Development Of Relative Quantification Of Sugar Chains Using Isotope Cysteine Derivatization Reagents

Glycosylation is one of the most common and complex forms of post-translational modification of proteins.Glycosylation proteins are involved in many life activities of organisms.When glycosylation is defective,organisms will develop diseases.Therefore,the sugar chain plays a very important role in life activities.By studying the structure of sugar chains and elucidating the functions of glycosyltransferases and glycosylase-expressing genes involved in the synthesis of sugar chains,it is helpful to explain the mechanism of disease production.At present,the qualitative research of sugar chains has made great progress.However,due to the complex and diverse structure of sugar chains and the difficulty in the synthesis of standard products,there is still no effective method for quantitative analysis of sugar chains.Therefore,quantitative studies of sugar chains have important significances for the screening of sugar chain markers and their clinical application.Currently,chemical markers based on HPLC-MS detection are effective methods for quantifying sugar chains,and there are two types,absolute and relative quantification.Absolute quantification requires the standard product of the sugar chain to be measured,but it is difficult for the standard product to be synthesized and obtained for the complex sugar chain composition in the biological sample.At present,the internal standard method is mainly used for relative quantification of isotope labeling.Because they have the same ionization efficiency,the relative abundance of this pair of peaks can be compared to analyze the changes in the expression level of sugar chains under different physiological conditions.Quantitative methods for stable isotopes as an internal standard in proteomics and metabonomics have been widely used,but quantitative analysis of sugar chains is still in its infancy.The non-specific Pronase E enzyme can cleave the N-/O-linked sugar chain on the glycoprotein into a glutamic acid with one amino acid active group(N-Glycan-Asn,O-Glycan-Ser/Thr),but Since the sugar compound does not have a UV group and cannot be detected directly,precolumn derivatization is required.Therefore,we have synthesized a novel derivatization reagent,d0/d5-BZC-OTZD,which can be used for the derivatization and relative quantitative analysis of sugar chains and the sugar chain..In this study,L-2-Thiazolidinone-4-carboxylic-acid was used as the precursor and reacted with benzoyl chloride(d0/d5-BZC)with isotopes.A chiral light-and heavy-isotope-labeled mass spectrometry derivatization reagent,d0/d5-BZC-OTZD,was synthesized with a carboxyl functional targeting recognition ability.On the basis of this compound,we synthesized the reagent d0-BZC-OTZD-NHS with the same quantitative method.Firstly,D-glucosamine was used instead of the sialyl glycopeptide model(SGP)and d0-BZC-OTZD at 40℃ for 1.5 h to give a derivatization product with a retention time of 8.060 min and sialyl glycopeptide model(SGP)was used to verify the labeling and detection of sugar chains.The reagent was finally applied to the analysis of the actual fetuin and ovalbumin proteins.In fetuin,five sugar chain products can be obtained,and in ovalbumin,eight sugar chain products can be obtained,demonstrating the effectiveness of the method.The d0/d5-BZC-OTZD reagent can effectively label sugar chains in glycoproteins.It can be used for sugar chain analysis of actual samples of carbohydrate chains such as fetuin and ovalbumin,and the derivatized sugar chains have a difference in m/z of Δm=5,the relative quantification of sugar chains in LC-MS can be performed,providing a new derivatization reagent for the simultaneous quantification of sugar chains.