| As one of the most important post-translational modifications,protein glycosylation plays crucial roles in various biological processes.Recently,aberrant alterations of serum N-glycans have been observed in various cancer studies,indicating that N-glycans may be the novel biomarkers for diagnosis of cancers.In view of characteristics for native N-glycans,many challenges may limit the discovery for N-glycan biomarkers.Firstly,the microscale and low ionization efficiency of native N-glycans makes demand for the development of derivative strategies with high sensitivity.Moreover,the non-template driven synthesis pattern increases the microheterogeneity and diversity of N-glycans,and isomeric N-glycan analysis may provide more information for branching and anomeric linkages.Therefore,novel strategies which may significantly improve the separation of isomers are indeed required.Furthermore,accurate quantitation of N-glycans is conducive for the identification of specific biomarkers.However,strategies based on quantitative analysis are still deficient.In view of the problems described above,a series of dual-derivatized strategies have been applied for qualitative and quantitative analysis of N-glycans.In addition,the established strategies have also been used for screening N-glycan biomarkers for cancers,and the main works are as following:NanoLC-ESI-MS has been a powerful tool for isomeric N-glycan analysis.In our work,the incorporation of nanoLC-ESI-MS and a dual-derivatized method with methylamidation and reduction has firstly been applied for the analysis of isomeric N-glycans obtained from serum with pancreatic cancer.Among the established methods,methylamidation can not only neutralize sialylated N-glycans,but also improve detection sensitivity.Additionally,N-glycans can be reduced to alditols by ammonia-borane complex,eliminating the effects of α-and β-types of anomers.By statistical analysis,25 specific isomers have been identified with significant difference between pancreatic cancer and healthy controls.Among which,AUCs of 4 isomers have exceeded 0.90,showing a highly accurate performance for diagnosis.Furthermore,these specific isomers have also been used for early diagnosis of pancreatic cancer.In the studies described above,the alteration in abundance of N-glycans are presented by relative abundance.Data processing method based on normalization requires analyzing abundance of each N-glycan.In contrast,a dual-derivatized method with d0/d5-benzoyl chloride and methylamidation has been applied for quantitative analysis of N-glycans,the strategy based on stable isotopic labeling can show aberrant changes of N-glycans more intuitively.Compared with traditional overnight digestion,glycosylamine can be effectively released in 20 min by microwave assisted rapid digestion,and the whole analytical process has been reduced to 4 h.In addition,although MALDI-MS shows soft-ionization in detection of N-glycans,the in-and post-source decay of sialylated N-glycans may occur,and the damage to N-glycans is irreversible.Methylamidation has been successfully applied for MALDI-MS analysis of sialylated N-glycans.The neutralization method can not only stabilize sialic acid,but also improve signal response of N-glycans.Eventually,the simultaneous quantitation of both neutral and sialylated N-glycans have been achieved.Moreover,glycoproteins of biological samples usually contain a relatively broad dynamic range,which makes demands for the quantitative capabilities of analytical methods.By quantitative analysis of high mannose N-glycans of RNase B with different molar ratios,the linear correlation coefficients have exceeded 0.9992,showing good capability for quantification.Furthermore,the dual-derivatized method has also been applied for quantitative analysis of serum glycomics,and 5 specific N-glycans which can be used for early diagnosis of myeloma have been identified.On the foundation of dual-derivatized method with d0/d5-benzoyl chloride and methylamidation,nanoLC-ESI-MS coupled with PGC separation has been applied for quantitative analysis of isomeric N-glycans.Since the isotope labeled N-glycans are derivatized and analyzed simultaneously,the effect of differences in ionization efficiency,charge distribution,ion suppression and signal response bias to MS can be eliminated effectively.By analyzing N-glycans of standard glycoproteins,the feasibility of the dual-derivatized method has been validated and the isomers of both neutral and sialylated N-glycans can be separated effectively.Moreover,by quantitative analysis of isomers from H5N2(RNase B)with different molar ratios,the linear correlation coefficients have exceeded 0.9998.Meanwhile,the reproducibility and accuracy of the method have also been evaluated,and the average CV and RE values haven’t exceed 4.88% and 3.02% respectively,indicating good stability and accuracy in quantitative glycomics.Furthermore,the strategy has also been applied for quantitative analysis of isomeric N-glycans obtained from one myeloma donor with different drug treatment stages,and some specific isomers which can be used for predicting drug effect have been identified.On the basis of studies described above,the quantitative analysis of isomeric N-glycans obtained from human serum transferrin has been carried out.Focused on the specific glycoprotein,interference caused by other glycoproteins can be eliminated.In addition,N-glycans with different linkage types of sialic acid may regulate different physiological processes.Therefore,the analysis of linkage types for sialic acid is very important.In this work,nanoLC-ESI-MS coupled with PGC was also applied for the separation of stable isotope labeled isomeric N-glycans.Meanwhile,sialic acid linkage types of isomeric N-glycans of transferrin have been identified with α 2-3 neuraminidase.By analyzing isomers of H5N4S2 from standard transferrin,a broader linear quantitative range has been achieved,which is effective than compositional analysis by MALDI-MS.Ultimately,the strategy has been used for screening specific biomarkers for myeloma,providing reference for predicting drug effect.The above-mentioned labeling reagents generally lack fluorophores group,which is not suitable for fluorescence detection by HPLC.In this work,a strategy based on Fmoc-OSu(Fmoc N-hydroxysuccinimide ester)has been established,providing a convenient way for fluorescent labeling.By microwave assisted rapid digestion,the time of glycoprotein deglycosylation have been largely reduced,inhibiting the hydrolysis of glycosylamines.In addition,the labeling of Fmoc can also enhance signal response,which is conducive for detecting N-glycans with low abundance.It is noteworthy that current methods which is available for preparation of standard glycans are still deficient.Although some methods have been established for it,the procedure is too complicated,which is not suitable for preparing standard glycans with high-purity.In our work,standard N-glycans have been successfully prepared by releasing Fmoc residue,providing possibilities for interaction studies of biomoleculars.Furthermore,the method has also been applied for analyzing serum N-glycans,aiming to screen specific biomarkers for early diagnosis of lung cancer.Overall,qualitative and quantitative studies for N-glycans have been carried out from the perspective of compositional glycan profiling and isomeric profiling.On the foundation of methylamidation,sialic acid residues can be stabilized,and simultaneous quantitative analysis of both neutral and sialylated N-glycans have been achieved.In addition,we not only analyzed linkage types of sialic acid on sialylated isomers but also prepared standard N-glycans successfully.Eventually,N-glycans obtained from serum with various cancers have been analyzed,providing prospects for discovery of specific N-glycan biomarkers. |
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