N-Glycans Release And Derivatization Coupling With HPLC-MS Analysis And Spearation

Glycosylation, an important process of the post translation of protein, plays a key role in folding,positioning and the interaction of proteins. In order to study the function and control mechanism of glycans, glycans released is necessary. O-linked glycans could be released from glycoprotein using chemical method while emzyme digestion is an available method used for N-linked glycans. In this study a non-specific enzymatic digestion of N-glycoprotein was developed, Under the optimized digestion conditions N-glycans bearing a single asparagine residue, keeping the close ring structure at reducing end,was released. ESI-MS was used to analyze the glycan-acid. New enzyme was used to digest glycan-Asn in order to recovery the reducing glycans. A brief summary is described as following:1. A non-specific enzymatic digestion of glycopeptides to release N-glycans from glycoprotein:Pronase E instead of the traditional PNGase F was used to release glycopeptides from Ribo B and Chicken Albumin, Under the optimized digestion conditions that the quantity ratio of the Pronase E to glycoprotein was 1:1, the closed-ring oligosaccharides named glycans-Asn with a single amino acid (Asn) were obtained. This method not only remains the native structure of N-glycans, but also provides possibility of fluorescent derivation with primary amine as function group.2. A modified 9-Fluorenylmethyl chloroformate (Fmoc-Cl) pre-column derivatization procedure has also been successfully applied to these glycans-Asn:the Fmoc-Cl labeled glycans-Asn products were characterized by high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI/MS). to prepare pured glycans we collected glycans respectively after HPLC analysis, then a new enzyme was introduced to digest the collected glycans to get reducing glycans.3. Glycans and glycan-acid derivatized by Fluorescein Isothiocyanate:For some reducative glycans without natural amine group, we used an indirect method which p-Phenylenediamine was chosen as a linker and introduced an free amino group to oligosaccharide through reductive amination reaction, which was ready for further derivatized with FITC. The conditions for FITC labeling were optimized as derivative reaction at 40℃for 6 h (pH=11), FITC dissolved in acetone. Using this procedure, we have prepared fluorescein derivatives of Chitoosaccharides and Glycan-Asn (Chicken Albumin digested by Pronase E)In conclusion, we established a new method about which glycoprotein was digested by Pronase E to release glycans, glycans was derivated by Fmoc-Cl/FITC to analyze with on-line HPLC and glycan-acid derivatives were digested by new enzyme to recovery free glycans. This enzyme digestion has exhibited a high efficiency in terms of glycoprotein digestion and fluorescent derivation, and HPLC separation is in favor of the reducing glycans recovering. Futherly, it was very applicable and easy to operate, which lead to the convenience of separation and preparation of pure N-glycans and explore the interaction between glycan and proteins.