The Effects Of Enrichment And Derivatization On The Analysis Of N-glycans By Electrospray Ionization Mass Spectrometry

Compared with genome and proteome, glycome contains more complex and diverse biological information. Research on its structure and function has became one of the hot spot in the field. At present, studies have shown that most disease occurrences are accompanied with the abnormal expression of the glycans. Therefore, glycans as potential highly specific biomarkers have great significance for early diagnosis of malignant disease.Biological mass spectrometry is a powerful tool for quantitative and qualitative analysis of glycans, however, the mass spectrometry has a poor tolerance of impurities, and different glycans enrichment processes and derivative methods have a greater impact on the sensitivity of mass spectrometry analysis. This study investigated the effect of three types of commonly used derivatization reagents and enrichment methods such as graphite carbon column for the analysis of electrospray ionization mass spectrometry (ESI-MS), it is impotant to the development of high sensitivity mass spectrometry method. The conclusion showed as follows:1. Release the N-linked glycans of glycoprotein samples using PNGase F, investigating two kinds of enrichment methods, graphite carbon column combined with microcrystalline cellulose column and cationic exchange column combined with microcrystalline cellulose column. The results indicate that the former procession is more stable. Prior to releasion N-glycans from human serum, treatment with dialysis and PVDF film, the sample under dialysis condition give better MS profile.2. To examine the sensitivity of the former derivative methods, we choose lactose as standard. The β-cyclodextrins internal standard was also adding to estimate the ionization efficiency. Further, application on biological and complex samples were also evaluated, such as N-glycans of chicken egg albumin, human serum glycoprotein. As expected, the Girard’s reagent and PMP gives better sensitivity in MS detection. On top of them, the Girard’s reagent P derivative shows best results, and the procession is not time-consuming.3. The Girard’s reagent P (d0) and its stable isotopes (d5) were preliminary applied to analysis of human serum N-glycans in different pathological states.19peaks of Δm=5Da, most of them with intensity ratio close to1:1, appeared in the range of m/z1200～2000. The results showed Girard’s reagent P derivative method can be further developed into a reducing glycan relative quantitative method, and it has good feasibile and practical prospect in quantitation and qualification of glycans from human serum glycoprotein.To sum up, our study based on ESI-MS detection, has improved the N-glycan enrichment method of the complex bio-sample. At the same time, comparing the effect of three types of derivative methods for mass spectrometric analysis, determine that Girard’s reagent P has the highest sensitivity of MS detection. This method proves simple, economic and stability, offers a novel tool in serum glycan biomarkers searching.