Development And Applications For High Sensitive Analysis Of N-Glycans Using MALDI-MS

As one of the most important and common post-translational modification,glycosylation participates in many physiological and pathological processes.Due to the anomeric linkage and branching of the glycans,the diversity of glycans moieties is much more than the linear nucleic acids and proteins.In addition,nontemplate-driven biosynthesis of glycans resulted in diverstiy and microheterogeneity.For these reasons,the difficulty and complexity of glycan analysis was improved,which making it a very challenging task.It has been an important technique for the analysis of glycans by using MALDI-MS,offering advantages of ease of operation,short analysis time and impurity tolerance.However,the hydrophilic nature of oligosaccharides resulting from the hydroxyl groups leads to lower ionization efficiency in the MALDI process.In addition,it was difficult for direct analysis of underivatized sialylated glycans because of the preferential cleavage of the sialic acid moiety by in-and postsource decay.Moreover,oligosaccharides that lack of basic sites were readily ionized by the addition of metal ions,which resulted in a decrease of sensitivity and an increase of difficulties in data interpretation.In this thesis,nonspecific proteolysis in coupling with co-derivatization of TMPP-Ac and methylamidation and rapid digestion in combined with co-derivatization were employed for high sensitive analysis of N-glycans using MALDI-MS.Then we successfully used the above method for the trance sample analysis from standard glycoproteins,human serum and cellular glycoproteins,which paves the way to the analysis of limited biological samples and discovery of bio markers.Firstly,extensive pronase E digestion in coupling with co-derivatization of TMPP-Ac and methylamidation was employed for glycomic analysis with enhanced sensitivity.TMPP-Ac-OSu,which bears a stable permanent positive charge that increasing the ion ionization efficiency in the positive mode of MS,was introduced to the amino acid residue of Asn-glycans.Methylamidation was subsequently attempted in combination with TMPP-Ac-OSu derivatization to stabilize and neutralize the carboxyl groups.Approximately 20 and 50-fold enhancement in the detection sensitivity was achieved for sialo-Asn and disialo-Asn respectively in comparison to their ative counterparts.Finally,this novel strategy was successfully applied to three model glycoproteins,including ribonuclease B,ovalbumin and transferrin.Secondly,a simple,rapid and highly sensitive method for N-glycan MALDI-MS analysis was presented.The new method involves the rapid deglycosylation by using an enzyme-friendly detergent and commonly used denaturing agents and derivatization with TMPP-Ac-OSu in a one pot reaction.For the analysis of sialoglycoproteins,methylamidation was also employed to neutralize the sialic acid.The total reaction time has been reduced from several days to several hours.With the developed strategy,more than 50 glycans was detected with only 25 nL of human serum applied.Therefore,the described method shows promise in the large-scale profiling of N-linked glycans as well as trace analysis when the sample amounts is highly limited.Thirdly,an optimized co-derivatization method was employed for rapid analysis of cellular N-glycan profiling with limited cell amounts.We found that CHCA was suitable as the matrix for the MALDI-MS analysis of derivatized glycans.Then the optimized method was successfully applied to the comprehensive analysis of cellular N-glycans obtained from small numbers of HeLa cells(～105).Moreover,three heterogeneous live cell lines,human normal liver cells(L-02),human hepatocyte carcinoma cells(HepG2 and Bel-7402),were also applied to validate the optimized method.We found that sialylated glcyans of membrane proteins and secreted proteins were all elevated in two hepatocyte carcinoma cells when it was compared to the normal liver cells.The results shown that it was able to reveal the difference in cellular N-glycan profiling of these two cell lines.In summary,a nonspecific proteolysis in coupling with co-derivatization of TMPP-Ac and methylamidation was employed for glycomic analysis with enhanced sensitivity.Then,we developed a simple,rapid and highly sensitive method for N-glycan MALDI-MS analysis,which involves the rapid deglycosylation by using an enzyme-friendly detergent and commonly used denaturing agents and derivatization with TMPP-Ac-OSu in a one pot reaction.Finally,we optimized the rapid and sensitive method for the analysis of N-glycans from small amout of cells,which was further used for the discovery of biomarkers from different cell lines.In conclusion,the methods presented in this study could lay a foundation for the analysis of N-glycans from trance samples.