012,1224,70-78; ), the building-up process of these materials or operating process are more loaded down with trivial details comparatively speaking, and the enrichment that glycopeptide is carried out in off-line operation simultaneously can bring inevitable sample loss and time loss.And then researcher utilizes the device of a lot of glycopeptide enrichment of different Materials to overcome the problems referred to above, comprises (Lam, the M.P. such as commercial packed column, open tubular column and solid-phase micro-extracting device (SPEtips), Siu, S.O., Lau, E., Mao, X., Sun, H.Z., Chiu, P.C., Yeung, W.S., Cox, D.M., Chu, I.K., Anal.Bioanal.Chem.2010,398,791-804; Luo, Q., Rejtar, T., Wu, S.L., Karger, B.L., J.Chromatogr.A.2009,1216,1223 – 1231; Zhu, J.Wang, F.Chen, R.Cheng, K.Xu, B.Guo, Z.Liang, X.Ye, M.Zou, H., Anal.Chem.2012,84,5146-5153).
Summary of the invention
For studying Problems existing at present, the object of the invention is to develop the acid amide type integral post that a kind of glycopeptide enrichment of quick preparation is special, realize the selective enrichment good to glycopeptide by hydrophilic interaction mechanism, can realize post operates, reduce loss and the experimental period of sample.
For achieving the above object, the technical solution used in the present invention is:
With NVP and acrylamide for monomer, N, N'-methylene-bisacrylamide is crosslinking agent, under the condition of the pore-foaming agent of proper proportion and in appropriate solvent system, the mode of being polymerized by illumination forms integral material in UV transparent quartz capillary, thus prepares a kind of acid amide type integral post based on hydrophilic interaction mechanism enrichment glycopeptide fast.
Concrete steps prepared by described acid amide type integral post are as follows:
1) using the NVP (NVP) of mol ratio 0.5:1-1.5:1 and acrylamide (AM) as monomer, add the N that mole is the 50%-200% of amount of monomer, N'-methylene-bisacrylamide (MBA) is as crosslinking agent, add the pore-foaming agent 1 of mass ratio 0.5:1-2:1 simultaneously, 4-butanediol and lauryl alcohol, in mixed solution, the mass ratio of crosslinking agent and pore-foaming agent is 1:5-1:10, and initator azodiisobutyronitrile (AIBN), its addition is the 0.5%-2% of monomer and crosslinking agent gross mass, after mixing, again dimethyl sulfoxide (DMSO) is joined in above-mentioned solution, its volume fraction is made to be 30%-70%, vibration is to clarification, pass into the N of 30 seconds to 90 seconds
2,
2) mixed solution is passed in printing opacity capillary, under UV-irradiation condition, reaction 5-20 minute;
3) reaction terminates rear acetonitrile (ACN) and cleans cylinder in capillary, can obtain acid amide type integral post.
Obtained acid amide type integral post may be used for the hydrophilic mechanism selective enrichment of glycopeptide, and the concrete steps of its technical method are as follows:
1) take the protein sample of 1mg, after sex change, reductive alkylation and enzymolysis, be dissolved in 1mL85%-75%ACN(containing 0.1%FA) in solution;
2) 10cm acid amide type integral post is intercepted, by the above-mentioned 1mg/mL protein hydrolysate of 2 μ L, with 85%-75%ACN(containing 0.1%FA) with the flow velocity loading of 0.25-2 μ L/min, rinse 10-30min;
3) adopt 70%-50%ACN(containing 0.1%FA) solution wash-out is carried out to glycopeptide, collect the enriched product eluted, directly carry out mass spectral analysis.
Tool of the present invention has the following advantages:
1. the acid amide type integral post in the present invention, prepare simple and quick, Stability Analysis of Structures is homogeneous, and permeability is good, has good hydrophily and the compatibility with LC-MS system simultaneously.
2. two kinds of monomers using of the present invention and the hydrophily of crosslinking agent all relatively good, amide group is present in adopted monomer and crosslinking agent simultaneously, the integral post that ensure that preparation can there be enough acid amides functional groups for the hydrophilic mechanism enrichment of glycopeptide, improve that it is selective.
Accompanying drawing explanation
Fig. 1 is acid amide type integral post preparation flow figure.
Fig. 2 is the ESEM phenogram of integral post, and the multiplication factor of a and b is respectively 1000 and 5000 times.
Fig. 3-1 is acid amide type integral post to glycopeptide concentration effect figure in 2 μ g human serum immunoglobulins (IgG), a and b is respectively former state and enriched product.
Fig. 3-2 is that in Fig. 3-1, enriched product is the enlarged drawing within the scope of 2500-3500 in mass-to-charge ratio.
Fig. 4-1 is acid amide type integral post to the concentration effect figure of (mass ratio is 1:1) glycopeptide in the mixed solution of glycoprotein (human serum immunoglobulin, IgG) and non-glycoprotein (bovine serum albumin(BSA), BSA), a and b is respectively former state and enriched product.
Fig. 4-2 is that in Fig. 4-1, enriched product is the enlarged drawing within the scope of 2500-3500 in mass-to-charge ratio.
Fig. 5-1 is acid amide type integral post to the concentration effect figure of (mass ratio is 1:100000) glycopeptide in the mixed solution of glycoprotein (human serum immunoglobulin, IgG) and non-glycoprotein (bovine serum albumin(BSA), BSA), a and b is respectively former state and enriched product.
Fig. 5-2 is that in Fig. 5-1, enriched product is the enlarged drawing within the scope of 2500-3500 in mass-to-charge ratio.
Detailed description of the invention
Embodiment 1
1. the preparation of acid amide type integral post
As shown in Figure 1, take 11.5mg acrylamide, 22.2mgN-vinyl pyrrolidone and 56mgN, N'-methylene-bisacrylamide, add 420 μ L dimethyl sulfoxide (DMSO)s, after vibration is dissolved to clarification, then add 0.9mg azodiisobutyronitrile, be mixed to clarification; Add 0.15g1,4-butanediol and 0.3g lauryl alcohol, after mixing, pass into N
2o in 30 seconds removing solution
2, be circulated into by this solution in the long UV transparent quartz capillary of 12cm, two ends silica gel seals.Under 365nm UV-irradiation, reaction 15min.Take out capillary, pass into acetonitrile about 1h, remove unreacted material, thus obtained acid amide type integral post.
2. Monolithic Columns internal attribute
As shown in Figure 2, the integral post Stability Analysis of Structures of preparation is homogeneous, and integral material is closely adherent for scanning electron microscope (SEM) photograph, can observe the existence of hierarchical porous structure from the figure after amplification.
Embodiment 2
1. standard protein sample pretreatment
Taking 1mg human serum immunoglobulin (IgG) is dissolved in 1mL20mM ammonium bicarbonate soln, thermal response 10min is added at 90 DEG C, after being cooled to room temperature, then add 4 μ L1M dithiothreitol (DTT) solution (with the preparation of 20mM ammonium bicarbonate soln), at 56 DEG C, add thermal response 1.5h; After being cooled to room temperature, add 8 μ L1M iodoacetamide solution (with the preparation of 20mM ammonium bicarbonate soln), at room temperature lucifuge reaction 40min.After reaction terminates, add the trypsin solution (with the preparation of 20mM ammonium bicarbonate soln) of 40 μ L1mg/mL, add thermal response 20h at 37 DEG C, add appropriate formic acid (FA) afterwards and make the pH of solution at about 2-3 with cessation reaction, finally obtain 1mg/mL standard protein enzymolysis product.
Adopt C18 pre-column to carry out desalination, freeze-drying to above-mentioned enzymolysis product, finally sample dissolution contained 0.1%FA at 1mL80%ACN() in.
2. the on-column enrichment of glycopeptide
Intercept 10cm acid amide type integral post, by 2 μ L above-mentioned 1mg/mL standard protein enzymolysis product, with 80%ACN(containing 0.1%FA) with the flow velocity loading of 1 μ L/min, rinse 20min, then adopt 70%ACN(containing 0.1%FA) condition wash-out 10min is carried out to glycopeptide, collect the enriched product eluted, directly carry out mass spectral analysis.
3.MALDI-TOFMS(Matrix-assisted laser desorption ionization) analyze
By 1 μ L enriched product and 1 μ L2,5-dihydroxy-benzoic acid (DHB) matrix (20mg/mLDHB is dissolved in 60% acetonitrile solution containing 0.1% trifluoroacetic acid) is put successively on MALDI target plate, after sample spot drying, carry out Mass Spectrometric Identification.MALDI-TOFMS experiment carries out on Ultraflex III TOF/TOF (BrukerDaltonics, Bremen, Germany), adopts linear reflective positive ion mode during detection.As shown in Fig. 3-1b, the glycopeptide signal strength signal intensity in IgG comparatively stoste (Fig. 3-1a) significantly improves, by documents (Yu, L.Li, X.Guo, Z.Zhang, X.Liang, X.Chem.Eur.J.2009,15,12618-12626), after the enrichment of acid amide type integral post, obtain altogether the glycopeptide 18 (Fig. 3-2) that signal to noise ratio is greater than 3, and most of non-glycopeptide is obtained for removal, shows that material has good glycopeptide accumulation ability and selective.
Embodiment 3
Glycopeptide selective enrichment under the interference of 1.BSA enzymolysis product
By 1mg/mLIgG and the BSA enzymolysis product prepared (being dissolved in the 80%ACN containing 0.1%FA) 1:1 and 1:100000 mixing in mass ratio, intercept 10cm acid amide type integral post, the mixed solution of the above-mentioned standard protein enzymolysis product of loading 2 μ L, with 80%ACN(containing 0.1%FA) rinse 30min, then adopt 70%ACN(containing 0.1%FA) condition wash-out 10min is carried out to glycopeptide, collect the enriched product eluted, directly carry out MALDI-TOFMS analysis.
2.MALDI-TOFMS analyzes
1 μ L enriched product and 1 μ LDHB matrix (20mg/mLDHB is dissolved in 60% acetonitrile solution containing 0.1% trifluoroacetic acid) are put successively on MALDI target plate, after sample spot drying, carries out Mass Spectrometric Identification.As shown in Fig. 4-1,4-2 and Fig. 5-1,5-2, mass ratio is that the biased sample of 1:1 is after the enrichment of acid amide type integral post, this integral material can selective enrichment to the glycopeptide of 10 in IgG (Fig. 4-2), eliminate the interference of most of BSA peptide hydrolysis simultaneously.Further increase interference ratio to 1:100000, still can selective enrichment to the glycopeptide of 6 in IgG (Fig. 5-2), demonstrate the good antijamming capability of this integral post and using value in actual sample.
Such integral post has the advantages such as making is simple and quick, Stability Analysis of Structures is homogeneous, hydrophily is better, acid amides functional group is many, and integral post of the present invention can realize the on-column enrichment based on hydrophilic interaction mechanism to glycopeptide in micro-glycoprotein sample.In the structure of glycoprotein sample pretreatment and online glycoprotein assay platform, there is good practical value and application prospect.
Claims (5)
1. based on the preparation method of the acid amide type integral post of hydrophilic interaction mechanism enrichment glycopeptide, it is characterized in that: adopt monomer and the crosslinking agent with acid amides functional group, add pore-foaming agent and initator, in UV transparent quartz capillary, prepare the homogeneous and stable integral post of structure by the method for light initiation polymerization, with amide group there is good hydrophily;
Concrete steps are:
1) join in dicyandiamide solution by the mixed solution of monomer, crosslinking agent, pore-foaming agent and initator, vibration mixing, mixed liquor passes into the N of 30 seconds to 90 seconds
2;
Described monomer is NVP (NVP) and the acrylamide (AM) of mol ratio 0.5:1-1.5:1, described crosslinking agent is N, N'-methylene-bisacrylamide (MBA), in mixed solution, the mol ratio of monomer and crosslinking agent is 0.5:1-2:1;
Described dicyandiamide solution is dimethyl sulfoxide (DMSO), and in reaction solution, shared volume is the 30%-70% of mixed liquor cumulative volume;
2) mixed liquor is passed in UV transparent capillary, under UV-irradiation condition, reaction 5-20 minute;
3) reaction terminates rear acetonitrile (ACN) and cleans cylinder in capillary, can obtain the acid amide type integral post having hollow porous and tie.
2. according to preparation method according to claim 1, it is characterized in that: described UV transparent quartz capillary is the UV transparent quartz capillary of 50-150 μm of internal diameter.
3. according to preparation method described in claim 1, it is characterized in that: described initator azodiisobutyronitrile (AIBN), its addition is the 0.5%-2% of monomer and crosslinking agent gross mass.
4. according to preparation method described in claim 1, it is characterized in that: described pore-foaming agent is BDO and lauryl alcohol, and mass ratio is between the two 0.5:1-2:1, in mixed solution, the mass ratio of crosslinking agent and pore-foaming agent is 1:5-1:10.
5. a preparation method according to claim 1, is characterized in that: the acid amide type integral post prepared is for the enrichment of glycopeptide in biological sample.
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| CN108169392A (en) * | 2016-12-07 | 2018-06-15 | 中国科学院大连化学物理研究所 | A kind of method of the amino-containing capillary copolymer material enrichment glycopeptide of richness |
| CN108168992B (en) * | 2016-12-07 | 2020-08-25 | 中国科学院大连化学物理研究所 | Glycopeptide enrichment and separation method based on two-dimensional porous crystal nitrogen-doped carbon material |
| CN110872383B (en) * | 2018-08-29 | 2021-06-22 | 中国科学院大连化学物理研究所 | Preparation and application of penicillamine modified hierarchical pore hybrid material |
| CN109633031A (en) * | 2019-01-23 | 2019-04-16 | 洛阳理工学院 | A kind of detection method of Ractopamine |
| CN111821961A (en) * | 2020-07-24 | 2020-10-27 | 福州大学 | High-hydrophilicity aptamer functional extraction material and application thereof |
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