CN109012611A - A kind of cellulose microsphere, cellulose microtrabeculae and its preparation method and application - Google Patents
A kind of cellulose microsphere, cellulose microtrabeculae and its preparation method and application Download PDFInfo
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- CN109012611A CN109012611A CN201810837166.7A CN201810837166A CN109012611A CN 109012611 A CN109012611 A CN 109012611A CN 201810837166 A CN201810837166 A CN 201810837166A CN 109012611 A CN109012611 A CN 109012611A
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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Abstract
The invention discloses a kind of cellulose microspheres, cellulose microtrabeculae and its preparation method and application, wherein, enrichment and/or purifying pretreatment of the cellulose microsphere for glycopeptide and/or glycan, the cellulose microsphere is solidified by cellulose aqueous solution through solution~gel transition method, and diameter is 5~20 μm, and cellulose microsphere, and cylinder both ends open are filled in the microtrabeculae, one end is equipped with the circular gasket of high molecular weight hydrophilic material, and gasket aperture is less than cellulose microsphere diameter.Cellulose microsphere synthesis process of the invention is simple, quick, green, pollution-free, at low cost, large specific surface area, uniform particle sizes, the cellulose microsphere of synthesis shows the spherical shape of rule, surface has porous structure, can preferably come into full contact with glycopeptide or glycan in enrichment or purification process, synthesized cellulose microsphere is filled in liquid transfer gun head, a microtrabeculae system is formed, efficiently purifying and the enrichment of glycan and glycopeptide can be realized in 2min.
Description
Technical field
The present invention relates to a kind of cellulose microspheres, cellulose microtrabeculae and its preparation method and application, belong to biochemical analysis neck
Domain.
Background technique
As one of the modification after most important protein translation, the glycosylation of protein is related to many biological mistakes
Journey, including immune response, molecular recognition, protein folding and receptor for stimulating etc..It is reported that the exception during protein glycosylation
Variation may cause serious human diseases.Therefore, by characterization glycopeptide and glycan, to deeply be ground to protein glycosylation
Study carefully and has great importance.In the development of analytical chemistry, mass spectrum (MS) has been acknowledged as in glycan proteome analysis
One preferred technique.It has lower sample consumption, higher flux and higher sensitivity when analyzing glycan and glycopeptide.But
It is, it is mass spectrographic to apply while being also limited by complicated glycosylation heterogeneity, low-abundance glycan/glycopeptide and produced from non-purpose
The signal of object inhibits.Therefore, in order to solve these problems, glycan/glycopeptide is effectively pre-processed before Mass Spectrometer Method
It is essential.
In recent years, researcher has explored various preprocess methods to be enriched with glycopeptide or purifying glycan, main packet
The affine method of agglutinin is included, method that boric acid is affine, hydrazine chemical coupling method and hydrophilic Interaction Chromatography (HILIC) etc..In these methods, it coagulates
Although it is high to the specificity of specific glycopeptide that collection element is affine, it is only effective to the glycopeptide containing certain a kind of end glycan, to other
Glycopeptide is not effectively combined effect；When being captured using boric acid chemical method to glycan albumen or glycopeptide, with glycopeptide
In conjunction with the inhibition that will receive a large amount of non-glycopeptides, in addition to this, boric acid with the reaction between two diameter base of cis- ortho position or meta position not only
It is limited to glycans, other may also equally be captured with the cis- component through base such as nucleotide, to cause to analysis result
Interference；It is derivative by multistep when hydrazine chemical method is enriched with glycopeptide, it is cumbersome, and when digestion release peptide, glycan chain part is also protected
It stays in solid phase, limits application of this method in polysaccharide chains structure elucidation in this way.In these methods, hydrophilic interaction color
Spectrometry because its to glycosylation coverage rate height, method favorable reproducibility, be easy to be concerned with mass spectrometry the advantages that.Pass through utilization
The method based on HILIC of hydrophilic interaction between glycan moiety and matrix is at low cost, high-efficient, favorable reproducibility.Therefore,
Development of Novel water wetted material will be helpful to further increase the efficiency of glycopeptide enrichment.In recent years, a variety of different hydrophilic materials
Material has been widely used in glycopeptide enrichment.However, the synthesis of these hydrophilic materials usually requires complicated synthesis program, multistep
Rapid functionalization and longer incubation time.The cost for the analysis method that this cumbersome process substantially increases, greatly limits
Their applications in actual sample are made.It would therefore be highly desirable to which it is hydrophilic to design that a program is simple, high sensitivity, the rate of recovery are good
Property glycopeptide enrichment material.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, micro- the purpose of the present invention is obtaining a kind of cellulose microsphere, cellulose
Column and its preparation method and application.
For achieving the above object, it the present invention uses cellulose microsphere and cellulose microtrabeculae and preparation method thereof and answers
Technical solution is as follows:
A kind of cellulose microsphere, enrichment and/or purifying pretreatment of the cellulose microsphere for glycopeptide and/or glycan,
The cellulose microsphere is solidified by cellulose aqueous solution through solution-gel transformation approach, and diameter is 5~20 μm.
Cellulose microsphere volume in the present invention is uniform, and preferred cellulose microsphere average diameter is 12 μm, and cellulose is micro-
Ball shows the spherical shape of rule, and size distribution curve meets Gaussian Profile.The surface of cellulose microsphere has porous structure, this
So that microballoon can preferably come into full contact with glycopeptide or glycan in enrichment or purification process.In the infrared light of cellulose microsphere
In spectrum, cellulose microsphere shows the feature absorption of cellulose, in 1065cm-1The peak that place observes is attributed to stretching for C-O key
Contracting vibration；In -3447cm-1Another strong peak that place occurs corresponds to the stretching vibration of hydroxyl, it was confirmed that cellulose microsphere contains
A large amount of hydroxyl.Pass through the N of cellulose microsphere2Absorption-desorption curve is up to it is known that measuring its BET specific surface area
209.7m2g-1, corresponding aperture is 10nm.
The preparation method of the cellulose microsphere includes the following steps:
I. cellulose-containing raw material is dissolved in the pre- sodium hydroxide/urea/water solution for being cooled to -10~-15 DEG C, machinery stirs
Mix clear cellulose aqueous solution, the cellulose mass concentration of cellulose aqueous solution is 3.0~4.0%；After dissolution from
The heart；
Ii. the cellulose aqueous solution after being centrifuged in step i is taken to instill the atoleine suspension containing Span 80 dropwise
In, dropping temperature is 40~50 DEG C, and it is uniform to suspending to continue 40~50 DEG C of stirrings after completion of dropwise addition；
Iii. it is stirred into step ii in the suspension of end and dehydrated alcohol is added, after mixing evenly, slow cooling to room
Temperature, suspension solidify to obtain cellulose microsphere.
Due to cellulose in sodium hydroxide aqueous solution of urea have good dissolubility, selection with sodium hydroxide/
Urea/water solution is as solvent, and weight ratio is sodium hydroxide: urea in preferred sodium hydroxide/urea/water solution: water=7~
9:6~12:79~87.
Cellulose containing raw material is preferably cotton linter, the cellulose microsphere uniform particle sizes extracted, and shape is uniform.Other are containing fibre
The raw material such as timber, stalk, plant residue etc. for tieing up element can also be used as raw material use.
Preferably, the weight ratio of each component is sodium hydroxide: urea: water=7:12 in sodium hydroxide/urea/water solution:
81。
Preferably, it is cooled to -15 DEG C when preparation in advance using 4g cotton linter is added in 110g sodium hydroxide/urea/water solution, obtains
The mass concentration of the cellulose aqueous solution arrived is 3.5%.
Centrifugal condition in step i specifically: be centrifuged 10min under conditions of 6000r/min, 15 DEG C.
Preferably, the preparation step of the atoleine suspension containing Span 80 includes: to be dissolved in the Span 80 of 2~3g
In the atoleine of 100mL, mechanical stirring is to uniform under conditions of 800~1200r/min, 40~50 DEG C of water-baths.Further
Preferably, centrifugal rotational speed 1000r/min, bath temperature are 45 DEG C.
Preferably, step ii specifically: the Span 80 of 2~3g is dissolved in the atoleine of 100mL, 800~
1200r/min, mechanical stirring 30min under conditions of 40~50 DEG C of water-baths；The cellulose solution of 16.9g is weighed at 45 DEG C dropwise
It instills in suspension, is dripped off in 30min, continue to be stirred until homogeneous in suspension.
It is further preferred that centrifugal rotational speed is 1000r/min, bath temperature is 45 DEG C.It is preferably added in atoleine
2.54g Span 80。
Preferably, step iii specifically: in the suspension for stirring end into step ii, the anhydrous second of 150mL is added
Alcohol, after stirring, slow cooling to room temperature, suspension solidifies to obtain cellulose microsphere.
Preferably, cured cellulose microsphere further includes washing step, and when washing successively uses water and ethyl alcohol to wash respectively,
More preferably wash 3 times respectively.
Preferably, the cellulose microsphere after washing is freeze-dried or saves for future use in 50% ethanol/water.
The present invention also provides a kind of preparation method of above-mentioned cellulose microsphere, preparation method mainly uses solution~gel
Transformation approach, the specific steps are as follows:
I. cellulose-containing raw material is dissolved in the pre- sodium hydroxide/urea/water solution for being cooled to -10~-15 DEG C, machinery stirs
Mix clear cellulose aqueous solution, the cellulose mass concentration of cellulose aqueous solution is 3.0~4.0%；After dissolution from
The heart；
Ii. the cellulose aqueous solution after being centrifuged in step i is taken to instill the atoleine suspension containing Span 80 dropwise
In, dropping temperature is 45 DEG C, and it is uniform to suspending to continue 45 DEG C of stirrings after completion of dropwise addition；
Iii. it is stirred into step ii in the suspension of end and dehydrated alcohol is added, after mixing evenly, slow cooling to room
Temperature, suspension solidify to obtain cellulose microsphere.
Due to cellulose in sodium hydroxide aqueous solution of urea have good dissolubility, selection with sodium hydroxide/
Urea/water solution is as solvent, and weight ratio is sodium hydroxide: urea in preferred sodium hydroxide/urea/water solution: water=7~
9:6~12:79~87.
Cellulose containing raw material is preferably cotton linter, the cellulose microsphere uniform particle sizes extracted, and shape is uniform.Other are containing fibre
The raw material such as timber, stalk, plant residue etc. for tieing up element can also be used as raw material use.
Preferably, the weight ratio of each component is sodium hydroxide: urea: water=7:12 in sodium hydroxide/urea/water solution:
81。
Preferably, preparation when using be pre-chilled in -15 DEG C of 110g sodium hydroxide/urea/water solution be added 4g cotton linter,
The mass concentration of obtained cellulose aqueous solution is 3.5%.
Centrifugal condition in step i specifically: be centrifuged 10min under conditions of 6000r/min, 15 DEG C.
Preferably, the preparation step of the atoleine suspension containing Span 80 includes: to be dissolved in the Span 80 of 2~3g
In the atoleine of 100mL, the mechanical stirring 30min under conditions of 800~1200r/min, 40~50 DEG C of water-baths.
Preferably, step ii specifically: the Span 80 of 2~3g is dissolved in the atoleine of 100mL, 800~
1200r/min, mechanical stirring 30min under conditions of 40~50 DEG C of water-baths；The cellulose solution of 16.9g is weighed at 45 DEG C dropwise
It instills in suspension, is dripped off in 30min, continue to stir 30min in suspension.
Preferably, step iii specifically: in the suspension for stirring end into step ii, the anhydrous second of 150mL is added
Alcohol, after stirring 5min, slow cooling to room temperature, suspension solidifies to obtain cellulose microsphere.
Preferably, cured cellulose microsphere further includes washing step, and when washing successively uses water and ethyl alcohol to wash respectively.
More preferably wash 3 times respectively.
Preferably, the cellulose microsphere after washing is freeze-dried or saves for future use in 50% ethanol/water.
Mixing time in above-mentioned steps is empirical value, can not be used as limitation of the present invention, as long as stirring
Uniform to suspension system, any time increase and decrease carried out under the premise of stirring evenly is deemed to fall of the invention
In protection scope.
As a preferred embodiment, the preparation process of the cellulose microsphere specifically:
110g sodium hydroxide/urea/water (weight ratio 7/12/81) solution is cooled to -15 DEG C by 1.1 in advance；
1.2 are immediately dissolved in 4g cotton linter in the solution being pre-chilled, and after mechanical stirring 5min, obtain the fiber of clear
Plain aqueous solution, mass concentration 3.5%；
Obtained cellulose solution is centrifuged 10min under conditions of 6000r/min, 15 DEG C by 1.3, to remove in solution
Bubble；
1.4 are dissolved in the Span 80 of 2.54g in the atoleine of 100mL, under conditions of 1000r/min, 45 DEG C of water-baths
Mechanical stirring 30min；Then the cellulose solution for weighing 16.9g instills in suspension dropwise at 45 DEG C, drips off in 30min；
Continue to stir 30min with identical condition after 1.5 completion of dropwise addition, is subsequently added into the dehydrated alcohol of 150mL, continues to stir
After mixing 5min, reaction is slowly cooled to room temperature, and suspension solidifies to obtain cellulose microsphere；
After 1.6 remove the atoleine on upper layer, obtained cellulose microsphere is successively washed 3 times with water and ethyl alcohol respectively
To remove residual liquid paraffin or Span 80；
1.7 obtained cellulose microsphere freeze-dryings save for future use in 50% ethanol/water.
Another goal of the invention of the invention is to provide a kind of cellulose using any of the above-described kind of cellulose microsphere micro-
Column, for glycan or the enriching and purifying of glycopeptide, and cylinder both ends open, one end are equipped with the circular pad of high molecular weight hydrophilic material
Piece, circular gasket aperture are less than cellulose microsphere diameter.In other words, at this point, cellulose microsphere is by high molecular weight hydrophilic material
Circular gasket is fixed and is filled in cylinder.
Preferably, the circular gasket of the high molecular weight hydrophilic material is porous polyethylene circular gasket.
In addition to porous polyethylene material, other high molecular weight hydrophilic materials such as cellulose acetate film, polypropylene, polytetrafluoro
Ethylene and its stretching material etc. can be used as the use of the circular gasket in the present invention.It is any that can achieve water phase micro- with cellulose
The material of ball separation, porous polyethylene material are not construed as limitation of the present invention.
Cellulose microtrabeculae in the present invention is generally used for the detection work of protein glycosylation, therefore generallys use the micro- of small size
Column carries out enriching and purifying work, and microtrabeculae volume is generally less than 10mL.It, can will be of the invention when needing to carry out extensive enriching and purifying
In cellulose microsphere be loaded into large-scale Filter column, equally can achieve identical enriching and purifying effect.No matter therefore using ring
The filter device of the variation in border, any enriching and purifying that glycan and glycopeptide are carried out using cellulose microsphere is deemed to fall this Shen
Within protection scope please.
Preferably, it is used for the ease of the independent preparation in laboratory, the microtrabeculae cylinder in the present invention is typically chosen liquid-transfering gun
Head generally uses the liquid-transfering gun pipette tips of 100 μ L on a small scale when detection.
4th goal of the invention of the invention is to provide a kind of preparation method of above-mentioned cellulose microtrabeculae, the cellulose
Microtrabeculae is made through following steps method:
A) cellulose microsphere and pure water are dispersed into cellulose microsphere into pure water with the ratio that mass ratio is 2~3:1；
B) cellulose microsphere scattered in step a) is moved in microtrabeculae cylinder, pressurization discharge pure water.
Preferably, the cylinder is liquid transfer gun head.
Preferably, the mass ratio of cellulose microsphere and pure water is 2.5:1.
When laboratory, selection liquid-transfering gun pipette tips are voluntarily prepared, the cellulose microtrabeculae is through following steps method system
: the cellulose microsphere and pure water are dispersed into cellulose microsphere into pure water to realize with the ratio that mass ratio is 2~3:1,
Scattered cellulose microsphere is inserted in cylinder by the separate gasket-end again, then liquid-transfering gun is cooperated to complete draining.
Preferably, when the cellulose microtrabeculae is multiple, cooperation liquid relief volley of rifle fire preparation.In other words, disposable when needing
When handling multiple samples, multiple microtrabeculaes are disposably prepared, and cooperate the volley of rifle fire to use, it can be achieved that the high-throughput purpose being enriched with.
In other words, the preparation method of the cellulose microtrabeculae in the present invention includes the following steps:
A) cellulose microsphere and pure water are dispersed into cellulose microsphere into pure water with the ratio that mass ratio is 2~3:1；
B) cellulose microsphere scattered in step a) is moved in microtrabeculae cylinder, pressurization discharge pure water.
Cellulose microtrabeculae, which can according to need, to be prepared separately one or prepares multiple, the Capacity Selection of microtrabeculae when preparation simultaneously
It is determined with the amount of sugar to be treated and glycopeptide.When prepared by laboratory, the pipette tips for being typically chosen orthobaric volume are tested, and are convenient for
Match corresponding liquid-transfering gun or the liquid relief volley of rifle fire.But this can not be considered limitation of the present invention, and the microtrabeculae of other volumes can also
To be used for the cellulose microtrabeculae in the present invention.
Preferably, the mass ratio of cellulose microsphere and pure water is 2.5:1.
As a preferred embodiment, preparing the preparation side that cellulose microtrabeculae is used in mass spectral analysis pretreatment in laboratory
Method includes the following steps:
It is 3mm that a diameter is equipped in the tip of the liquid transfer gun head of 2.1 100 μ L, with a thickness of the pad of the rounded porous of 1mm
Piece；Gasket is porous polyethylene material；
2.2 weigh the cellulose microsphere of 500 μ g and are dispersed in the pure water of 200 μ L, are transferred to liquid-transfering gun filled with pad
In the liquid transfer gun head of piece；
After 2.3 are got the water in pipette tips with liquid-transfering gun, a microtrabeculae filled with cellulose microsphere, which is prepared, to be completed.
The present invention also provides a kind of cellulose microsphere as described in above-mentioned any preferred embodiment or cellulose microtrabeculaes
Application, the pretreatment of the cellulose microsphere or cellulose microtrabeculae for glycan and/or glycopeptide before Mass Spectrometer Method.
The cellulose microsphere or cellulose microtrabeculae can be also used for the glycan and/or glycopeptide before high performance liquid chromatography detection
Pretreatment.
The cellulose microsphere or cellulose microtrabeculae are also used for protein glycosylation analysis pretreatment.
The present invention provides a kind of glycan and/or the preprocess method of glycopeptide, the preprocess method uses above-mentioned fibre
It ties up plain microballoon or above-mentioned cellulose microtrabeculae carries out glycan purification and/or glycopeptide enrichment.
The present invention also provides a kind of detection methods of protein glycosylation, which is characterized in that the albumen is adopted after digestion
With above-mentioned cellulose microsphere or above-mentioned cellulose microtrabeculae carry out glycan purification and/or glycopeptide enrichment after, then carry out mass spectrum or
Efficient liquid phase chromatographic analysis.
Cellulose microtrabeculae when the pretreatment of glycan and glycopeptide before cellulose microtrabeculae is used for Mass Spectrometer Method is known as CM-
PTs.The experiment proved that CM-PTs is 125mg g to the load capacity of glycopeptide after being enriched with by CM-PTs~1(IgG/ cellulose
Microballoon), non-glycopeptide noisy to glycopeptide and other impurity almost all are removed, and glycopeptide is all retained.High sensitivity,
The rate of recovery is high, and load capacity is big, and selectivity is good, and bioaccumulation efficiency is high and sample consumption is lower.Using CM-PTs after purification, Ke Yiqing
Chu glycan is detected, almost without the interference of other any impurities.Especially for TMPP-Ac- glycan, passing through CM-PTs
After removing excessive derivatization reagent and other impurities, the mass spectrogram of target product does not have any impurity peak, intensity and signal-to-noise ratio
It has all obtained greatly enhancing.
Compared with prior art, the present invention synthesized a kind of porous hydrophilic material-cellulose microsphere for glycan and
The enriching and purifying of glycopeptide, cellulose microsphere synthesis process is simple, quick, green, pollution-free, at low cost, large specific surface area, partial size
Uniformly.Cellulose microsphere is used for the purifying and enrichment of glycan and glycopeptide for the first time.The cellulose microsphere of synthesis shows rule
Spherical shape, surface have porous structure, can preferably come into full contact with glycopeptide or glycan in enrichment or purification process.It will be closed
At cellulose microsphere be filled in liquid transfer gun head, form a microtrabeculae system, the height of glycan and glycopeptide can be realized in 2min
Effect purifying and enrichment, not only raw material is cheap and easy to get environmental-friendly, and preparation process is simple and efficient, easy to use, is suitble to extensive
It promotes the use of.The enrichment of cellulose microtrabeculae in through the invention, can greatly shorten the preprocessing process of glycan and glycopeptide, mention
The accuracy of high detection provides more accurate data and supports for clinical detection and scientific research.
Detailed description of the invention
Fig. 1 is the effect schematic diagram of cellulose microtrabeculae of the invention in glycopeptide, glycan enriching and purifying；
Fig. 2 a is cellulose microsphere aqueous solution schematic diagram of the invention；
Fig. 2 b is the electronic scanner microscope image of cellulose microsphere of the invention；
Fig. 2 c is cellulose microsphere particle diameter distribution situation prepared by the present invention；
Fig. 2 d is the electronic scanner microscope enlarged drawing of cellulose microsphere of the invention；
Fig. 2 e is cellulose microsphere microscopic appearance figure of the invention；
Fig. 3 is the MALDI-TOF mass spectrum comparison diagram of different enrichment modes after IgG digestion provided in an embodiment of the present invention；
Fig. 4 is the MALDI-TOF mass spectrum of enrichment front and back after human IgG provided in an embodiment of the present invention and BSA mixture digestion
Comparison diagram；
Fig. 5 is the mass spectral intensities pair for the human IgG that different cellulose microsphere contents provided in an embodiment of the present invention are enriched with digestion
Than figure；
MALDI- after the enrichment that Fig. 6 is the IgG after the tryptic digestion of three low concentrations provided in an embodiment of the present invention
TOF mass spectrum comparison diagram；
Fig. 7 is MALDI- of the glycan provided in an embodiment of the present invention discharged through RNase B after cellulose microsphere is enriched with
TOF mass spectrum comparison diagram.
Specific embodiment
It to a kind of cellulose microsphere provided by the invention, cellulose microtrabeculae and preparation method thereof and is answered below with reference to embodiment
In detail, completely illustrate as further.The embodiments described below is exemplary, for explaining only the invention, and cannot
It is interpreted as limitation of the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples
It tests material unless otherwise specified, is that market is commercially available.
One, the synthesis of cellulose microsphere
The present embodiment carries out the synthesis of cellulose microsphere using the method that solution-gel is converted.
110g sodium hydroxide/urea/water (weight ratio 7/12/81) solution is cooled to -15 DEG C by 1.1 in advance；
1.2 are immediately dissolved in 4g cotton linter in the solution being pre-chilled, and after mechanical stirring 5min, obtain the fiber of clear
Plain aqueous solution, mass concentration 3.5%；
Obtained cellulose solution is centrifuged 10min under conditions of 6000r/min, 15 DEG C by 1.3, to remove in solution
Bubble；
1.4 are dissolved in the Span 80 of 2.54g in the atoleine of 100mL, under conditions of 1000r/min, 45 DEG C of water-baths
Mechanical stirring 30min；Then the cellulose solution for weighing 16.9g instills in suspension dropwise at 45 DEG C, drips off in 30min；
Continue to stir 30min with identical condition after 1.5 completion of dropwise addition, is subsequently added into the dehydrated alcohol of 150mL, continues to stir
After mixing 5min, reaction is slowly cooled to room temperature, and suspension solidifies to obtain cellulose microsphere；
After 1.6 remove the atoleine on upper layer, obtained cellulose microsphere is successively washed 3 times with water and ethyl alcohol respectively
To remove residual liquid paraffin or Span 80；
1.7 obtained cellulose microsphere freeze-dryings save for future use in 50% ethanol/water.
The range estimation form of cellulose aqueous solution is as shown in Figure 2 a, and Fig. 2 b, 2d and 2e show cellulose microsphere under Electronic Speculum
Structural form, Fig. 2 c be microballoon particle diameter distribution situation, it can be seen that cellulose microsphere uniform diameter made from the present embodiment
Porous surface, cellulose microsphere show the spherical shape of rule, and size distribution curve meets Gaussian Profile.
Two, the preparation of cellulose microtrabeculae (CM-PTs)
One diameter is 3mm by 2.1, and the tip of the liquid transfer gun head into 100 μ L is disclosed with a thickness of the permeable gasket of circle of 1mm；
Round permeable gasket is porous polyethylene material；
2.2 weigh the cellulose microsphere of 500 μ g and are dispersed in the pure water of 200 μ L, are transferred to liquid-transfering gun filled with pad
In the liquid transfer gun head of piece；
After 2.3 are got the water in pipette tips with liquid-transfering gun, a microtrabeculae filled with cellulose microsphere, which is prepared, to be completed.
When needing disposably to handle multiple samples, multiple microtrabeculaes are disposably prepared, and cooperate the volley of rifle fire to use, it can be achieved that high
The purpose of flux enrichment.
The cellulose microtrabeculae and its use process prepared is as shown in Figure 1, pass through the hydrophily of cellulose microsphere and glycopeptide
Interaction, can accurately distinguish glycopeptide molecule after enrichment, be convenient for subsequent mass spectral analysis.
Using IgG and HRP as standard glycan albumen, IgG is used to assess this method and is applied to sugar the cellulose microtrabeculae prepared
Effect when peptide is enriched with, including sensitivity, load factor etc., HRP are used to verify the generality that this method is applied to glycopeptide enrichment.
BSA is non-glycan albumen, and when carrying out glycopeptide enrichment to enrichment glycan protein standard substance IgG, non-glycan albumen mark is artificially added
Quasi- product BSA, to assess the anti-interference ability that this method resists non-glycopeptide in glycopeptide enrichment.Gathered by changing glycan albumen with non-
The molar ratio of glycoprotein studies the concentration effect to glycopeptide of this method under different degrees of interference.Specific cellulose is micro-
The verification process of column is as follows.
Three, the tryptic digestion of standard glycan albumen
Accepted standard glycan albumen is IgG or HRP in the present embodiment, and bovine serum albumin(BSA) (BSA) is non-glycan albumen
The tryptic digestion of standard items, IgG or HRP are shown in steps are as follows:
3.1 take standard glycan protein I gG (HRP) 500 μ g, are dissolved in 500 μ L 50mM, the ammonium bicarbonate buffers that pH is 8.0
In；
Glycan albumen is boiled 10min by 3.2 makes its denaturation；Later with enzyme: substrate is that the ratio of 1:40 (w/w) is added centainly
The trypsase of amount；
3.3 react the mixture in step 3.2 16~18 hours under 37 DEG C of water-baths；After reaction, by mixture
5min is boiled again so that unreacted trypsin inactivation.
The digestion of non-glycan protein standard substance bovine serum albumin(BSA) (BSA) is shown in steps are as follows:
3.1.1 500 μ g of standard glycan protein B SA is taken, 500 μ L 50mM, the ammonium bicarbonate buffers that pH is 8.0 are dissolved in
In；
3.2.1 the poly- sugar alcohol of two sulphur Soviet Union of 75 μ L200mM is added into above-mentioned solution, and is reacted 1 hour at 37 DEG C, with also
The disulfide bond of former albumen；
3.3.1 after reaction, the iodoacetamide for adding 75 μ L200mM is protected from light and reacts 1 hour at room temperature；
3.4.1 with enzyme: substrate is that a certain amount of trypsase is added in the ratio of 1:40 (w/w)；This mixture is in 37 DEG C of water
Bath lower reaction 16~18 hours；
3.5.1 after reaction, mixture is boiled into 5min again so that unreacted trypsin inactivation.
Obtained enzymolysis product is placed in -20 DEG C of preservations, in case using.
Four, the quick release and label of glycan
The quick release and label of glycan in the present embodiment use the microwave-assisted quick digestion of this development in laboratory
Method is to realize the quick release of N- glycan.Detailed process are as follows:
4.1 by the standard glycan pyrenoids polysaccharide-mucleic acid enzyme B (RNase B) of 20 μ g, and being dissolved in 40 μ L, 10mM, pH is 8.5
In phosphate buffer；
Sample is boiled 10min by 4.2 makes its denaturation；Temperature is cooled to room temperature, and the NP- of 4.8 μ L10% is added into solution
40, stand 10min；
4.3 are added the poly- glycosidase F of inscribe of 1 μ L；Mixture after the addition low fire reaction 20min in micro-wave oven carries out enzyme
It cuts；
It is two parts by sample aliquot, portion is directly purified with CM-PTs after 4.4 digestions；Another is by derivative reagent
After label, then purified with CM-PTs.Sample after label is for carrying out high performance liquid chromatography detection analysis, wherein for glycan
Derivative label, seven sugar of malt are marked with 2- aminobenzoic acid, and RNase B is marked with TMPP.
The present embodiment is efficiently quick using microwave-assisted digestion, it is only necessary to which digestion process can be completed in 20min, and traditional
37 DEG C of digestions need at least 12h to compare the preparation duration for enormously simplifying test sample overnight.
Five, glycopeptide enrichment and glycan purification
The step of glycopeptide enrichment is with glycan purification is identical, includes the following steps: load sample, washs impurity, elutes sample
Product.Specifically:
The tryptic digestion product of the 10 μ g standard proteins loading eluent of 100 μ L is diluted.Glycopeptide enrichment is poly-
When sugar purifying, CM-PTs is washed three times with the pure water of 100 μ L by the continuous suction of liquid-transfering gun first；It is slow with 100 μ L loadings
Fliud flushing balances microtrabeculae three times；The digestion protein sample diluted is constantly aspirated on CM-PTs using liquid-transfering gun, thus
Adsorb glycopeptide/glycan in sample sufficiently with cellulose microsphere；Microtrabeculae is washed three times with 100 μ L sample-loading buffers, to wash away
Non- glycopeptide and other impurity on unadsorbed；Finally microtrabeculae is eluted twice with the eluent of 50 μ L, it is micro- by cellulose is adsorbed on
Glycopeptide/glycan of ball elutes, and is directly used in MALDI TOF-MS detection.In addition, material (the ZIC- of several commercializations
HILIC, microcrystalline cellulose, DPA-6S) also it is received in liquid transfer gun head, for assessing its effect to glycopeptide enrichment.
Six, mass spectral analysis
Using MALDI TOF-MS (Applied Biosystems/MDS Sciex), reflective-mode is analyzed by mass spectrometry.
Its repetitive rate and acceleration voltage are respectively 200Hz and 20kV.The sample eluent of 2 μ L is added drop-wise on MALDI steel plate and makes it
Then naturally dry covers sample with the 2,5-dihydroxybenzoic acid (DHB, 10mg/mL) of 2 μ L, after natural drying upper machine testing.
Obtained mass spectrometric data by Data explorer 4.0 (Applied Biosystems/MDS Sciex, Concord,
Canada) software is further processed.
Seven, it is enriched with result verification
The effect of different enrichment modes compares after 7.1 IgG tryptic digestions
By 0.1 μ g μ L-1IgG after (100 μ L) tryptic digestion is directly used in mass spectral analysis, mass spectrogram such as Fig. 3 a institute
Show, in the sample before being enriched with as seen from the figure, the signal in mass spectrogram is only capable of detecting 2 intensity almost entirely from non-glycopeptide
Very low glycopeptide.
After IgG after 10 μ g tryptic digestions is enriched with CM-PT, the enriched sample obtained after elution is used for matter
Spectrum analysis, mass spectrogram is as shown in Figure 3b, occurs 27 intensity and the very high glycopeptide peak of signal-to-noise ratio on mass spectrogram as seen from the figure, together
When, the signal of non-glycopeptide is almost completely removed.Thus it proves, after being enriched with by CM-PTs, to the noisy non-saccharide of glycopeptide
Peptide and other impurities almost all are removed, and glycopeptide is all retained.
The concentration effect for being commercialized material (including ZIC-HILIC, MCC and DPA-6S) for three kinds is compared with CM-PTs, figure
3c is shown as ZIC-HILIC concentration effect, wherein there is 10 glycopeptides to be detected；Fig. 3 d is shown as MCC concentration effect, in MCC
After enrichment, only 8 glycopeptides are detected, and are accompanied by the very strong interference from non-glycopeptide；Fig. 3 e is shown as DPA-6S
Concentration effect only detects 4 kinds of low intensive glycopeptide peaks, even with attempt to three kinds of different elutions after being enriched with DPA-6S
Condition, enrichment result are still very poor.
In contrast, after CM-PTs is enriched with, from 0.1 μ g μ L-1IgG enzymolysis product in can identify 27 kinds of glycopeptides, demonstrate,prove
Bright this method has better bioaccumulation efficiency and sample consumption is lower.In addition, the entire enrichment process of CM-PTs method is to pass through
Liquid-transfering gun constantly aspirates tens of completions, and compared with pervious HILIC method, this method is more convenient time saving.
7.2 CM-PTs verify the selectivity of glycopeptide
As shown in figure 4, by the enzymolysis product that non-glycopeptide BSA is artificially added into IgG tryptose enzymolysis product, to come
CM-PTs is studied to the selectivity of glycopeptide.The result shows that when the molar ratio of IgG and BSA is 1:10, before Fig. 4 a is enrichment,
Due to the serious inhibition of the non-glycopeptide of BSA, glycopeptide is not detected；Fig. 4 b is the intensity of non-glycopeptide after being enriched with using CM-PTs
It significantly reduces, and 13 glycopeptides can be identified.Even if when the molar ratio of IgG and BSA is up to 1:100, before Fig. 4 c is enrichment
For glycopeptide without clear band, Fig. 4 d is after CM-PTs is enriched with, still to have 7 glycopeptides that can be clearly detected after enrichment, Qiang You
Demonstrate to power the good anti-interference ability of CM-PTs and the high selectivity to glycopeptide.In addition, load capacity of the CM-PTs to glycopeptide
For 125mg g~1(IgG/ cellulose microsphere) is higher than many reported glycopeptide enrichment methods.
Eight, the performance verification of CM-PTs
Fig. 5 show the calculating of the load capacity and the rate of recovery of CM-PTs, in order to calculate CM-PTs for when glycopeptide is enriched with
For the load capacity of glycopeptide, the IgG of 10 μ g is respectively with fine filled with different number (50 μ g, 60 μ g, 80 μ g, 100 μ g and 150 μ g)
The microtrabeculae of plain microballoon is tieed up to be enriched with, the mass spectral intensities of six kinds of glycopeptides in the sample after measuring each enrichment, and by mass spectrographic
Signal strength determines its specific load capacity.Calculating for the load capacity of glycan, selecting seven sugar of malt is standard protein
Cellulose microsphere is studied for the load capacity of glycan.In addition, we also studied the rate of recovery of the CM-PTs in glycan purification.Make
It combines multiple microtrabeculaes to carry out Simultaneous purification to seven sugar of malt of the aminobenzoic acid label of different quality with a volley of rifle fire, then uses
High performance liquid chromatography to equivalent purified and without purified aminobenzoic acid label seven sugar of malt detect,
The peak area that both compares calculates the rate of recovery with this.Fig. 5 Plays deviation is to calculate institute by five duplicate experimental results
?.
Fig. 6 is the experiment test that sensitivity is enriched with for CM-PTs, is illustrated as cellulose microsphere and is enriched with respectively through tryptose
Tri- kinds of various concentration human IgG sample MALDI-TOF mass spectrograms of 100ng/ μ L, 10ng/ μ L, 1ng/ μ L of enzyme enzymatic hydrolysis, by can in figure
Clearly to obtain, in time in the case where sample concentration to be measured is extremely low, through enrichment still can detecte be more than 50% sugar
Peptide, it was demonstrated that enrichment process is effectively and from the interference of impurity.
Fig. 7 is the glycan (a) discharged after RNase B (0.1 μ g/ μ l) enzymatic hydrolysis and TMPP-Ac- glycan (b) through cellulose
MALDI-TOF mass spectrogram after microballoon enrichment, wherein square represents N-acetyl-glucosamine, and circle represents mannose.As shown in Figure 7
After cellulose microsphere enrichment in through the invention, glycan can be by accurate detection, and band is clearly noiseless.Explanation passes through
The enrichment of cellulose microtrabeculae in the present invention, can greatly shorten the preprocessing process of glycan and glycopeptide, improve the accurate of detection
Degree provides more accurate data and supports for clinical detection and scientific research.
Be it is necessary to described herein finally: above embodiments are served only for making technical solution of the present invention further detailed
Ground explanation, should not be understood as limiting the scope of the invention, those skilled in the art's above content according to the present invention
The some nonessential modifications and adaptations made all belong to the scope of protection of the present invention.
Claims (10)
1. a kind of cellulose microsphere, it is characterised in that: the cellulose microsphere is for the enrichment of glycopeptide and/or glycan and/or pure
Change pretreatment, the cellulose microsphere is solidified by cellulose aqueous solution through solution-gel transformation approach, and diameter is 5~20 μ
m。
2. cellulose microsphere according to claim 1, it is characterised in that: the average diameter of the cellulose microsphere is 12 μ
m。
3. cellulose microsphere according to claim 1, which is characterized in that cellulose microsphere method as follows
It is made:
I. cellulose-containing raw material is dissolved in the pre- sodium hydroxide/urea/water solution for being cooled to -10~-15 DEG C, mechanical stirring obtains
The cellulose aqueous solution of clear, the cellulose mass concentration of cellulose aqueous solution are 3.0~4.0%；It is centrifuged after dissolution；
Ii. it takes the cellulose aqueous solution after being centrifuged in step i to be instilled in the atoleine suspension containing Span 80 dropwise, drips
Heating degree is 40~50 DEG C, and it is uniform to suspending to continue 40~50 DEG C of stirrings after completion of dropwise addition；
Iii. it is stirred into step ii in the suspension of end and dehydrated alcohol is added, after mixing evenly, slow cooling to room temperature hangs
Supernatant liquid solidifies to obtain cellulose microsphere.
4. a kind of cellulose microtrabeculae, it is characterised in that: filled with any cellulose of claims 1 to 3 in the microtrabeculae
Microballoon, and cylinder both ends open, one end are equipped with the circular gasket of high molecular weight hydrophilic material, and gasket aperture is less than cellulose microsphere
Diameter.
5. cellulose microtrabeculae according to claim 4, it is characterised in that: the cellulose microtrabeculae is through following steps method system
:
A) cellulose microsphere and pure water are dispersed into cellulose microsphere to pure water to realize with the ratio that mass ratio is 2~3:1
In；
B) cellulose microsphere scattered in step a) is moved in the cylinder, pressurization discharge pure water.
6. the preprocess method of a kind of glycan and/or glycopeptide, it is characterised in that: the preprocess method uses such as claim 1
~3 any any cellulose microtrabeculaes of the cellulose microsphere or claim 4~5 carry out glycan purification and/or sugar
Peptide enrichment.
7. the application of cellulose microsphere or cellulose microtrabeculae described in one kind according to claim 1~3 or 4~5, feature exist
In the pretreatment of the cellulose microsphere or cellulose microtrabeculae for glycan and/or glycopeptide before Mass Spectrometer Method.
8. the application of cellulose microsphere or cellulose microtrabeculae described in one kind according to claim 1~3 or 4~5, feature exist
In the pretreatment of the cellulose microsphere or cellulose microtrabeculae for glycan and/or glycopeptide before high performance liquid chromatography detection.
9. the application of cellulose microsphere or cellulose microtrabeculae described in one kind according to claim 1~3 or 4~5, feature exist
In the cellulose microsphere or cellulose microtrabeculae are for protein glycosylation analysis pretreatment.
10. a kind of detection method of protein glycosylation, which is characterized in that the albumen is after digestion using such as claims 1 to 3
Any any cellulose microtrabeculae of the cellulose microsphere or claim 4~5 carries out glycan purification and/or glycopeptide is rich
After collection, then carry out mass spectrum or efficient liquid phase chromatographic analysis.
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