Derivatization And Separation Analysis Of Glycoprotein N-/O-glycan Aminopyrazolone Chromogenic Reagent

As the third type of biomacromolecule besides nucleic acid and protein,carbohydrate plays an important role in signal transduction,molecular recognition and immune response.Glycosylation is one of the most important modifications in the post-translational modification of proteins,which is important for the structure and function regulation of protein.Abnormal changes of glycan from glycoprotein is associated with the development of many diseases.Therefore,functional glycomics,which mainly focuses on the structure-function of glycans,has attracted a great deal of attention in the life sciences.Sugar chains need to be derived in the process of analysis as the chromogenic groups was lost,which can not only make the glycans with UV or fluorescent groups,improving the detection sensitivity,and reducing the polarity of glycans,facilitating the chromatographic separation.However,the existing regents generally only have one active functional group,producing the corresponding glycan derivatives without free active groups for further preparation and functional analysis of glycans.Just reported a few bifunctional reagents,applies the reducing N-glycans analysis,but it is difficult to obtain the reducing glycoprotein O-glycans,hence not used widely application.Therefore,it is of great significance to develop a method that the derivation of bifunctional group reagent with reducing glycans,which is suitable for glycoprotein N-glycans and O-glycans.In the study,the reagent of aminopyrazolone hetero-functional group,3-amino-l-phenyl-2-pyrazoline-5-ketone(PAP),was used as a generalized derivation reagent for glycoprotein N-/O-glycans.The regent not only can occur Reductive amination reaction with reducing glycans,producing single PAP-labeled glycans with active methylene in the acidic medium,and can occur with reducing glycans of Michael addition reaction in alkaline medium,generating double PAP-labeled glycans with an active amino,and can also realize the release of glycoprotein O-glycans and simultaneous labeling PAP in alkaline medium.The glycan derivatives were purified by graphite carbon or C18 solid phase extraction small column,which can be directly detected and quantitatively analyzed by high performance liquid chromatography(HPLC)and electrospray mass spectrometry(ESI-MS)and hydrophilic liquid chromatography-mass spectrometry online combination separation(HILIC-MS),establishing the foundation for further the immobilization of glycoprotein glycans and function research.The main results are as follows:1.A new method for the reducing N-glycans were derived PAP by Reductive amination and Michael addition was proposed.After gradual optimization,the best conditions as follows:First,500 ?L of 0.625 M PAP in DMSO and 150 ?L of glacial acetic acid were added to a screw-capped tube with N-glycans released from ovalbumin and human milk,and mixed well by shaking.Then,the tube was heated and maintained at 70? for 2 h.After the completion of the reaction,and 500 ?L of 0.75 M NaBH3CN solution were added to the above solution,and then,the tube was heated and maintained at 70 ? for 1 h.Secondly,500 ?L of 0.75 M PAP in DMF and 500 ?L of 0.3 M NaOH were added to a screw-capped tube with N-glycans released from ovalbumin and human milk,and mixed well by shaking.Then,the tube was heated and maintained at 50? for 1.5 h.This two methods of derivatization of glycans completely,which have high reaction specificity.The PAP-labeled glycans of Reductive amination reaction have active methylene,and the PAP-labeled glycans of Michael addition reaction have two active aminos.2.A new method of HPLC separation and analysis of PAP-labeled glycans was established by different reaction modes.Maltodextrin mixture as a modal glycan.The separation conditions of HPLC for the PAP-labeled glycans were optimized,and the optimal separation conditions were as follows:column:TSK-GEL Amide-80 column(250mm×4.6mm,5?m);column temperature of 20?,the detection wavelength was set to 254 nm,the flow rate was set to 1 mL min-1;Solvents A,B,and C were ACN,100 mM ammonium acetate(pH=6.0),and Milli-Q water,respectively.The separation gradient was as follows:t=0 min,75%A,25%B;t=120 min,55%A,45%.The separation conditions have good generality to the two kinds of PAP-labeled glycans,and the separation degree of the glycans is well.3.The PAP derivation method was used to analyze the N-glycans in ovalbumin and human milk.The results are as follows:the PAP-labeled N-glycans prepared by Reductive amination were analyzed via on-line HILIC-MS in the negative ion mode.18 PAP-labeled N-glycans of ovalbumin were observed,among them,two glycans,H5N6 and H6N6,have two isomers.A total of 14 PAP-labeled N-glycans of human milk were discovered,two glycans,H3N4F1 and H4N5F1,have two isomers.Glycans,H3N5F1,have three isomers.The PAP-labeled N-glycans prepared by Michael addition were analyzed via on-line HILIC-MS in the positive ion mode.Totally,22 PAP-labeled N-glycans were discovered,Glycans,H3N6,have three isomers,and two glycans,H4N6 and H6N6,have two isomers.A total of 19 PAP-labeled N-glycans of human milk were discovered,two glycans were found in all PAP-labeled N-glycans,H4N5F1 and H4N5SA2,have two isomers.These results shown that the proposed method to different glycoprotein and complex biological samples with good applicability,and the mass spectrum detection sensitivity of PAP-labeled glycans detected by Michael addition reaction higher than the Reductive amination reaction.4.A new method for the dissociation of glycoprotein O-glycans and the simultaneous labeling of PAP was proposed and established.Asialifetuin as a modal glycans,and the reaction conditions were optimized by the system.The optimum reaction condition as follows:glycoprotein was dissolved in 500 ?L 1.2 M PAP/DMF solution,and 500 ?L 1 M NaOH/H2O solution was added to 16 h at 65?.On this basis,the O-glycans of pig stomach mucins and fetal bovine serum were successfully released by the new method,and further processing HILIC-MS analysis.The pig stomach mucin was dissociated from 30 O-glycans via the new method.A total of 4 O-glycans,2 neutral glycans and 2 acid glycans were dissociated from the fetal bovine serum through the method.The results are show that all kinds of neutral and acidic O-glycans of glycoprotein and complex biological samples have good applicability.