New Sample Preparation Methods Of N-Glycan Analysis And Their Applications

N-glycosylation of proteins plays an important role in biological processes including cell signaling,fertilization,proliferation,differentiation and so on.More and more studies have shown that N-glycans are closely associated with the occurrence and development of many diseases such as cancer,diabetes and heart failure.Therefore,studies of N-glycans have shown great potential in further understanding and effective treatment of human diseases.Due to the micro-heterogeneity and structural diversity of N-glycans,it is extremely difficult to study N-glycans comprehensively.The methods used for N-glycan analysis mainly consist of high-performance liquid chromatography(HPLC),capillary electrophoresis(CE),mass spectrum(MS)and the combination of above technologies.The sample preparation for N-glycan analysis includes a series of steps such as enzyme digestion,purification,labeling,and repurification.However,in the existing methods,complicated sample preparation and limited analytical sensitivity hinder its wide application in clinic.Therefore,fast,efficient and highly sensitive N-glycan analysis is becoming more and more meaningful,and also has a large research and development space.The specific research work is as follows:First,we establish a microwave-assisted rapid digestion method by peptide N-glycosidase F(PNGase F),which successfully achieves the complete release of N-glycans in only 20 minutes.After the glycoprotein is digested by PNGase F,the reducing end of the released N-glycan will exist as glycosylamine in a short time.While the direct glycosylamine labeling can remove the purification of N-glycan after digestion,significantly shortening the sample preparation process for N-glycan analysis.By double strategies of glycosylamine labeling with the(N-succinimidyloxycarbonyl-methyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide(TMPP-Ac-OSu)and methylamidation,MALDI-MS analysis sensitivity of N-glycans has been enhanced by 35 times,and a quantitative detection limit as low as 10 fmol is achieved;54 N-glycan structures are detected from only 50 n L of the serum sample,which exceeds the 10-fold sensitivity of the commonly used analysis method for serum N-glycans.However,MALDI-MS is slightly insufficient in the quantitative analysis.So,a method for the quantitative analysis of N-glycans by a glycosylamine fluorescent labeling reagent of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC)combined with hydrophilic interaction liquid chromatography fluorescence detection(HILIC-FLD)is further proposed.Compared with the traditional N-glycan fluorescence analysis method,the entire N-glycan preparation time is shortened from 20 h to 50 min,and the detection sensitivity is increased by 10 times.This method has good quantitative linearity(R~2>0.991)and reproducibility(RSD<8.7%).By application of the established method and a series of statistical analysis,it is revealed that the core-fucosylation,mono-and disialylated N-glycans in human serum are significantly related to the occurrence and development of lung cancer.HILIC has become a powerful N-glycan analysis technique,and the derivatized N-glycan can also be analyzed using reversed phase liquid chromatography(RPLC)and normal phase liquid chromatography(NPLC).We therefore comparatively analyze the chromatographic behavior and fluorescence response of N-glycan AQC derivatives(including neutral and acidic structures)during the separation of HILIC,NPLC and RPLC under different conditions.Results show that for acidic derivatives,NPLC is better than HILIC for separation,but it is somewhat limited in the reproducibility and applicability of the analysis;RPLC has low selectivity and retention for the both two types of derivatives,resulting in serious co-elution;HILIC has good separation and selectivity for both types of derivatives,especially in the analysis of complex samples(such as serum).But in the sample preparation process of HILIC analysis of N-glycans,a series of steps such as multi-step purification after AQC labeling and concentration and reconstitution before analysis limit its analysis efficiency.In order to solve this problem,we have established an ultrafiltration tube-assisted purification method and successfully achieved direct HILIC analysis of the target.While obtaining enhanced recovery of derivatives,it significantly reduces the sample preparation time by 25 and 6 times compared to existing general analytical method and N-glycan AQC labeling method with multi-step purification,respectively.The specific glyco-biomarker A2G2S1 has identified for the diagnosis of oral diseases and to predict their development:it significantly increased from the healthy state to the oral inflammation state,and significantly decreased when the oral inflammation state changed to oral cancer.The 96-well plate is furthermore used as a carrier to explore the sample preparation of N-glycan and obtain good stability(RSD<10%)providing a technical basis for high-throughput analysis.On-line and automated analysis of sample preparation for N-glycan analysis can increase analysis throughput and reduce analysis costs.In order to achieve this goal,based on the above research,combined with the microfluidic chip platform,sample preparations of digestion,derivatization and purification process are explored and verified through different types of N-glycan structures.Finally,an online N-glycan sample preparation of 30min has been successfully achieved and is then proved to be a simple and highly applicable method by the analysis of N-glycans in human serum.In this paper,we systematically studied the sample preparation process of N-glycan analysis by using technologies of microwave-assisted digestion,glycosylamine labeling,microfluidic chip combined with platforms of HPLC and matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS).A variety of efficient sample preparation strategies have been established,and their related applications in biology are studied as well.Fast and efficient N-glycan qualitative and quantitative methods and online preparation of N-glycans have been established,and studies of their biological applications,provid a new way for clinically efficient glycomic analysis.