| Glycosylation is one of the most common and important biological phenomena of post-translational modification processes in eukaryotic cells, and playing a vital role in the implementation of proteins'biological functions. Studies have shown that many diseases are associated with abnormal glycans expression. Whether the glycans expressed abnormally may be potential biomarkers of a certain disease.In this paper, we conducted a preliminary study of the glycans quantitation by the isotopic labeling combined with modern biological mass spectrometry, and make a relative quantitative and qualitative analysis of different sourse of glycans to study whether the glycans were varied expressed.Two different relative quantitative and qualitative methods established here were employed to study reducing glycans and N-glycans:1. Analysis method for relative quantitation and qualitation of reducing glycans via aniline stable isotopic labeling and ESI-MS. The experiment has established a method for quantitation and qualitation of reductive glycans based on aniline stable isotopic labeling, and optimized the various conditions which affect the derivatization efficiency. Under the condition that the saccharide, aniline and sodium cyanoborohydride in a molar ratio of 1:1.2:10, reducing oligosaccharide can be quantatively derivatized by aniline at 70℃within 15 min, In order to test the linearity and dynamic range of this method, aliquots of standard oligosaccharide (Maltodextrin) were labeled with d0/d5-aniline separately. Results indicate that the isotopic labeling method employed here was providing relative quantitative data with a dynamic range of 15-fold, adequate linearity (R=0.9986) and reproducibility with a coefficient of variation that was 10.30% on average. The established method was sensitive and reproducible for the candidate glycans and was successfully used for comparing the differences between free human milk oligosaccharides (HMOs) and free bovine milk oligosaccharides (BMOs). The results indicated that the lactose in human milk was more than that from bovine milk, and the varieties of oligosaccharides from human milk were more complex than that of bovine milk. The relative quantitative and qualitative method we established here will provide an alternative option for studying the changes of the glycans and further understand the machinery of the glycosylation, which associate with a wide assortment of diseases intimately.2. A Novel Method for Relative Quantitation of N-Glycans via RABA(Reductive Alkylation by d0/d6-Acetone) and ESI-MS Analysis. An established method for quantitation of N-glycans bearing single Asn residue based on acetone stable isotopic labeling and ESI-MS analysis was reported. The N-glycoproteins were digested as glycan with an amino acid(Glycans-Asn) by non-specific protease Pronase E instead of the traditional PNGase F. Consequently, an amino group was introduced to N-glycans, and the amino group was labeled with actone. In order to test the linearity and dynamic range of this method, aliquots of N-glycan-Asn(M5-M8-Asn) obtained from Ribo B which hydrolyzed by Pronasre E were labeled with d0/d6-acetone seperately. The results indicated that RABA relative quantitative method employed here could provide relative quantitative data with a dynamic range of 10-fold, adequate linearity (R>0995) and reproducibility with a coeffcient of variation that was less than 8.7%. The established method was sensitive and reproducible for candidate glycans and was successfully used for comparing the differences between different purity of chicken egg albumin.In summary, this study is based on mass spectrometry and stable isotopic labeling to establish the relative quantitative and qualitative methods. The method is low cost, simple operation, detective sensitivity and high throught, which can be employed to study trace changes in the expression of glycans and laid a foundation for further study the glycans biomarker.
|
PDF Full Text Request