CN107560920A - A kind of new material of sialylated glycopeptide of concentration and separation and its application - Google Patents
A kind of new material of sialylated glycopeptide of concentration and separation and its application Download PDFInfo
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Abstract
The invention discloses a kind of method of the sialylated glycopeptide of specific enrichment.Claimed g C3N4Application in sialylated glycopeptide and/or sialylated glycan and/or sialylated glucosides is enriched with.A kind of method for being enriched with sialylated glycopeptide of present invention protection, comprises the following steps：(1) by g C3N4Dispersion liquid is added in the digestion products of sialydated glycoproteins, and vibration is enriched with sialylated glycopeptide；(2) after pregnant solution centrifugation, to g C3N4Eluent is added in precipitation, obtains saliva acidizing sugar peptide solution.Method provided by the invention is based on g C environment-friendly, that preparation is easy, cost is cheap3N4Material, enrichment condition is gentle, specificity is high, keeps the structural information of sialylated sugar chain to the full extent.This method can truly reflect the natural glycosylation state of protein, have important application value in terms of the detection of sialylated glycopeptide and the examination of related neoplasms mark in complex samples.
Description
Technical field
The present invention relates to the new material and new method of a kind of sialylated glycopeptide of concentration and separation.Based on graphite phase carbon nitride
(g-C3N4) a large amount of N atoms can form the characteristic of hydrogen bond with sialylated sugar chain O-H in material uniqueness triazine ring structure, and
Conjugated structure distinctive π interaction absorption properties, on the premise of glycoprotein structure is not destroyed, realize sialylated glycopeptide,
Sialylated glycan or the efficient, inexpensive of sialylated glucosides, specific enrichment separation.For saliva acidizing sugar in complex samples
The detection of peptide and the examination of related neoplasms mark etc. provide new approaches and new method.
Background technology
In the vital movement of organism, sialylated sugar chain plays specific recognition and mediation, to protein
26S Proteasome Structure and Function has particularly important influence.Research finds, in human body the expression of sialic acid have with many pathological states extremely close
The contact cut, as progress, bacteriovirus infection, neurogenic disease and interior occur for autoimmune disease, angiocardiopathy, tumour
Diacrisis etc..Sialydated glycoproteins are modified after a kind of important protein translation, disease marker can be used as, for depth
Entering mechanism and diagnoses and treatment of study of disease etc. has very important meaning.Sialic acid in complicated biological sample system
Abundance dynamic range is very big, significant component of low abundance sialylated glycopeptide, sialylated glycan or saliva acidizing sugar
Glycosides is highly susceptible to the non-suppression of other compositions.Therefore the matter of utmost importance that the research institute of sialic acid faces is that it is efficiently separated
Enrichment.
The methods of a variety of sialylated glycopeptides of enrichment existing at present, mainly include agglutinin method, chemical reaction method and hydrophilic
Material chromatography etc..Wherein the affine method of agglutinin is most widely used side in current sialydated glycoproteins enrichment research
Method.Agglutinin can be with specific enrichment sialic acid, but cost is higher, and adhesion is weak, is also unfavorable for carrying out high flux tumour mark
The work such as the examination of will thing.Conventional chemical reaction concentration method includes boric acid chemical reaction method and hydrazide chemistry reaction method etc.,
In the especially N-type glycosylation site identification of high-throughout glycosylation site, good application is obtained.But such method
Reactions steps are more, and condition is difficult to control, and chemical reaction process destroys the structure of sugar chain, is unfavorable for further carrying out
The identification and analysis of sugar chain type and structure.And amphoteric ion type hydrophily chromatography and strong ion-exchange chromatography non-can select
The various types of glycopeptides of enrichment of selecting property, but specificity is poor, and bioaccumulation efficiency has much room for improvement, and easily causes sialic acid information and lose
Lose.
The content of the invention
It is an object of the invention to provide a kind of new material for being enriched with sialylated glycopeptide and its new enrichment method.
Application of the claimed carbon nitride material in sialylated glycopeptide is enriched with.
The present invention also protects a kind of method for being enriched with sialylated glycopeptide, comprises the following steps：
(1) by g-C3N4Dispersion liquid is added in the digestion products of sialydated glycoproteins, vibration enrichment saliva acidizing sugar
Peptide；
(2) after pregnant solution centrifugation, to the g-C for being enriched with sialylated glycopeptide3N4Eluent is added in precipitation, obtains sialic acid
Change glycopeptide solution.
In above-mentioned enrichment method, digestion products are the products for obtaining sialydated glycoproteins progress digestion.The enzyme
Cut specially tryptic digestion.
In above-mentioned enrichment method, in step (1), g-C3N4Material is calcined by dicyandiamide or melamine or urea and is made.
It is 1~10g, such as 5g to calcine presoma dosage.Obtained g-C3N4Powder can pre-process as follows：g-C3N4Washing.It is described
The specific method of washing is：With 0.1M hydrochloric acid, ultra-pure water and 80% acetonitrile solution supersound washing, (ultrasound parameter specifically may be used respectively
For 20KHz, 5min).
In above-mentioned enrichment method, in step (1), g-C3N4Dispersion liquid is the mixed solution of acetonitrile/water solution, acetonitrile
Volume fraction is 70%~80%, such as 80%.
In above-mentioned enrichment method, in step (1), vibration enrichment time is 0.5~4 hour, such as 1 hour.
In above-mentioned enrichment method, in step (2), the solution centrifugal power after enrichment is 13500~21000g, such as
13500g。
In above-mentioned enrichment method, in step (2), eluent is the aqueous solution of the pH 5.5~12, such as 0.025% ammonia
The aqueous solution (pH=11.13).
In above-mentioned enrichment method, in step (2), vibration elution time is 0.5~4 hour, such as 45min.
In above-mentioned enrichment method, in step (2), after the completion of elution, the water of sialylated glycopeptide is obtained using centrifugal process
Solution, centrifugal force are 13500~21000g, such as 13500g.
In above-mentioned enrichment method, sialydated glycoproteins concretely fetuin, transferrins, and ox tire ball
Albumen, transferrins, the mixture of albumin and ribonuclease B.
G-C in the present invention3N4Have to sialylated glycopeptide and/or sialylated glycan and/or sialylated glucosides good
Concentration effect, enriched product purity is close to 100%, the rate of recovery 100%.In method provided by the invention, using gentle elution
Liquid elutes object, compared with titanium dioxide concentration method, method conditional provided by the invention gentle (preventing sialic acid from coming off),
Material is cheap and easy to get, is enriched with specificity height, significant effect.The enrichment is carried out using method provided by the invention, can be kept
The structural information of sialylated sugar chain and/or glycan, retain complete particular peptide segment information, be advantageous to follow-up mass spectral analysis, instead
The natural glycosylation state of protein is reflected, to biomarker screen research and early warning, process detection, the prognosis of major disease
Assessment etc. is respectively provided with important meaning.
Brief description of the drawings
Fig. 1 is the pregnant solution after myosin enzymolysis liquid (a), pregnant solution (b), enrichment raffinate (c), sialidase processing
(d) mass spectrogram of the pregnant solution (e) and after PNGase ferment treatments, wherein matter/lotus refer to the ratio between molecular mass and charge number.◆ table
Show and a sialylated residue is differed between sialylated glycopeptide mass spectra peak, be 291Da.
Fig. 2 is the pregnant solution after transferrins enzymolysis liquid (a), pregnant solution (b), enrichment raffinate (c), sialidase processing
(d) mass spectrogram of the pregnant solution (e) and after PNGase ferment treatments, wherein matter/lotus refer to the ratio between molecular mass and charge number.◆ table
Show and a sialylated residue is differed between sialylated glycopeptide mass spectra peak, be 291Da.
Fig. 3 is g-C3N4To the concentration effect mass spectrogram of the different sialylated glycopeptides of applied sample amount myosin, wherein matter/lotus
Refer to the ratio between molecular mass and charge number.
Fig. 4 is different elution requirements for g-C3N4The influence of the sialylated glycopeptide of myosin is enriched with, matter/lotus refers to point
The ratio between protonatomic mass and charge number.
Fig. 5 is the mass spectrogram that titanium dioxide is enriched with the sialylated glycopeptide of myosin,
Fig. 6 is g-C3N4The stereoscan photograph of material.
Fig. 7 is g-C3N4The transmission electron microscope photo (a) and high power transmission electron microscope photo (b) of material.
Fig. 8 is g-C3N4Structural representation (a illustrations), x-ray photoelectron energy spectrum diagram (a) and the infrared spectrogram of material
(b)。
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Result of the test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average." % " in following examples, unless otherwise specified, represents volumn concentration.
The present inventor is had found with g-C during long term test3N4As enrichment material can efficiently, it is special,
The leniently sialylated glycopeptide of separation and concentration.
Suction pipette head (Tip) (maximum loading volume is 200 μ L) is purchased from Axygen Scientific companies, product mesh
Record number is T-200-Y.Fetuin, transferrins, bovine albumin, ammonium hydrogen carbonate, iodoacetamide, formic acid, 2,5 dimethyl benzenes
Formic acid, titanium dioxide are purchased from Sigma-Aldrich companies, catalog number be followed successively by F3004, T3309, A5503,09830,
V900355、14265、85707、798517.Dithiothreitol (DTT) is purchased from Merck companies, catalog number 8011.Acetonitrile is purchased from
Thermo Fisher companies, catalog number A998.Trifluoroacetic acid is purchased from Tedia companies, and catalog number is TS 4295.
Trypsase is purchased from Roche companies, catalog number 11418025001.Sialidase, PNGase F enzymes are purchased from New
Englan Biolabs companies, catalog number are P0720S, P0704S respectively.Ultra-pure water comes from Merck Millipore
Synergy ultrapure water machines.
Embodiment 1, using g-C3N4It is enriched with sialylated glycopeptide
First, g-C3N4The step of being enriched with sialylated glycopeptide
Step 1: g-C3N4The preparation of material
It is added in crucible, is put into Muffle furnace after presoma (can be dicyandiamide or melamine or urea) is ground
550 DEG C of cappings are calcined 4 hours, and faint yellow product is taken out, and grinding obtains g-C3N4Powder.
Step 2: the preparation of enzymolysis sample solution
It (can be fetuin or transferrins or fetuin, transferrins and ox to take 20 μ L1 μ g/ μ L albumen
Albumin mixed protein), add 2.2 μ L250mM ammonium hydrogen carbonate and 0.6 μ L1M dithiothreitol (DTT)s (preparation of 25mM ammonium hydrogen carbonate)
In, 56 DEG C are incubated 60 minutes (reduction reaction, it is therefore an objective to opened disulfide bond), then add 5.6 μ L iodoacetamides containing 500mM
(preparation of 25mM ammonium hydrogen carbonate), after fully mixing, 25 DEG C of (room temperature) lucifuges are reacted 45 minutes (to be opened in closing reduction reaction
Disulfide bond), 1.5 μ L100mM dithiothreitol (DTT)s (preparation of 25mM ammonium hydrogen carbonate) are then added with terminating reaction, then by system
Freeze-drying, be stored in -80 DEG C it is standby.
Step 3: sialylated glycopeptide is enriched with from sample solution
1st, g-C is taken3N4Powder, respectively with 0.1M hydrochloric acid, ultra-pure water and 80% acetonitrile solution supersound washing (ultrasound parameter
Concretely 20KHz, 5min).
2nd, by 2mg g-C3N4It is scattered in 1mL acetonitrile/water solution, obtains g-C3N4Dispersion liquid.Acetonitrile/water dissolved in acetonitrile
Volume fraction be 80%.
3rd, packing enzymolysis sample, takes 2 μ g protein zymolytes, adds 60 μ L g-C after freeze-drying thereto3N4Dispersion liquid.
Constantly vibration is enriched with sialylated glycopeptide.It is 1 hour to vibrate enrichment time.
4th, after completing step 3, by the g-C after the completion of enrichment3N4Dispersion liquid centrifuges, centrifugal force 13500g.After the completion of centrifugation
Take out supernatant and be enriched with raffinate.Obtain g-C3N4Precipitation, that is, adsorb the enrichment material of sialylated glycopeptide.
5th, after completing step 4, to the g-C for adsorbing sialylated glycopeptide3N4Ammoniacal liquor eluent, constantly vibration are added in precipitation
It will adsorb in g-C3N4The sialylated glycopeptide on surface elutes.The concentration of eluent ammoniacal liquor is 0.025%.The body of eluent
Product is 60 μ L.It is 45 minutes to vibrate elution time.
6th, after completing step 5, by the solution centrifugal after the completion of elution, centrifugal force 13500g.On being taken out after the completion of centrifugation
Clear liquid is the eluent rich in sialylated glycopeptide.
7th, after completing step 6, eluent freezing is drained, carries out mass spectral analysis.
2nd, contrast test
1st, in step 2, fetuin enzymolysis stoste C18 posts (1.7 μm) desalination is obtained.
2nd, 4 obtained enrichment raffinate in step 3, with C18 posts (1.7 μm) desalination.
3rd, the pregnant solution that step 3 1~6 obtains.
4th, the fetuin enzymolysis stoste that step 2 obtains, every 20 μ g albumen is molten with 16 μ L water weight, and 2 μ L10 of addition ×
G1 reaction buffers and 2 μ L sialidases, 37 DEG C of incubation 16h.It has been digested that, be enriched with according to 1-6 in step 2.
5th, the fetuin enzymolysis stoste that step 2 obtains, every 20 μ g albumen is molten with 16 μ L water weight, and 2 μ L10 of addition ×
Reaction buffer and 2 μ L10 × NP-40,0.5 μ L PNGase enzymes, 37 DEG C of incubation 16h.It has been digested that, entered according to 1-6 in step 2
Row enrichment.
3rd, test limit experiment (different sample concentrations)
Step 1 is to 2 with the 1 to 2 of step 3.
3rd, packing enzymolysis sample, takes 0.2 μ g, 0.5 μ g, 1 μ g, 2 μ g, 4 μ g and 8 μ g protein zymolytes respectively, adds g-C3N4
Dispersion liquid.Constantly vibration is enriched with sialylated glycopeptide.
Step 4 is to 7 with the 5 to 7 of step 3.
4th, check experiment (elution requirement selection)
Step 1 is to 4 with the 1 to 4 of step 3.
5th, complete step 4 after, respectively with 0.1% trifluoroacetic acid, 0.2% formic acid, ultra-pure water, 25mM ammonium hydrogen carbonate,
0.025% ammonia spirit elutes, and collects efflux respectively, obtains five different pregnant solutions, carries out mass spectral analysis.
5th, check experiment
Titanium dioxide is enriched with
1st, post is filled：Titanium dioxide is scattered in 100% acetonitrile, filled to Tip, it is about 4-5mm to make its packed height,
It is preferred that 4mm, as titanium dioxide Tip posts.
2nd, pillar is activated：50 μ L acetonitriles are added, 2500g centrifuges 5~10min；Add 50 μ L ultra-pure waters, 2500g centrifugations 10
~20min；Add 20 μ L loading buffers (100mg DHB is added in the aqueous solution of the trifluoroacetic acid of 80% acetonitrile/5%,
It is configured to the loading buffer that concentration is 100mg/mL), 2500g centrifugations 10min.
3rd, loading：2 μ g fetuins are taken to digest sample, it is molten with 20 μ L sample-loading buffers weight, sample of the weight after molten is added
Enter into Tip posts, 1500g centrifugation 10min, efflux loading again again, in triplicate.
4th, (being respectively repeated 2 times) is rinsed：Adding the aqueous solution of the 80 μ L containing 80% acetonitrile and 2% trifluoroacetic acid, (2500g is centrifuged
20min, abandon efflux), adding the aqueous solution of the 80 μ L containing 20% acetonitrile and 0.1% trifluoroacetic acid, (2500g centrifuges 20min, abandoned stream
Go out liquid).
5th, after completing step 4,80 μ L0.8% phosphate buffers is added, 2500g centrifugation 20min, efflux is collected and contains saliva
Liquid is acidified the eluent of glycopeptide.
6th, eluent freezing is drained, mass spectral analysis.
6th, concentration effect compares
Mass-spectrogram is shown in Fig. 1.Fig. 1 a are to use the fetuin enzymolysis stoste mass spectrogram after C18 posts (1.7 μm) desalination, ox
Can detect 11 sialylated glycopeptide mass spectra peaks in the mass spectrogram of myosin enzymolysis stoste, and the peak intensity of glycopeptide compared with
It is weak.Fig. 1 b are g-C3N4It is enriched with the mass spectrogram of fetuin enrichment eluent, g-C3N445 are found that in eluent after enrichment
Individual sialylated glycopeptide mass spectra peak, glycopeptide peak is very strong, and almost without the interference of non-glycopeptide in figure.Fig. 1 c are g-C3N4Enrichment
Enrichment raffinate afterwards, g-C3N4The signal of glycopeptide and non-glycopeptide after enrichment in remaining solution only without sialic acid, shows
g-C3N4Sialylated glycopeptide can be specifically enriched with.After Fig. 1 d are fetuin enzymolysis, then handled with sialidase, entered
Row g-C3N4Enrichment.After sialidase processing, the sialic acid of glycopeptide end is cut off, and original sialylated glycopeptide peak all disappears
, leave behind some without sialic acid glycopeptides and non-glycopeptide peak.After Fig. 1 E are fetuin enzymolysis, then with PNGase enzymes
Processing, carry out g-C3N4Enrichment.N sugar is cut off after PNGase ferment treatments, and obtained signal all is from non-glycopeptide.Comparison diagram 1d
Found with Fig. 1 e, they have the peak of the same matter/lotus in part, and it is non-glycopeptide peak to show these peaks.And had more in Fig. 1 d than Fig. 1 e
The peak come, it should be some non-liquefied glycopeptide peaks of saliva.Fig. 1 b, 1d and 1e are contrasted and found, non-sialylated glycopeptide is not
Sialylated glycopeptide from sialidase processing, shows g-C3N4For the concentration effect of non-saliva glycopeptide and non-glycopeptide not
Substantially.When saliva glycopeptide, non-saliva glycopeptide and non-glycopeptide be present simultaneously, priority enrichment combination saliva glycopeptide, enter one
Step shows g-C3N4To the high degree of specificity of sialylated glycopeptide enrichment.
Fig. 2 a are to use the transferrins enzymolysis stoste mass spectrogram after C18 post desalinations, and transferrins digests the mass spectrogram of stoste
In can detect 9 sialylated glycopeptide mass spectra peaks, and the peak intensity of glycopeptide is weaker.Fig. 2 b are g-C3N4It is enriched with ox tire ball egg
The mass spectrogram of white enrichment eluent, g-C3N446 sialylated glycopeptide mass spectra peaks, glycopeptide peak are found that in eluent after enrichment
It is very strong, almost without the interference of non-glycopeptide.Fig. 2 c are g-C3N4Enrichment raffinate after enrichment, g-C3N4It is remaining after enrichment
The signal of glycopeptide and non-glycopeptide in solution only without sialic acid.After Fig. 2 d are transferrins enzymolysis, then with sialidase
Reason, carry out g-C3N4Enrichment.Original sialylated glycopeptide peak all disappears, and leaves behind some without the glycopeptide of sialic acid and non-
The peak of glycopeptide.After Fig. 2 e are transferrins enzymolysis, then with PNGase ferment treatments, carry out g-C3N4Enrichment, obtained signal all come
From non-glycopeptide.It is similar to the enrichment discipline of myosin in Fig. 1, g-C3N4Also the sialylated glycopeptide of transferrins can be carried out
Specific enrichment.Illustrate for the less transferrins of sialylated modification, g-C3N4Still have to sialylated glycopeptide very strong
Accumulation ability.
Contrast g-C3N4To the enrichment mass spectrum (Fig. 3) of different amounts of fetuin enzymolysis liquid, it can be found that：Ox tire ball egg
The information of sialylated glycopeptide is not seen when being in vain 20fmol.During 50fmol, there is the information of sialylated glycopeptide, still
Its signal to noise ratio and peak intensity are less high.And there is more rich sialylated glycopeptide information during 0.4pmol, during 0.8pmol
Glycopeptide peak reaches very high intensity.And continue to increase the amount of protein enzymatic hydrolyzate, with saliva after 0.8pmol proteolysis liquid enrichment
Acidifying glycopeptide peak intensity compares no significant change with the quantity at peak.Show g-C3N4The minimum detectability of enrichment up to 50fmol,
0.8pmol is optimum protein applied sample amount.
Contrast utilizes different elution solution elution g-C3N4On sialylated glycopeptide mass spectrogram (Fig. 4 a, 4b, 4c, 4d and
4e) it can be found that：Strong acid eluent can not elute sialylated glycopeptide, with eluent pH rise, obtained saliva
Liquid acidifying glycopeptide peak number amount is more and more, and peak intensity also gradually increases, and the ammonia spirit that optimal condition is 0.025% elutes.Say
Bright weakly alkaline environment is advantageous to the elution of sialylated glycopeptide.
In the mass spectrogram of titanium dioxide method enrichment fetuin enzymolysis liquid (Fig. 5), it can be found that titanium dioxide is enriched to
Sialylated glycopeptide be concentrated mainly on 3000~5000Da, it is bad to the glycopeptide bioaccumulation efficiency at HMW end.And titanium dioxide
Titanium enrichment condition is not gentle enough, and there occurs molecular dehydration, makes mass spectrogram more complicated.For example a water is taken off corresponding to m/z 4729
The signal peak m/z 4711 of molecule.By contrast, g-C3N4Not only (can mainly it be distributed with the sialylated glycopeptide of enriched
Region is located at m/z=3000~7000), and appearance of the mild condition without dehydration peak, significant effect is better than conventional titanium dioxide
Titanium concentration method.
Embodiment 2, g-C3N4The pattern and structural characterization of enrichment material
To g-C in the present embodiment3N4Enrichment material carries out pattern and structural characterization, further speculates g-C3N4It is enriched with saliva
It is acidified the mechanism of glycopeptide.
Fig. 6 is g-C3N4Scanning electron microscope (SEM) photograph, it can be found that g-C3N4For the micron order bulk structure of stratiform accumulation, part
Position can see below 50nm layer structure.Fig. 7 a and Fig. 7 b are g-C respectively3N4Transmission electron microscope and high-resolution transmission electricity
Mirror figure.As illustrated, g-C3N4It is to be formed by the accumulation of unformed individual layer.
Fig. 8 a illustrations are g-C3N4Theoretical construct figure, it can be seen that have substantial amounts of triazine ring and N-H key in its structure.Figure
8a is g-C3N4X-ray photoelectron energy spectrum diagram, the peak at 286ev and 397ev has corresponded to C1s and N1s combination energy, has shown institute
The material of preparation is mainly made up of C and N atoms, consistent with its theoretical construct.Fig. 8 b are g-C3N4Infrared spectrogram, wherein
1200~1650cm-1Absworption peak in wave-number range has corresponded to the typical stretching vibration of C-N heterocycles, and 807cm-1Absworption peak
The normal mode vibration of triazine structure is corresponded to.3000~3600cm-1Between absworption peak corresponded to N-H and O-H stretching vibration, point
Do not come from oxygen defect a small amount of in the unpolymerized amino of edge of materials and structure.Substantial amounts of triazine ring and N-H structures in material
Hydrogen bond can be formed with sialic acid, be advantageous to the specific enrichment to sialic acid.
Claims (8)
1.g-C3N4Apply in the sialylated glycopeptide of concentration and separation.
2. a kind of kit for being used to be enriched with sialylated glycopeptide, including g-C3N4, 80% acetonitrile/water solution and 0.025% ammoniacal liquor
Solution.
3. a kind of method for being enriched with sialylated glycopeptide, comprises the following steps：
(1) by g-C3N4Dispersion liquid is added in the digestion products of sialydated glycoproteins, and vibration is enriched with sialylated glycopeptide；
(2) after pregnant solution centrifugation, to the g-C for being enriched with sialylated glycopeptide3N4Eluent is added in precipitation, obtains saliva acidizing sugar
Peptide solution.
4. the enrichment method described in claim 3, it is characterised in that：In step (1), g-C3N4Dispersion liquid is the mixed of acetonitrile/water
Solution is closed, the wherein volume fraction of acetonitrile is 70%~80%, g-C3N4Concentration be 0.5~4mg/mL.
5. the enrichment method described in claim 3, it is characterised in that：Using the method for vibration enrichment and vibration elution, during vibration
Between be 0.5~4 hour.
6. the enrichment method described in claim 3, it is characterised in that：Obtain being enriched with the g- of sialylated glycopeptide using centrifugal process
C3N4Precipitation, centrifugal force is 13500~21000g.
7. the method described in claim 3, it is characterised in that：In step (2), made using the aqueous solution of the pH between 5.9~12
For the ammonia spirit of eluent, such as 0.025%.
8. the method described in claim 3, it is characterised in that：The aqueous solution of sialylated glycopeptide is obtained using centrifugal process, centrifuged
Power is 13500~21000g.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104374848A (en) * | 2013-08-14 | 2015-02-25 | 中国科学院大连化学物理研究所 | Method for enriching glycopeptide by phenylboronic acid material |
| CN104807927A (en) * | 2014-01-24 | 2015-07-29 | 中国医学科学院基础医学研究所 | Method for enriching sialylaglycopeptide, sialylated glycans or sialylated glycoside |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104374848A (en) * | 2013-08-14 | 2015-02-25 | 中国科学院大连化学物理研究所 | Method for enriching glycopeptide by phenylboronic acid material |
| CN104807927A (en) * | 2014-01-24 | 2015-07-29 | 中国医学科学院基础医学研究所 | Method for enriching sialylaglycopeptide, sialylated glycans or sialylated glycoside |
Non-Patent Citations (1)
| Title |
|---|
| GANG-TIAN ZHU等: "Magnetic graphitic carbon nitride anion exchanger for specific enrichment of phosphopeptides", 《JOURNAL OF CHROMATOGRAPHY A》 * |
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