CN107796889A - The different two functional group reagents derivatization of amino-pyrazol quinoline ketone and method for separating and analyzing of reproducibility sugar chain and glycoprotein O sugar chains - Google Patents

Abstract

Present invention relates particularly to the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain, specially reproducibility sugar chain and different two functional group reagent of amino-pyrazol quinoline ketone, by two kinds of reaction pattern derivatizations, first, reduction amination generates the sugar chain derivative of active methylene in acid condition；Second, Michael addition reaction occurs in the basic conditions, sugar chain derivative of the generation with primary amino radical.Two kinds of derivatives caused by the derivatization method of the present invention carry different activities group, can carry out the further analysis and research of sugar chain, derivatization efficiency high is versatile.The present invention additionally provides in the basic conditions simultaneously, release of different two functional group reagent of amino-pyrazol quinoline ketone to glycoprotein O sugar chains and the method marked simultaneously, obtains the O sugar chain derivatives with primary amino radical, enormously simplify Sample Preparation Procedure.

Description

The different two functional groups examination of the amino-pyrazol quinoline ketone of reproducibility sugar chain and glycoprotein O- sugar chains Agent derivatization and method for separating and analyzing

Technical field

The invention belongs to glycobiology technical field, and in particular to the amino-pyrazol of reproducibility sugar chain and glycoprotein O- sugar chains The different two functional group reagents derivatization of quinoline ketone and method for separating and analyzing.

Background technology

Threeth class large biological molecule of the glucide as structure life entity, has particularly significant biological function, is such as Organism provide energy matter, be form plant cell wall chief component, the differentiation of cell, development, tumour hair It is raw to play an important role with intercellular signal transduction, identification, immune response etc..Glycosylation is protein post-translational modification In it is most important modification one of, play very important effect during protein translation regulation and control, protein degradation etc..50% Protein above exists in the form of glycoprotein, and more and more research shows, glycosylation is abnormal can to cause mankind's disease Disease.In treatment clinical course, the antibody drug used is with glycosylation.

At present, substantial amounts of research shows that the anomalous variation of the sugar chain on glycoprotein is relevant with many diseases, therefore with research The 26S Proteasome Structure and Function of sugar chain has attracted life science widely to pay close attention to for the functional sugar group of main contents, especially with sugar Chain screens the research of cancer serum mark as research object, is early diagnosis, prognosis, treatment, the reaction of human cancer Prediction and population screening open new approach, for example, sialylated modification can usually occur for the sugar-chain end of glycoprotein, it is not Sugar chain structure can only be stablized, and it is closely related with the occurrence and development of many malignant diseases；Therefore sugar chain structure is separated Seem very important with structure elucidation.

But because naturally occurring glucide does not contain chromophoric group in itself, it is divided with high performance liquid chromatograph Met difficulty from detection, and derivatization can not only make ultraviolet on sugar chain band or fluorophor, improve detection sensitivity, again can be with Sugar chain polarity is reduced, sugar chain is retained on a column, is easy to sugar chain to separate.Derivative to sugar chain C-1 ends half by contracting Hydrophobic chemical group on aldehyde structure band, improve the hydrophobicity of sugar chain structure, improve the Ionization Efficiency of glucide, Jin Erke Improve sensitivity of the glucide in MS detection and analysis.

Mainly have currently for the pre-column derivatization technology of reproducibility sugar chain C-1 terminal aldehyde groups：(1) reductive amination method, example Such as AEC (abbreviation AEC) derivatization；(2) alkaline condensation with pyrazolone reacts, e.g., 1- phenyl -3- first Base -5- pyrazolones (abbreviation PMP)；(3) hydrazine reagent commonly uses phenylhydrazine, Gerald T reagents etc. into hydrazone reaction.But above-mentioned spread out Biochemical reagents all only carry a functional group, and after to sugar chain derivatization, derivative products lack free active function groups, nothing Method is used for the further analysis of sugar chain, such as carbohydrate chip structure, the immobilization of magnetic ball and fluorescence labeling.

With the development that sugar group is learned, for the ease of the further analysis of sugar chain, the difunctional for sugar chain derivatization spreads out Biochemical reagents exploitation is extremely important, and this kind of reagent has two active function groups, and one of functional group can be used for mark sugar The reducing end of chain, another functional group can be that follow-up carbohydrate chip structure, the immobilization of magnetic ball and fluorescence labeling etc. provide reaction site, For functional selection.This kind of reagent report is less, mainly there is DAP (abbreviation DAP) and N- amino-ethyls -2- Aminobenzamide (abbreviation AEAB) both, but they carry two amino, are also easy to produce Competition, are reacted with sugar chain When selectivity it is not strong, and can be only applied to the derivatization of reproducibility sugar chain, for the O- sugar chains on glycoprotein and do not apply to.

In view of problem above, developing utilization new, that derivatization is more efficient has the different of Liang Ge different activities functional group It is significant that two functional group reagents organize the research learned to the method that reproducibility sugar chain performs the derivatization for sugar.

The content of the invention

In order to solve the method severe reaction conditions performed the derivatization in the prior art to reproducibility sugar chain, derivatization efficiency It is not high, and derivatization reagent only contains an active function groups, it is impossible to the defects of realizing the further analysis of sugar chain, a side of the invention Face provides the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain, real by the following technical programs It is existing：

The different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain, specially reproducibility sugar chain with The different two functional group reagents 3- amino -1- phenyl-2-pyrazoline-5-ketones of amino-pyrazol quinoline ketone, are reduced in acid condition Aminating reaction, generate the sugar chain derivative of active methylene；Or Michael addition reaction occurs in the basic conditions, generate Sugar chain derivative with primary amino radical；

The reproducibility sugar chain is the sugar chain discharged on free reproducibility sugar chain or glycoprotein, wherein, the free reduction Property sugar chain be the reproducibility sugar chain that is extracted from plant, animal or reproducibility sugar chain that synthesis obtains.

Further, the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of reproducibility sugar chain of the present invention Method, comprise the following steps：

Step (1A)：Reproducibility sugar chain is added into 3- amino -1- phenyl-2-pyrazoline-5-ketones to prepare with protic In obtained solution, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is less than 1mol/L, the reaction The ratio between molar concentration of 3- amino -1- phenyl-2-pyrazoline-5-ketones and reproducibility sugar chain is more than or equal to 10 in system:1；

Step (2A)：Organic monoacid, volume basis of the organic monoacid in reaction system are added into reaction system Than being more than 3 and less than 6 for the pH of 7%~25%, reaction system, 65 DEG C~75 DEG C are heated to, is reacted 1 hour~2 hours；

Step (3A)：After reaction terminates, sodium cyanoborohydride, the cyano group boron are added into step (2A) reaction system The ratio between amount of material of sodium hydride and reproducibility sugar chain is more than or equal to 20:1；

Step (4A)：65 DEG C~75 DEG C are maintained, is reacted 0.5 hour~1 hour, post processing is obtained with active methylene group Sugar chain derivative.

Further, the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of reproducibility sugar chain of the present invention Method, comprise the following steps：

Step (1A)：Reproducibility sugar chain is added into 3- amino -1- phenyl-2-pyrazoline-5-ketones with DMF or DMSO with obtained To solution in, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is 0.625mol/L, the reaction The ratio between molar concentration of 3- amino -1- phenyl-2-pyrazoline-5-ketones and reproducibility sugar chain is 25 in system:1；

Step (2A)：Glacial acetic acid is added into reaction system, percent by volume of the glacial acetic acid in reaction system is 15%, the pH of reaction system is more than 3 and less than 6, is heated to 70 DEG C, reacts 2 hours；

Step (3A)：After reaction terminates, sodium cyanoborohydride is added into step (2A) reaction system, wherein the cyanogen The ratio between amount of material of base sodium borohydride and reproducibility sugar chain is 30:1；

Step (4A)：70 DEG C are maintained, is reacted 1 hour, post processing obtains the sugar chain derivative of active methylene.

Further, the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of reproducibility sugar chain of the present invention Method, comprise the following steps：

Step (1B)：Reproducibility sugar chain is added into 3- amino -1- phenyl-2-pyrazoline-5-ketones to prepare with protic In obtained solution, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is less than 1mol/L, the reaction The ratio between molar concentration of 3- amino -1- phenyl-2-pyrazoline-5-ketones and reproducibility sugar chain is more than or equal to 5 in system:2；

Step (2B)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH is more than 9.5, is heated to 40 DEG C~70 DEG C, react 1 hour~1.5 hours；

Step (3B)：After reaction terminates, regulation reactant mixture pH to neutrality, post processing obtains the sugar chain with primary amino radical Derivative.

Further, the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of reproducibility sugar chain of the present invention Method, comprise the following steps：

Step (1B)：Reproducibility sugar chain is added into 3- amino -1- phenyl-2-pyrazoline-5-ketones with DMF or DMSO with obtained To solution in, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is 0.25mol/L~0.75mol/ L, the ratio between 3- amino -1- phenyl-2-pyrazoline-5-ketones and molar concentration of reproducibility sugar chain are 3 in the reaction system:1；

Step (2B)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH=10, is heated to 50 DEG C, react 1.5 hours；

Step (3B)：After reaction terminates, regulation reactant mixture pH to neutrality, post processing obtains the sugar chain with primary amino radical Derivative.

Further, the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of reproducibility sugar chain of the present invention In method, reproducibility sugar chain is with the different two functional group reagents 3- amino -1- phenyl-2-pyrazoline-5-ketones of amino-pyrazol quinoline ketone in acid The sugar chain derivative for the active methylene that derivatization obtains carries out preliminary purification with graphitic carbon solid-phase extraction column under the conditions of property；

The graphitic carbon solid-phase extraction column purge process is：Graphitic carbon solid-phase extraction column is first activated with acetonitrile, then is steamed with double Water balance, then by sample loading, desalination first is eluted with distilled water after loading, then, is entered with the acetonitrile solution of suitable concn Row elution, collects eluent, the sugar chain derivative of methylene that be concentrated under reduced pressure after purification, active；

Reproducibility sugar chain exists with the different two functional group reagents 3- amino -1- phenyl-2-pyrazoline-5-ketones of amino-pyrazol quinoline ketone The sugar chain derivative with primary amino radical that derivatization obtains under alkalescence condition C18 solid-phase extraction column preliminary purifications；

The C18 solid-phase extraction columns purge process is：C18 solid-phase extraction columns are first activated with acetonitrile, then distilled water balance, so Afterwards by sample loading to be purified, desalination first is eluted with distilled water after loading, then, is washed with the acetonitrile solution of suitable concn It is de-, collect eluent, sugar chain derivative that be concentrated under reduced pressure after purification, that there is primary amino radical.

On the other hand, amino-pyrazol quinoline ketone different two functional group reagent of the invention for also providing described reproducibility sugar chain spreads out The separation method for the sugar chain derivative that biochemical method is prepared, the separation method are divided using one-dimensional normal phase column HPLC From specific chromatographic condition is as follows：

Chromatographic column：TSK-GEL Amide-80 posts；

20 DEG C, Detection wavelength 254nm, flow velocity 1.0mL/min of column temperature；

Mobile phase A is acetonitrile, and B is pH=6 100mM ammonium acetate solution, and C is distilled water,

Sample separation condition：T=0min, 75%A, 25%B；T=120min, 55%A, 45%B；

The eluent of each component is collected respectively, and is concentrated under reduced pressure；

The derivatization product of a variety of sugar chains contained in sugar chain derivatization sample is obtained through above separation process.

In yet a further aspect, present invention also offers different two functional group reagent of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains Derivatization method, comprise the following steps：

Step (1C)：Glycoprotein sample is added into 3- amino -1- phenyl-2-pyrazoline-5-ketones to prepare with protic In obtained solution, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution be 0.75mol/L~ 1.2mol/L, per 5mg protein samples, add the solution of 3- amino -1- phenyl-2-pyrazoline-5-ketones described in 0.5mL；

Step (2C)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH=9~11, are heated to 50 DEG C~75 DEG C, react 8 hours~24 hours；

Step (3C)：After reaction terminates, regulation reactant mixture pH to neutrality, post processing obtains the O- sugar with primary amino radical Chain derivative.

Further, the different two functional group reagents derivatization of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains of the present invention Method, comprise the following steps：

Step (1C)：By glycoprotein sample add 3- amino -1- phenyl-2-pyrazoline-5-ketones and DMF prepare to obtain it is molten In liquid, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is 1mol/L, per 5mg protein samples, is added The solution of 3- amino -1- phenyl-2-pyrazoline-5-ketones described in 0.5mL；

Step (2C)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH=10, is heated to 65 DEG C, react 12 hours；

Step (3C)：After reaction terminates, reactant mixture pH is to neutrality for regulation, and organic solvent washes away impurity, is passed through after concentration C18 solid-phase extraction columns purify to obtain the O- sugar chain derivatives with primary amino radical.

Further, the different two functional group reagents derivatization of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains of the present invention In method, the C18 solid-phase extraction columns purifying detailed process in the step (3C) is：

C18 solid-phase extraction columns are first activated with acetonitrile, then distilled water balance, then by sample loading to be purified, after loading first Desalination is eluted with distilled water, then, is eluted with the acetonitrile solution of suitable concn, collects eluent, being concentrated under reduced pressure to have There are the O- sugar chain derivatives of primary amino radical.

Compared with prior art, beneficial effects of the present invention：

1st, the present invention has used different two functional group reagent of amino-pyrazol quinoline ketone to perform the derivatization reproducibility sugar chain first Method, by taking 3- amino -1- phenyl-2-pyrazoline-5-ketones as an example, there is structure shown below, abbreviation PAP,

This kind of derivative reagent has the different functional group of two activity, is active amino and active methylene group respectively.For With sugar chain reducing end reductive amination process occurs for reproducibility sugar chain, in acid condition, active amino, and active methylene group is herein Under the conditions of do not react；In the basic conditions, with sugar chain reducing end Michael addition reaction occurs for active methylene group, and active Amino does not react on this condition；Therefore, by the sugar after the different two functional group reagents derivatization of this kind of amino-pyrazol quinoline ketone Chain all carries an one's share of expenses for a joint undertaking active amino or active methylene group, the further analysis available for sugar chain.And this derivatization method Derivatization efficiency high, the reaction time is short, is adapted to various reproducibility sugar chains, versatile, simpler and more direct, efficient.

2nd, the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain of the invention, in acid bar Under part, the reproducibility sugar chain derivatization product after PAP reagent derivatizations, the mass concentration scope of solution is in 0.006mg/L In the range of~2.5mg/L, all have preferably linear；Sugar chain derivatization after PAP reagent derivatizations in the basic conditions Product, the mass concentration scope of solution all have preferably linearly, can quantified in the range of 0.002mg/L~2.5mg/L Analysis；Quantitative test under the conditions of two kinds repetition experiment RSD be below 5%, reacted this method have well repeatability, can By property.

3rd, the present invention also provides the different two functional group reagents derivatization of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains first Method, in the basic conditions, in O- sugar chains reducing end and different two functional group reagent of amino-pyrazol quinoline ketone that glycoprotein discharges Active methylene group Michael addition reaction occurs, O- sugar chain derivative of the generation with primary amino radical, and active amino is at this Do not reacted under part.

" mass concentration " of the present invention refers to that the quality of certain component in unit volume mixture is referred to as the matter of the component Measure concentration.

" substance withdrawl syndrome " of the present invention is also known as " molar concentration " and refers to contained solute in unit volume solution The amount of material.

Brief description of the drawings

Fig. 1 is two kinds of experiment course of reaction figures that different two functional group reagents PAP performs the derivatization to reproducibility sugar chain.

Fig. 2 is that the ESI-MS collection of illustrative plates (A) of the maltodextrin PAP derivatives of embodiment 1 and the maltodextrin PAP of embodiment 2 derive The ESI-MS collection of illustrative plates (B) of thing.

Fig. 3 is the selection result of PAP derivative reaction conditions under the acid condition of embodiment 3.

Fig. 4 is the selection result of PAP derivative reaction conditions under the alkalescence condition of embodiment 4.

Fig. 5 is the HILIC-HPLC collection of illustrative plates (A) and alkalescence condition of maltodextrin PAP derivatives under the acid condition of embodiment 5 The HILIC-HPLC collection of illustrative plates (B) of lower maltodextrin PAP derivatives.

Fig. 6 is for the ESI-MS collection of illustrative plates (A) of the PAP derivatives of the egg albumin N- sugar chains of embodiment 6 under alkalescence condition and in fact Apply the ESI-MS collection of illustrative plates (B) of the PAP derivatives under the people lactoprotein's N- sugar chain alkalescence conditions of example 7.

Fig. 7 is for the ESI-MS collection of illustrative plates (A) of the PAP derivatives of the egg albumin N- sugar chains of embodiment 6 under acid condition and in fact Apply the ESI-MS collection of illustrative plates (B) of the PAP derivatives under the people lactoprotein's N- sugar chain acid conditions of example 7.

Fig. 8 is the ultraviolet figures of online HILIC-MS of the PAP derivatives under the egg albumin N- sugar chain alkalescence conditions of embodiment 6 The online HILIC-MS for composing (A), EIC collection of illustrative plates (C) and the PAP derivatives under the people lactoprotein's N- sugar chain alkalescence conditions of embodiment 7 is purple Outer collection of illustrative plates (B), EIC collection of illustrative plates (D).

Fig. 9 is the ultraviolet figures of online HILIC-MS of the PAP derivatives under the egg albumin N- sugar chain acid conditions of embodiment 6 Compose the online HILIC-MS of the PAP derivatives under people lactoprotein's N- sugar chain acid conditions of (A), EIC collection of illustrative plates (C) and embodiment 7 Uv-spectrogram (B), EIC collection of illustrative plates (D).

Figure 10 is the ESI-MS collection of illustrative plates of the PAP derivatives of the pig stomach mucin O- sugar chains of embodiment 9.

Embodiment

Further detailed description is done to the present invention with reference to specific embodiment, but embodiments of the present invention are not limited to This.

The embodiments described below, unless other aspects show that all temperature are set to degree Celsius, unless otherwise specified The error range of temperature is that reaction temperature is room temperature, and room temperature refers to 25 DEG C ± 5 DEG C, all temperature without specified otherwise in embodiment Error is ± 5 DEG C.

3- amino -1- phenyl-2-pyrazoline-5-ketones (PAP) are bought in Alpha Ai Sha companies；Maltodextrin, Egg-white Albumen, pig stomach mucin, sodium cyanoborohydride (NaBH₃CN), sodium hydroxide, N-N dimethylformamides (DMF) buy in Sigma-Aldrich companies；PNGase F are purchased from New England BioLabs companies；Lauryl sodium sulfate (SDS), two Sulphur threitol (DTT), NP-40 are purchased from Aladdin Industrial Inc companies；Human milk is contributed from Xijing hospital volunteer Offer；Solid phase extraction column Sep-Pak C18 (100mg/1mL) are purchased from Waters companies；Solid phase extraction column porous graphitic carbon post (150mg/4mL) is purchased from Alltech Associates companies；Trifluoroacetic acid aqueous solution is purchased from Fisher Scientific companies.Its Remaining is domestic AR；High performance liquid chromatograph (HPLC) is bought in Japanese Shimadzu Corporation；LTQ-XL types electron spray electricity From mass spectrum (ESI-MS, Thermo Finnigan companies of the U.S.).Solid phase extraction column Sep-Pak C18 (100mg/1mL) are purchased from Waters companies；Solid phase extraction column porous graphitic carbon post (150mg/4mL) is purchased from Alltech Associates companies；Crystallite Cellulose pillar is made by oneself with disposable pipette tips.Trifluoroacetic acid aqueous solution is purchased from Fisher Scientific companies, and other reagents are Analyze pure.Concentrated ammonia liquor is directly bought, and percent mass concentration is 26%~28%.

Distilled water is that laboratory is prepared with automatic dual pure water distiller.

Mass Spectrometric Identification (ESI-MS) uses electron spray ionisation linear ion hydrazine mass spectrum (LTQ XL, Thermo in the present invention Scientific, USA) detected, unless otherwise instructed, testing conditions are as follows：

One-level ESI-MS parameter settings are as follows：Sample size, the control of 2 μ L injection annulus；Load sample mobile phase be methanol/water (50%/ 50%, v/v)；Flow velocity is 50 μ L/min；Operating voltage is 4kV；Sheath gas is 20arb；Auxiliary gas flow speed is 10arb；Hair Tubule voltage is 37V；Capillary lens voltage is 250V；Capillary temperature is 300 DEG C；Scan type is one-level full scan；Most Big injection length is 1000ms；Micro scanning is 3 times；Data acquisition uses LTQ Tune softwares, and the analysis software of sugar chain structure is Glycoworkbench。

The use of brief word or foreign language term below is through the present invention：

ACN acetonitriles

DMSO dimethyl sulfoxide (DMSO)s

FBS hyclones

Lac, Lactose lactose

Int% relative abundances

MeOH methanol

ML milliliters

Min minutes

Ms milliseconds

M mol/L

H hours

Retention time retention times

Reducing sugar reducing sugars

Ovalbmin egg albumins

Peak area peak areas

Relative Abundance relative abundances

SDS lauryl sodium sulfate

Samples samples

V is lied prostrate

Specific embodiment：

Embodiment in the present invention is the different two functional group reagents derivatization of amino-pyrazol quinoline ketone on reproducibility sugar chain Method, and the preliminary purification of derivatization product, separation and authentication method.Embodiment in the present invention also describes glycoprotein O- The different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of sugar chain.

By taking PAP as an example, performed the derivatization to reproducibility sugar chain two kinds examinations of different two functional group reagent of amino-pyrazol quinoline ketone The process for testing reaction is as shown in Figure 1.Firstly, for free reproducibility sugar chain, by taking maltodextrin oligosaccharide mixture as an example, pass through two Kind differential responses pattern derivatization.First, in acid condition, first sugar chain is dissolved in PAP/DMF and weak acid system and being reacted, then NaBH is added into above-mentioned system₃After the completion of CN reactions, by purifying, obtain carrying active methylene group (- CH₂- CO -) spread out Biology；Second, in the basic conditions, sugar chain is dissolved in PAP/DMF systems, regulation system pH is alkalescence, after the completion of reaction, warp Purifying is crossed, obtains carrying active amino (- NH₂) derivative；Again by HPLC respectively to two kinds of derivative separation analyses.It is right The derivative of sugar chain on glycoprotein, sugar chain first discharged and purified, and the sugar chain of release secondly is passed through into above two differential responses Pattern is derived, then derivative is separated respectively by HPLC and analyzed.Above-mentioned sugar chain derivative subsequently can be applied to carbohydrate chip Structure and functional analysis.

Reproducibility sugar chain includes the reproducibility sugar chain discharged on free reproducibility sugar chain or glycoprotein, wherein, dissociate reduction Property sugar chain be the reproducibility sugar chain that is extracted from plant, animal or reproducibility sugar chain that synthesis obtains.

Specific experiment process is described below.

Embodiment 1：The preliminary purification of maltodextrin (Maltodextrin) derivatization in acid condition and derivative And detection

(1) derivatization

PAP is dissolved in the solution for being configured to that concentration is 0.625mol/L in DMF, weighs maltodextrin 5mg (maltodextrins (Maltodextrin) it is a kind of macromolecular reproducibility oligosaccharides) add in 500 μ L PAP solution, then added into reaction system 150 μ L glacial acetic acids, reactant mixture react 2 hours at 70 DEG C.After reaction terminates, into mixture, addition concentration is 0.75mol/L NaBH₃The CN μ L of the aqueous solution 500, gained reaction system continue reaction 1 hour at 70 DEG C.After reaction terminates Room temperature is cooled to, is washed 3 times, each 1.5mL~2mL with dichloromethane, collects and arrives crude product after aqueous phase concentration.

(2) crude product of derivative graphitic carbon post preliminary purification desalination

Solid phase extraction column porous graphitic carbon post is activated with 3mL acetonitriles first, is then balanced, will slightly produced with 12mL distilled waters Thing loading, desalination then is eluted with 15mL distilled waters, elute objective sugar chain derivative with 35%ACN solution afterwards, collect elution Liquid, derivative is obtained after concentrate drying.

(3) detection and analysis of derivatization product

Derivative is tested and analyzed using ESI-MS；Positive ion mode detects, and scan type is one-level full scan, data Collection uses LTQ Tune softwares.

Specifically mass spectrum setup parameter is：

Sample size：2 μ L injection annulus control；

Load sample mobile phase：The methanol aqueous solution of volume ratio 50%, flow velocity are 50 μ L/min；

Operating voltage：4kV；

Sheath gas：20arb, auxiliary gas flow speed：10arb；

Capillary voltage：37V, capillary lens voltage：250V, capillary temperature：300℃；

Maximum injection length：1000ms, micro scanning：3 times；

Collision gas：Helium；

Isotope width m/z 3.00；

Ion collision energy is 35%~45%；

It is 0.25 to activate electric charge；

Activationary time is 30ms.

The matter of the maltodextrin PAP derivatives after reduction amination in acid condition is obtained according to above-mentioned testing conditions Spectrogram, as shown in Figure 2 A.As can be seen that maltodextrin and each mass spectrum after PAP derivatizations in Fig. 2A ESI-MS spectrograms The molecular weight at peak increases by 159, that is, connects a PAP fragment, it was demonstrated that maltodextrin complete derivatization.

Embodiment 2：The preliminary purification of maltodextrin (Maltodextrin) derivatization in the basic conditions and derivative And detection

(1) derivatization

PAP is dissolved in the solution for being configured to that concentration is 0.625mol/L in DMF, weighing maltodextrin 5mg, (maltodextrin is A kind of macromolecular reproducibility oligosaccharides) add in 500 μ L PAP solution, then 0.4mol/L hydroxide is added into reaction system The μ L of the aqueous solution 500 of sodium, gained reaction system react 1.5h at 50 DEG C.Reaction is cooled to room temperature after terminating, with 1M hydrochloric acid The aqueous solution is adjusted to pH=7, is then washed 1 time with dichloromethane, collect aqueous phase add 3~5 drop glacial acetic acids, then again with 1.5mL~ 2mL dichloromethane washs, and collects aqueous phase, is concentrated under reduced pressure, water-soluble after drying, then is washed 1 time with dichloromethane, obtains the thick of derivative Product.

(2) crude product of the derivative preliminary desalting purifying of C18 solid phase extraction columns

C18 solid phase extraction columns are activated with 3mL acetonitriles first, are then balanced with 12mL distilled waters, by crude product loading, so Afterwards with 12mL distilled waters elution desalination, objective sugar chain derivative is eluted with 15%ACN solution afterwards, collects eluent, concentration is dry Sugar chain derivative is obtained after dry.

(3) detection and analysis of derivatization product

Derivative is tested and analyzed using ESI-MS；Positive ion mode detects, and scan type is one-level full scan, data Collection uses LTQ Tune softwares.

Specifically mass spectrum setup parameter is：

Sample size：2 μ L injection annulus control；

Load sample mobile phase：The methanol aqueous solution of volume ratio 50%, flow velocity are 50 μ L/min；

Operating voltage：4kV；

Sheath gas：20arb, auxiliary gas flow speed：10arb；

Capillary voltage：37V, capillary lens voltage：250V, capillary temperature：300℃；

Maximum injection length：1000ms, micro scanning：3 times；

Collision gas：Helium；

Isotope width m/z 3.00；

Ion collision energy is 35%~45%；

It is 0.25 to activate electric charge；

Activationary time is 30ms.

The maltodextrin PAP after Michael addition reaction in the basic conditions is obtained according to above-mentioned testing conditions to derive The mass spectrogram of thing, as shown in Figure 2 B.In Fig. 2 B ESI-MS spectrograms as can be seen that maltodextrin with it is every after PAP derivatizations The molecular weight of individual mass spectra peak increases by 332, that is, connects two PAP fragments, it was demonstrated that maltodextrin complete derivatization.

It can be seen from embodiment 1~2, the different two functional group reagents PAP of amino-pyrazol quinoline ketone is to the reproducibility sugar chain that dissociates Derivatization method, not only the derivatization of sugar chain can be completed in acid condition, can also realize sugar chain in the basic conditions Derivatization, and derivatization efficiency high, the reaction time is short, more extensive compared to the applicable reaction condition of existing derivatization method, It is simpler and more direct, efficient.

Different two functional group reagent of amino-pyrazol quinoline ketone has the different functional group of two activity, be respectively active amino and Active methylene group.For reproducibility sugar chain, in acid condition, with sugar chain reducing end reductive amination process occurs for active amino, And active methylene group does not react on this condition；In the basic conditions, with sugar chain reducing end mikey occurs for active methylene group That addition reaction, and active amino does not react on this condition；Therefore, by different two functional group of this kind of amino-pyrazol quinoline ketone Sugar chain after reagent derivatization all carries an one's share of expenses for a joint undertaking active amino or active methylene group, the further analysis available for sugar chain.

Embodiment 3：The conditional filtering of different two functional group reagents PAP derivatization methods under acid condition

Using reducing disaccharides lactose as substrate, the derivative reaction condition of PAP under acid condition and sugar chain is screened, Sour environment is obtained by adding organic monoacid into reaction system, glacial acetic acid, benzoic acid or formic acid can be used to realize This reaction, it is important that control the pH value of reaction system to be not below 3, prevent acidity is too strong from causing sugar chain to degrade, wherein Glacial acetic acid reaction effect is preferable, therefore in following conditional filtering, selects glacial acetic acid.

Single factor test screening test is carried out to the derivatising condition in sour environment, investigates the mol ratio of PAP and sugar chain respectively, Cyano group in the addition of glacial acetic acid and reduction amination in the ratio between substance withdrawl syndrome namely in same solvent, condensation course Influence of the dosage of sodium borohydride to derivatization efficiency.

The use of sodium cyanoborohydride in fixed glacial acetic acid dosage, the temperature of condensation reaction, reaction time and reduction amination Amount, the temperature of reduction amination and reaction time, PAP/Lac molar concentration rate is investigated from 1 times (1:1) 25 times (25 is risen to:1) When, derivative yield, result of the test is as shown in Figure 3 a.

Result of the test is shown：PAP/Lac molar concentration rate is from 1 times (1:1) 40 times (40 is risen to:1) it is lactose-derived when Produce amount is in rising trend, shows that derivative can increase as PAP concentration increases, when molar concentration rate is more than 10 times (10:1) When, derivatization yield is more than 60%, and when molar concentration rate is more than 25 times (25:1) when, tend towards stability, derivatization yield highest. Therefore PAP/Lac molar concentration rates are 25 times, and reaction effect is preferable when PAP concentration is no more than 0.625mol/L.

Cyano group boron hydrogen in fixed PAP/Lac molar concentration rate, the temperature of condensation reaction, reaction time and reduction amination Change dosage, the temperature of reduction amination and the reaction time of sodium, the volume for investigating glacial acetic acid accounts for the reaction solution volume of condensation reaction Percentage (percent volume content) from 0% rise to 25% when, derivatization yield, result of the test is as shown in Figure 3 b.

Result of the test is shown：Glacial acetic acid main function is the pH for adjusting reaction system, the percent volume content of glacial acetic acid from When accounting for cumulative volume 0% and rising to 25%, lactose derivatives are in rising trend, show that target product can be with the increase of glacial acetic acid amount And increase, when the percent volume content (v/v) of glacial acetic acid is more than 7%, derivatization yield is more than 60%, and when the hundred of glacial acetic acid After partial volume content brings up to 15%, tend towards stability, derivatization yield highest.It is excessive in view of glacial acetic acid amount and easily make reactant The pH value of system is too low, is so as to be unfavorable for second step reductive amination process, therefore choose to add the percent volume content of glacial acetic acid Reaction effect is preferable when 15%.

Fixed PAP/Lac molar concentration rate, glacial acetic acid dosage, the temperature of condensation reaction, reaction time and reduction amine The temperature of reduction amination and reaction time in change, investigate the ratio between sodium cyanoborohydride and the Lac amount of material (namely reactant The ratio between substance withdrawl syndrome in system) from 1:1 rises to 60:When 1, derivatization yield, result of the test is as shown in Figure 3 c.

Result of the test is shown：NaBH₃CN/Lac substance withdrawl syndromes ratio is from 1 times (1:1) 60 times (60 is risen to:1) when, breast Sugar derivatives is in rising trend, shows that target product can be with NaBH₃CN dosage increases and increased, and works as NaBH₃CN/Lac materials Measure concentration ratio and be more than 20 times (20:1) when, derivatization yield is more than 60%, and works as NaBH₃CN/Lac substance withdrawl syndromes are than improving To after 30 times, tend towards stability, derivatization yield highest.NaBH₃CN/Lac substance withdrawl syndromes ratio is 30 times (30:1) reacted when Effect is preferable.Sodium cyanoborohydride is typically dissolved in water or alcohol and added as a solution, in test the concentration of lactose For 0.025mol/L, NaBH₃When CN concentration is 30 times of sugar chain, that is, concentration is 0.75mol/L.

In above-mentioned experiment, when PAP is reacted with sugar chain, reaction dissolvent can select suitable proton type molten Agent, in the art most commonly DMSO and DMF.

Embodiment 4：The conditional filtering of different two functional group reagents PAP derivatization methods under alkalescence condition

Using reducing disaccharides lactose as substrate, the derivative reaction condition of PAP under alkalescence condition and sugar chain is screened. Single factor test screening test is carried out to the derivatising condition in alkaline environment, respectively the mol ratio of investigation PAP and sugar chain, that is, In the ratio between substance withdrawl syndrome in same solvent, condensation course be hydrogenated with sodium oxide molybdena after the pH of system, the temperature of addition reaction and Influence of the reaction time to derivatization efficiency.

The pH (pH=10) of fixed reaction system, the temperature of addition reaction and reaction time, investigate the mole dense of PAP/Lac Ratio is spent from 1 times (1:1) 4 times (4 is risen to:1) when, derivative yield, result of the test is as shown in fig. 4 a.

Result of the test is shown：PAP/Lac molar concentration rate is from 1 times (1:1) 4 times (4 is risen to:1) when, lactose derivatives Yield is in rising trend, shows that derivative can increase as PAP concentration increases, when molar concentration rate is more than 2.5 times (5:2) When, derivatization yield is more than 60%, and when molar concentration rate is more than 3 times (3:1) when, tend towards stability, derivatization yield highest.By Precipitation is also easy to produce in PAP reagent concentrations are excessive, is unfavorable for purifying, therefore PAP/Lac molar concentration rates are 3 times, and PAP concentration is general Reaction effect is preferable during no more than 0.75mol/L.

When fixed PAP/Lac molar concentration rate, PAP concentration are 0.75mol/L, the temperature of addition reaction and reaction Between, investigate influences of the pH to derivatization of reaction system, sodium hydroxide can in solid form or be dissolved into solution add it is anti- Answer in system, for the simplicity of experiment, sodium hydroxide adds as an aqueous solution in this experiment, and investigation is to use sodium hydroxide The aqueous solution regulation reaction system pH from 8.5 rise to 14 when, derivative yield, result of the test is as shown in Figure 4 b.Verification experimental verification Reagent for regulation system pH can also select ammoniacal liquor.

Result of the test is shown：The pH of reaction system from 8.5 rise to 14 when, lactose derivatives yield is in rising trend, table Bright derivative can increase with the raising of alkalescence, and when pH is 9.5, derivatization yield is more than 60%, and when pH increases to 10, Derivative yield reaches highest, when pH continues to increase to more than 12, precipitation occurs in reaction system, makes product purification difficulty Increase, thus it is speculated that be due to that reaction solution alkalescence is too strong, caused by sugar chain is degraded, therefore it is 10 to choose reaction system pH.

Fixed PAP/Lac molar concentration rate, the pH (pH=10) of reaction system and addition reaction time, investigate reaction temperature Degree is from when rising to 70 DEG C for 30 DEG C, and derivative yield, result of the test is as illustrated in fig. 4 c.

Result of the test is shown：For reaction temperature from when rising to 70 DEG C for 30 DEG C, lactose derivatives yield is in rising trend, shows Derivative can increase as reaction temperature raises, and when reaction temperature is more than 40 DEG C, derivatization yield is more than 60%, and when anti- When answering temperature more than 50 DEG C, tend towards stability, derivatization yield highest.When being more than 70 DEG C due to reaction temperature, one is easily sloughed PAP, forms single PAP products, therefore reaction effect is preferable when reaction temperature is kept for 50 DEG C.

Fixed PAP/Lac molar concentration rate, the pH (pH=10) and addition reaction temperature of reaction system, when investigating reaction Between from when extending to 3 hours within 0.5 hour, derivative yield, result of the test is as shown in figure 4d.

Result of the test is shown：Reaction time, lactose derivatives yield was in rising trend from when extending to 3 hours within 0.5 hour, Show derivative can with the reaction time extend and increase, when reacted between be more than 1 hour when, derivatization yield be more than 60%, And when reacted between be more than 1.5 hours when, tend towards stability, derivatization yield highest.Therefore choose the reaction time be 1.5 hours when it is anti- Answer effect preferable.

In above-mentioned experiment, when PAP is reacted with sugar chain, reaction dissolvent can select suitable proton type molten Agent, in the art most commonly DMSO and DMF.

Embodiment 5：The HPLC separation methods of PAP derivatization products

The maltodextrin PAP derivatizations product that embodiment 1 and embodiment 2 are prepared can use one-dimensional normal phase column HPLC is separated, and HPLC instrument selection Shimadzu LC-2010AHT chromatographs, chromatographic column selects TSK-GEL Amide-80 normal phase columns (HILIC, 4.6mm × 250mm), specific chromatographic condition is as follows：

20 DEG C, Detection wavelength 254nm, flow velocity 1.0mL/min of column temperature；

Sample size is 10 μ L；

Mobile phase A is acetonitrile, and the ammonium acetate solution (pH=6) for the 100mM that B is, C is distilled water,

Sample separation condition：T=0min, 75%A, 25%B；T=120min, 55%A, 45%B；

The eluent of each component is collected respectively, and is concentrated under reduced pressure；

The derivatization product of a variety of sugar chains contained in sugar chain derivatization sample is obtained through above separation process.

The maltodextrin PAP derivatives under acid condition after condensation, reduction amination are obtained according to above-mentioned separation condition HILIC-HPLC collection of illustrative plates, as shown in Figure 5A.The HILIC- of maltodextrin PAP derivatives after Michael addition reaction HPLC collection of illustrative plates, as shown in Figure 5 B.

Embodiment 6：Separation, the detection of the PAP derivatizations and derivative of egg albumin N- sugar chains

(1) release of egg albumin N- sugar chains

The present invention prepares N- sugar chains using PNGase F enzyme process, and its standard step is as follows, by taking egg albumin as an example：

Enzymolysis：5mg egg albumins are dissolved in 540 μ L ultra-pure water, add 50 μ L albuminous degenerations liquid (0.5g SDS and 0.62g DTT are dissolved in 10mL distilled water), 10min is heated at 100 DEG C, is cooled to room temperature, is added 50 μ L phosphate buffers (1.90g sodium phosphates are dissolved in 10mL water, and pH=7.5 is adjusted to phosphoric acid) and 60 μ L 10%NP-40 (Nonidet P40s Used after being diluted with water 10 times), 1.2 μ L PNGase F enzymes (500unit/ μ L) are then added, 37 DEG C of insulations are dissociated 24 hours Afterwards, freezen protective, with to be purified.

Purifying：Obtained sample centrifugation will be digested, successively with C18 solid phase extraction columns and graphitic carbon solid phase extraction column pair The sample of dissociation is purified.C18 solid phase extraction columns purify, and purge process is as follows：10mL acetonitriles (CH₃CN pillar) is activated, 10mL ultra-pure waters balance pillar, and Aspirate supernatant is added dropwise in pillar, the ultrapure washing samples of 20mL；Water receiving elutes sample.Sample passes through C18 after purification, is and then purified, its process is as follows with graphitic carbon solid phase pillar：10mL acetonitriles (CH₃CN pillar, 10mL) are activated Ultra-pure water balance pillar, loading, with 25mL distilled waters remove it is miscellaneous, water lotion is not collected, then with 25% acetonitrile solution 4mL Elution, connects sample, will collect obtained sample normal temperature after purification and is spin-dried for, in -21 DEG C of freezen protectives, for follow-up derivatization, Detection and analysis.

(2) under acid condition the PAP derivatizations and product of egg albumin N- sugar chains separation, detection

The reaction condition for screening to obtain according to embodiment 3 carries out the PAP derivatizations of egg albumin N- sugar chains.PAP is molten Liquid concentration is 0.625mol/L, and (molecular weight of N- sugar chains is all higher than 342, therefore the egg albumin N- sugar chains that step (1) obtains The amount of its material is less than 0.0146mmol), the addition of all reaction reagents calculates according to sugar chain for 0.0146mmol, glacial acetic acid Percentage by volume in reaction system is 15%, the temperature 70 C of condensation reaction, 2 hours reaction time, then addition and PAP The isometric concentration of solution is 0.75mol/L NaBH₃The CN aqueous solution, continue reaction 1 hour at 70 DEG C.

Then ESI-MS detections are carried out according to the post processing in embodiment 1, purifying and detection method, obtains mass spectrogram as schemed Shown in 7A.By understanding that egg albumin N- sugar chains are increased by PAP derivatizations, the molecular weight of each mass spectra peak completely in Fig. 7 A 159, that is, connect a PAP fragment.

Egg albumin N- sugar chains PAP derivatizations product under acid condition according to embodiment 5 separation method, using one Normal phase column HPLC separation is tieed up, online HILIC-MS uv atlas is PAP and egg albumin N- such as Fig. 9 A as shown in Figure 9 A The online HILIC-MS collection of illustrative plates of derivatization product after sugar chain generation reduction amination, isolates to obtain 18 kinds of sugar chains, then more points Each eluting peak separated out, respectively extraction are obtained extracting ion flow chromatography (EIC) as shown in Figure 9 C, analyzed based on MS, identical molecule Appearance time of the sugar chain isomers of amount in liquid phase separation is different, finds that wherein double charge has 6 according to Fig. 9 C, Single electric charge 12, has 2 isomer to be present, is H5N6 and H6N6 respectively, and H5N6 and H6N6 have two isomerisms respectively Body, result above are consistent with document report.

(3) under alkalescence condition the PAP derivatizations and product of egg albumin N- sugar chains separation, detection.

The reaction condition for screening to obtain according to embodiment 4 carries out the PAP derivatizations of egg albumin N- sugar chains.PAP is molten Liquid concentration is 0.75mol/L, and 5mg egg albumins discharge to obtain N- sugar chains, all reaction examinations according to the same method of step (1) The addition of agent calculates that (molecular weight of N- sugar chains is all higher than 342, therefore 5mg egg albumins obtain according to 0.0146mmol The amount of N- sugar chain materials is less than 0.0146mmol), the concentration of sodium hydroxide solution is 0.3mol/L, continues to react at 50 DEG C 1.5 hour.

Then ESI-MS detections are carried out according to the post processing in embodiment 2, purifying and detection method, obtains mass spectrogram as schemed Shown in 6A.From in Fig. 6 A, egg albumin N- sugar chains are increased by PAP derivatizations, the molecular weight of each mass spectra peak completely 332, that is, connect two PAP fragments.

Egg albumin N- sugar chains PAP derivatizations product under alkalescence condition according to embodiment 5 separation method, using one Normal phase column HPLC separation is tieed up, online HILIC-MS uv atlas is PAP and egg albumin N- such as Fig. 8 A as shown in Figure 8 A The online HILIC-MS collection of illustrative plates of derivatization product after sugar chain generation Michael's addition, isolates to obtain 22 sugar chains；Then it is more Each eluting peak isolated, respectively extraction obtain extracting ion flow chromatography (EIC) as shown in Figure 8 C.Analyzed based on MS, identical point Appearance time of the sugar chain isomers of son amount in liquid phase separation is different, is found according to Fig. 8 C, egg albumin N- sugar In 22 sugar chains of chain, wherein double charge has 13, single electric charge 9, with the presence of 3 sugar chain isomers, be respectively H3N6, H4N6 and H6N6, wherein, H3N6 has 3 isomers, and H4N6 has two isomers, and H6N6 has two isomerisms Body, result above are consistent with document report.

Embodiment 7：Separation, the detection of the PAP derivatizations and derivative of people lactoprotein's N- sugar chains

(1) release of people lactoprotein N- sugar chains

According to the standard glycoprotein N- sugar chain preparation methods in embodiment 6, egg albumin is replaced with into people lactoprotein i.e. Human milk N- sugar chains can be prepared.

(2) under acid condition the PAP derivatizations and product of people lactoprotein N- sugar chains separation, detection

The reaction condition for screening to obtain according to embodiment 3 carries out the PAP derivatizations of people lactoprotein's N- sugar chains.PAP solution Concentration is 0.625mol/L, and the people lactoprotein N- sugar chains that 5mg people lactoproteins digest to obtain are as reaction substrate, all reaction reagents Addition calculate that (molecular weight of N- sugar chains is all higher than 342, therefore the N- sugar that 5mg people lactoproteins obtain according to 0.0146mmol The amount of chain material is less than 0.0146mmol), percentage by volume of the glacial acetic acid in reaction system is 15%, the temperature of condensation reaction 70 DEG C, in 2 hours reaction time, then add the NaBH with the isometric concentration of PAP solution for 0.75mol/L₃The CN aqueous solution, Continue reaction 1 hour at 70 DEG C.

Then ESI-MS detections are carried out according to the post processing in embodiment 1, purifying and detection method, obtains mass spectrogram as schemed Shown in 7B.By understanding that people lactoprotein N- sugar chains increase by 159 by PAP derivatizations, the molecular weight of each mass spectra peak completely in Fig. 7 B, Connect a PAP fragment.

According to the separation method of embodiment 5, use is one-dimensional for people lactoprotein's N- sugar chains PAP derivatizations product under acid condition Normal phase column HPLC is separated, and online HILIC-MS uv atlas as shown in Figure 9 B, isolates to obtain 14 kinds of sugar chains, such as Fig. 9 B, is PAP and the derivatization product after human milk N- sugar chains generation reductive amination process HILIC-MS collection of illustrative plates, are obtained 14 kinds of sugar chains.So Each eluting peaks isolated afterwards, respectively extraction obtain extracting ion flow chromatography (EIC) as shown in fig. 9d more.Analyzed based on MS, phase Appearance time of the sugar chain isomers in liquid phase separation with molecular weight is different, is found according to Fig. 9 D, wherein double charge There are 2, single electric charge 12, be H3N4F1, H3N5F1 and H4N5F1 respectively with the presence of 3 sugar chain isomers, wherein, H3N4F1 has two isomers, and H3N5F1 has three isomers, and H4N5F1 has two isomers, result above It is consistent with document report.

(3) under alkalescence condition the PAP derivatizations and derivative of people lactoprotein N- sugar chains separation, detection

The reaction condition for screening to obtain according to embodiment 4 carries out the PAP derivatizations of people lactoprotein's N- sugar chains.PAP solution Concentration is 0.75mol/L, and the people lactoprotein N- sugar chains that 5mg people lactoproteins digest to obtain are as the reaction substrate (molecule of N- sugar chains Amount is all higher than 342, therefore the amount of N- sugar chain materials that 5mg people lactoproteins obtain is less than 0.0146mmol), all reaction reagents Addition calculates according to 0.0146mmol, and the concentration of sodium hydroxide solution is 0.3mol/L, and it is small to continue reaction 1.5 at 50 DEG C When.

Then ESI-MS detections are carried out according to the post processing in embodiment 2, purifying and detection method, obtains mass spectrogram as schemed Shown in 6B.By understanding that people lactoprotein N- sugar chains increase by 332 by PAP derivatizations, the molecular weight of each mass spectra peak completely in Fig. 6 B, Connect two PAP fragments.

According to the separation method of embodiment 5, use is one-dimensional for people lactoprotein's N- sugar chains PAP derivatizations product under alkalescence condition Normal phase column HPLC is separated, and as shown in Figure 8 B, Fig. 8 B are that with human milk N- sugar chains mikey occurs for PAP to online HILIC-MS uv atlas The online HILIC-MS collection of illustrative plates of derivatization product after your addition reaction, isolates 20 sugar chains；What is be then demultiplex out respectively washes De- peak, respectively extraction are obtained extracting ion flow chromatography (EIC) as in fig. 8d, analyzed based on MS, the sugar chain of identical molecular weight is same Appearance time of the enantiomers in liquid phase separation is different, is found according to Fig. 8 D, in 20 kinds of obtained sugar chains, wherein double electricity Lotus has 12, single electric charge 8, is H4N5F1 and H4N5SA2 respectively with the presence of two sugar chain isomers, H4N5F1 has 2 Isomer, H4N5SA2 have 2 isomers, and result above is consistent with document report.

Embodiment 6~7 shows that the different two functional group reagents PAP of amino-pyrazol quinoline ketone of the invention is to reproducibility sugar chain Derivatization method is applicable not only to free reproducibility sugar chain, is equally applicable to the sugar chain discharged in glycoprotein, and through spreading out Obtained sugar chain derivative after biochemistry can be separated through HPLC, and then can carry out sugar chain analysis, experiment results proved , the PAP derivative analysis results of sugar chain are accurate, consistent with existing report result.

Embodiment 8：The quantitative analysis method of the PAP derivatization products of sugar chain and applicability are assessed

(1) quantitative analysis method of the PAP derivatization products of the sugar chain under acid condition is assessed

According to the derivative reaction condition for the optimization for screening to obtain in embodiment 3：PAP solution concentrations are in reaction system 0.625mol/L, lactose 0.025mol/L, percentage by volume is added as 15% glacial acetic acid, the temperature 70 C of condensation reaction, instead Answer time 2 h；Then the NaBH with the isometric concentration of PAP solution for 0.75mol/L is added₃The CN aqueous solution, at 70 DEG C Under continue reaction 1 hour.Then according to the post processing in embodiment 1, purification process, the lactose of active methylene is obtained PAP derivatives.

Quantitative analysis is carried out to lactose derivatives using C18-HPLC methods.Using lactose as standard items, by derivatization product Be configured to respectively concentration for 0.006,0.008,0.02,0.06,0.07,0.08,0.1,0.25,0.35,0.5,1,2.5mg/L The solution of 13 concentration gradients, HPLC analyses are carried out, so as to obtain a series of peaks.Then using the concentration of lactose as abscissa, inhale Receipts peak area is ordinate, draws quantitation curves, each concentration mensuration is three times.

When drawing standard curve, error during in order to reduce quantitative, with 0.006,0.008,0.02,0.06mg/L this four Individual concentration draws first curve；With 0.07,0.08,0.1,0.25mg/L this four concentration draw Article 2 curves；With 0.35, 0.5th, 1, this four concentration of 2.5mg/L draw Article 3 curves.

When the concentration of lactose is 0.006mg/L, absworption peak average area is 9534.4, when lactose concn is 0.06mg/L When, absworption peak average area is 101907.6, therefore when the absorption peak area of sugar chain in sample is at 9534.4~101907.6, It is applicable curvilinear equation y=0.18x；When lactose concn is 0.07mg/L, average peak is 317135.3, when lactose concn is During 0.25mg/L, absworption peak average area is 1258749, thus in the sample absorption peak area of sugar chain 317135.3~ At 1259749, curvilinear equation y=0.50x is applicable；When lactose concn is 0.35mg/L, absworption peak average area is 1969859.7, when lactose concn is 2.5mg/L, absworption peak average area is 13930597.Therefore when the absworption peak of sample sugar chain Area is applicable curvilinear equation y=0.55x at 1969859.7~13930597.The repeatability that accuracy passes through measuring method Rigorous analysis is carried out with the middle degree of accuracy, experiment is repeated 6 times per analysis site, calculates RSD values, lowest detection is limited to signal to noise ratio S/N For 3 when detection be limited to 0.006mg/L.Specific test result is shown in Table 1：

Table 1：The quantitative analysis method of PAP derivatizations product under the conditions of lactose acidity

As shown in Table 1, the calibration curve coefficient correlation of quantitative analysis method is above 0.99, has higher correlation, Proof can carry out quantitative analysis when derivatives concentration is in the range of 0.006~2.5mg/L；The RSD for repeating experiment is less than 5%, it was demonstrated that repeatability is preferably；Minimum detection limit as little as 0.006mg/L, there is higher sensitivity, suitable for quantitative analysis.

(2) quantitative analysis method of the PAP derivatization products of the sugar chain under alkalescence condition is assessed

The derivative reaction condition for the optimization for screening to obtain according to embodiment 4 carries out the PAP derivatizations of lactose：In system PAP solution concentrations are 0.75mol/L, lactose 0.3mol/L, isometric 0.3mol/L sodium hydroxide solution are added, at 50 DEG C Under continue reaction 1.5 hours.Then according to the post processing in embodiment 2, purification process, the lactose PAP with primary amino radical is obtained Derivative.

Quantitative analysis is carried out to lactose derivatives using C18-HPLC methods.Using lactose as standard items, by derivatization product Be configured to respectively concentration for 0.002,0.003,0.0055,0.006,0.008,0.01,0.015,0.05,0.08,0.15, 0.35th, 0.5,1,2.5mg/L 14 concentration gradients carry out HPLC analyses, so as to obtain a series of absworption peaks.Then with lactose Concentration be abscissa, absorption peak area is ordinate, draw quantitation curves.

In order to prove the reliability of curve, each concentration gradient is run three times respectively.Error during in order to reduce quantitative, with dense Spend for 0.002,0.003,0.0055,0.006,0.008mg/L this five concentration draw first curve；Using concentration as 0.01, 0.015th, 0.05, this four concentration of 0.08mg/L draw Article 2 curves；With concentration 0.15,0.35,0.5,1.0,2.5 this five Concentration draws Article 3 curve.

When lactose concn is 0.002mg/L, absworption peak average area is 8875.33, when lactose concn is 0.008mg/L When, average peak area is 36476, therefore when the absorption peak area of sugar chain in sample is at 8875.33~36476, it is applicable curve Equation y=0.41x；When lactose concn is 0.015mg/L, absworption peak average area is 89596.33, when lactose concn is During 0.15mg/L, absworption peak average area is 666222.3, thus in the sample absorption peak area of sugar chain 89596.33~ 666222.3 when, be applicable curvilinear equation y=0.44x；When lactose concn is 0.35mg/L, average peak area is 3009436, When lactose concn is 2.5mg/L, absworption peak average area is 32361465, therefore when the absorption peak area of sugar chain in sample exists At 3009436~32361465, curvilinear equation y=1.29x is applicable.

Accuracy carries out rigorous analysis by the repeated and middle degree of accuracy of measuring method, and examination is repeated 6 times per analysis site Test, calculate RSD values, lowest detection is limited to test limit 0.002mg/L when signal to noise ratio S/N is 3.Specific test result is shown in Table 2：

Table 2：The quantitative analysis method of PAP derivatizations product under lactose alkalescence condition

As shown in Table 2, the calibration curve coefficient correlation of quantitative analysis method is above 0.99, has higher correlation, Proof can carry out quantitative analysis when derivatives concentration is in the range of 0.002~2.5mg/L；The RSD for repeating experiment is less than 5%, repeatability preferably, minimum detection limit as little as 0.002mg/L, has higher sensitivity, suitable for quantitative analysis.

Embodiment 8 demonstrates, and different two functional group reagent of amino-pyrazol quinoline ketone of reproducibility sugar chain provided by the invention spreads out The PAP derivatives of biochemical method, in acid condition reproducibility sugar chain, mass concentration scope is in 0.006mg/L~2.5mg/L models In enclosing, all have preferably linear；The PAP derivatives of reproducibility sugar chain, mass concentration scope exist in the basic conditions In the range of 0.002mg/L~2.5mg/L, all have preferably linearly, quantitative analysis can be carried out；Quantitative test under the conditions of two kinds Repeat experiment RSD and be below 5%, having reacted this method has repeatability, reliability well.

The present invention also explores the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of the O- sugar chains in glycoprotein Method.Glycoprotein is dissolved in the solution that the different two functional group reagents PAP of amino-pyrazol quinoline ketone protic is configured to, added NaOH or ammoniacal liquor regulation pH, in a heated condition, stirring reaction, is obtained with active primary amino radical (- NH₂) O- sugar chains derive Thing.

Specific experiment process is as described in Example 8.

Embodiment 9：Different two functional group reagent of amino-pyrazol quinoline ketone of pig stomach mucin O- sugar chains discharges and derived simultaneously Change the detection with derivative

(1) release of pig stomach mucin O- sugar chains and derivatization

PAP is dissolved in the solution for being configured to that concentration is 1mol/L in DMF, 5mg pig stomach mucins is weighed, is dissolved in 500 μ L's In PAP/DMF solution, then add the 500 μ L 0.7mol/L NaOH aqueous solution, react 12 hours at 65 DEG C, after question response terminates, add 1mol/L hydrochloric acid solution adjusts pH to add 1.5mL~2mL dichloromethane, then 13000r/min centrifuges 3min, takes to neutrality Clearly, ice acetic acid 3~5 drips, and is adjusted to pH=4~5, then adds 1.5mL~2mL dichloromethane, centrifuges 3min in 13000r/min, takes Supernatant, hang and do, be re-dissolved in 500 μ L distilled waters, add 1.5mL~2mL dichloromethane, centrifuge 3min in 13000r/min, take supernatant, The crude product of outstanding dry derivative.

(2) O- sugar chain derivatives C18 solid phase extraction column preliminary purifications

C18 solid phase extraction columns are activated with 3mL acetonitriles first, are then balanced with 12mL distilled waters, by crude product loading, so Afterwards with 12mL distilled waters elution desalination, objective sugar chain derivative is eluted with 25%ACN solution afterwards, collects eluent, concentration is dry O- sugar chain derivatives are obtained after dry.

(3) detection and analysis of O- sugar chain derivatives

Derivative is tested and analyzed using ESI-MS；Positive ion mode detects, and scan type is one-level full scan, data Collection uses LTQ Tune softwares.Specific testing conditions are consistent with embodiment 2.Mass spectrogram is obtained, as shown in Figure 10.

It can be seen from fig. 10 that release obtains the O- sugar chains after 26 PAP derivatizations altogether, each O- sugar chains mass spectra peak Molecular weight increases by 332, that is, connects two PAP fragments, does not find to be marked with an one's share of expenses for a joint undertaking PAP products, it is [M+ that mass spectra peak is most of Na]⁺, a small amount of is [M+H]⁺[M+K]⁺Ionic species, this result are consistent with document report.

Above example 9 demonstrates the different two functional group reagents PAP of amino-pyrazol quinoline ketone and glycoprotein in the basic conditions Reaction, the O- sugar chains reducing end that glycoprotein discharges are sent out with the active methylene group in different two functional group reagent of amino-pyrazol quinoline ketone Raw Michael addition reaction, O- sugar chain derivative of the generation with primary amino radical, and active amino does not react on this condition, one Release and the mark of O- sugar chains are realized in individual course of reaction simultaneously, experiment process is enormously simplify, improves conventional efficient, this is right Organize to learn to study in sugar and there is significant application value.

Embodiment 10：Different two functional group reagent of amino-pyrazol quinoline ketone of pig stomach mucin O- sugar chains discharges and derived simultaneously Change reaction condition screening

The present invention also enters to the different two functional group reagents derivative reaction condition of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains Screening is gone.It is anti-to the derivatization of pig stomach mucin O- sugar chains specifically according to the reaction condition screening technique of embodiment 4 Condition is answered to be screened.

Using pig stomach mucin as substrate, PAP under alkalescence condition and pig stomach mucin reaction condition are screened.To alkali Property environment in derivatising condition carry out single factor test screening test, investigate PAP dosage respectively, be hydrogenated with sodium oxide molybdena in course of reaction The influence of the pH of system, the temperature and reaction time of reaction to derivative yield afterwards.

Result of the test is shown：In the case of keeping other reaction conditions constant, 500 μ L are added according to every 5mg protein samples PAP solution calculates, the concentration of PAP solution from 0.5mol/L rise to 1.2mol/L when, derivative can increase with PAP/DMF concentration Adding and increase, when PAP solution concentrations are more than 0.75mol/L, derivatization yield is more than 60%, and when concentration is more than 1mol/L, Tend towards stability, derivative yield highest.Precipitation is also easy to produce because PAP solution concentrations are excessive, is unfavorable for purifying, therefore, according to every The PAP solution that 5mg protein samples add 500 μ L calculates, and optimal PAP solution concentrations are 1mol/L.

The solvent of PAP solution can also use the suitable solvents such as DMSO instead in addition to DMF.

In the case of keeping other reaction conditions constant, sodium hydroxide is configured to the aqueous solution of various concentrations, or made With concentrated ammonia liquor, add in reaction system, adjust pH, when pH rises to 14 from 9, derivative yield is in rising trend, shows to spread out Biology can increase as alkalescence improves, and when pH is 9, derivatization yield is more than 60%, and when pH increases to 10, derivatization production Amount reaches highest, and when pH continues to increase to more than 11, impurity occurs in reaction system, makes the increase of product purification difficulty, pushes away Survey is due to that reaction solution alkalescence is too strong, and caused by sugar chain is degraded, therefore it is 10 to choose reaction system pH.

In the case of keeping other reaction conditions constant, from when rising to 75 DEG C for 40 DEG C, derivative yield is in reaction temperature Ascendant trend, show that derivative can increase as reaction temperature raises, when reaction temperature is more than 50 DEG C, derivatization yield is big In 60%, and when reaction temperature is more than 65 DEG C, tend towards stability, derivatization yield highest.When being more than 75 DEG C due to reaction temperature, A PAP easily is sloughed, forms single PAP products, therefore reaction effect is preferable when reaction temperature is kept for 65 DEG C.

Reaction time, derivative yield was in rising trend from when extending to 24 hours within 8 hours, showed that derivative can be with anti- Answer time lengthening and increase, when reacted between be more than 8 hours when, derivatization yield be more than 60%, and when reacted between be more than 12 During hour, tend towards stability, derivatization yield highest.Therefore choose the reaction time be 12 hours when reaction effect it is preferable.

Above example has filtered out the different two functional group reagents PAP of amino-pyrazol quinoline ketone in the basic conditions to O- sugar chains Derivatization optimum experimental condition, release and the derivatization of O- sugar chains are carried out under experimental condition after optimization, reaction efficiency is high, Impurity is few, is easy to purify.

Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to is assert The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention, On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's Protection domain.

Claims (10)

1. the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain, specially reproducibility sugar chain and ammonia The different two functional group reagents 3- amino -1- phenyl-2-pyrazoline-5-ketones of base pyrazoline ketone, reduction amination occurs in acid condition Reaction, generate the sugar chain derivative of active methylene；Or Michael addition reaction occurs in the basic conditions, generation has The sugar chain derivative of primary amino radical；

The reproducibility sugar chain is the sugar chain discharged on free reproducibility sugar chain or glycoprotein, wherein, the free reducing sugar Chain is the reproducibility sugar chain extracted from plant, animal or synthesizes obtained reproducibility sugar chain.

2. the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain according to claim 1, It is characterised in that it includes following steps：

Step (1A)：Reproducibility sugar chain addition 3- amino -1- phenyl-2-pyrazoline-5-ketones are prepared to obtain with protic Solution in, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is less than 1mol/L, the reaction system The ratio between molar concentration of middle 3- amino -1- phenyl-2-pyrazoline-5-ketones and reproducibility sugar chain is more than or equal to 10:1；

Step (2A)：Organic monoacid is added into reaction system, percent by volume of the organic monoacid in reaction system is 7%~25%, the pH of reaction system is more than 3 and less than 6, is heated to 65 DEG C~75 DEG C, reacts 1 hour~2 hours；

Step (3A)：After reaction terminates, sodium cyanoborohydride, the cyano group hydroboration are added into step (2A) reaction system The ratio between amount of material of sodium and reproducibility sugar chain is more than or equal to 20:1；

Step (4A)：65 DEG C~75 DEG C are maintained, is reacted 0.5 hour~1 hour, post processing obtains the sugar chain with active methylene group Derivative.

3. the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain according to claim 2, It is characterised in that it includes following steps：

Step (1A)：Reproducibility sugar chain is added into 3- amino -1- phenyl-2-pyrazoline-5-ketones and prepares what is obtained with DMF or DMSO In solution, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is 0.625mol/L, the reaction system The ratio between molar concentration of middle 3- amino -1- phenyl-2-pyrazoline-5-ketones and reproducibility sugar chain is 25:1；

Step (2A)：Glacial acetic acid is added into reaction system, percent by volume of the glacial acetic acid in reaction system is 15%, The pH of reaction system is more than 3 and less than 6, is heated to 70 DEG C, reacts 2 hours；

Step (3A)：After reaction terminates, sodium cyanoborohydride is added into step (2A) reaction system, wherein the cyano group boron The ratio between amount of material of sodium hydride and reproducibility sugar chain is 30:1；

Step (4A)：70 DEG C are maintained, is reacted 1 hour, post processing obtains the sugar chain derivative of active methylene.

4. the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain according to claim 1, It is characterised in that it includes following steps：

Step (1B)：Reproducibility sugar chain addition 3- amino -1- phenyl-2-pyrazoline-5-ketones are prepared to obtain with protic Solution in, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is less than 1mol/L, the reaction system The ratio between molar concentration of middle 3- amino -1- phenyl-2-pyrazoline-5-ketones and reproducibility sugar chain is more than or equal to 5:2；

Step (2B)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH is more than 9.5, is heated to 40 DEG C ~70 DEG C, react 1 hour~1.5 hours；

Step (3B)：After reaction terminates, regulation reactant mixture pH to neutrality, post processing obtains having the sugar chain of primary amino radical to derive Thing.

5. the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain according to claim 4, It is characterised in that it includes following steps：

Step (1B)：Reproducibility sugar chain is added into 3- amino -1- phenyl-2-pyrazoline-5-ketones and prepares what is obtained with DMF or DMSO In solution, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is 0.25mol/L~0.75mol/L, institute It is 3 to state the ratio between molar concentration of 3- amino -1- phenyl-2-pyrazoline-5-ketones and reproducibility sugar chain in reaction system:1；

Step (2B)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH=10, is heated to 50 DEG C, instead Answer 1.5 hours；

Step (3B)：After reaction terminates, regulation reactant mixture pH to neutrality, post processing obtains having the sugar chain of primary amino radical to derive Thing.

6. the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of reproducibility sugar chain according to claim 1, Characterized in that, reproducibility sugar chain and the different two functional group reagents 3- amino -1- phenyl -2- pyrazolines -5- of amino-pyrazol quinoline ketone Ketone in acid condition the active methylene that derivatization obtains sugar chain derivative with graphitic carbon solid-phase extraction column carry out just Step purifying；

The graphitic carbon solid-phase extraction column purge process is：Graphitic carbon solid-phase extraction column is first activated with acetonitrile, then is put down with distilled water Weighing apparatus, then by sample loading, desalination first is eluted with distilled water after loading, then, is washed with the acetonitrile solution of suitable concn It is de-, collect eluent, the sugar chain derivative of methylene that be concentrated under reduced pressure after purification, active；

Reproducibility sugar chain is with the different two functional group reagents 3- amino -1- phenyl-2-pyrazoline-5-ketones of amino-pyrazol quinoline ketone in alkalescence Under the conditions of the obtained C18 solid-phase extraction column preliminary purifications of the sugar chain derivative with primary amino radical of derivatization；

The C18 solid-phase extraction columns purge process is：C18 solid-phase extraction columns are first activated with acetonitrile, then distilled water balance, then will Sample loading to be purified, first desalination is eluted with distilled water after loading, then, is eluted with the acetonitrile solution of suitable concn, Collect eluent, sugar chain derivative that be concentrated under reduced pressure after purification, that there is primary amino radical.

7. the different two functional group reagents derivatization method system of amino-pyrazol quinoline ketone of the reproducibility sugar chain described in claim 1~6 The separation method of standby obtained sugar chain derivative, the separation method are separated using one-dimensional normal phase column HPLC, and its feature exists In specific chromatographic condition is as follows：

Chromatographic column：TSK-GEL Amide-80 posts；

20 DEG C, Detection wavelength 254nm, flow velocity 1.0mL/min of column temperature；

Mobile phase A is acetonitrile, and B is pH=6 100mM ammonium acetate solution, and C is distilled water,

Sample separation condition：T=0min, 75%A, 25%B；T=120min, 55%A, 45%B；

The eluent of each component is collected respectively, and is concentrated under reduced pressure；

The derivatization product of a variety of sugar chains contained in sugar chain derivatization sample is obtained through above separation process.

8. the different two functional group reagents derivatization method of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains, it is characterised in that including with Lower step：

Step (1C)：Glycoprotein sample is added into different two functional groups examination 3- amino -1- phenyl-2-pyrazoline-5-ketones and proton type Solvent is prepared in obtained solution, and the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is 0.75mol/L ~1.2mol/L, per 5mg protein samples, add the solution of 3- amino -1- phenyl-2-pyrazoline-5-ketones described in 0.5mL；

Step (2C)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH=9~11, are heated to 50 DEG C ~75 DEG C, react 8 hours~24 hours；

Step (3C)：After reaction terminates, regulation reactant mixture pH to neutrality, post processing obtains having the O- sugar chains of primary amino radical to spread out Biology.

9. the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains according to claim 8 Method, it is characterised in that comprise the following steps：

Step (1C)：Glycoprotein sample is added into different two functional groups examination 3- amino -1- phenyl-2-pyrazoline-5-ketones to prepare with DMF In obtained solution, the concentration of the 3- amino -1- phenyl-2-pyrazoline-5-ketones in the solution is 1mol/L, per 5mg albumen Sample, add the solution of 3- amino -1- phenyl-2-pyrazoline-5-ketones described in 0.5mL；

Step (2C)：Sodium hydroxide or ammoniacal liquor are added into reaction system, until reaction system pH=10, is heated to 65 DEG C, instead Answer 12 hours；

Step (3C)：After reaction terminates, regulation reactant mixture pH to neutrality, add organic solvent and wash away impurity, passed through after concentration C18 solid-phase extraction columns purify, and obtain the O- sugar chain derivatives with primary amino radical.

10. the different two functional group reagents derivatization side of amino-pyrazol quinoline ketone of glycoprotein O- sugar chains according to claim 9 Method, it is characterised in that the C18 solid-phase extraction columns in the step (3C) purify detailed process and are：

C18 solid-phase extraction columns are first activated with acetonitrile, then distilled water balance, then by sample loading to be purified, first with double after loading Water elution desalination is steamed, then, is eluted with the acetonitrile solution of suitable concn, collects eluent, primary must be had by being concentrated under reduced pressure The O- sugar chain derivatives of amino.