Analysis Of Serum N-glycan And Screening Of Novel Biomarkers Based On Mass Spectrometry

Glycosylation is a common post-translational modification of proteins,which is widely involved in various physiological activities,such as protein folding,cell communication,tumor metastasis,immune recognition and microbial invasion.More and more clinical studies had shown that the glycosylation degree of one or more proteins and their corresponding glycan chain structure would change in many diseases or abnormal physiological conditions.The admeasurement of these changes not only could understand the physiological functions of some proteins,but also could be used for the diagnosis of diseases.Therefore,searching for possible biomarkers by studying the structure and content of N-glycans has become one of the hotspots in the field of glycomics.Serum is the most widely studied sample in glycomics because of its wide source,easy to obtain and rich in various types of glycoproteins.However,in view of the complexity of serum samples and the glycosylation properties of proteins,the corresponding glycan structure diversity and microscopic heterogeneity contributes to the difficulty in analyzing the structure and low content of N-glycans in serum.Compared with traditional analytical methods,bio-mass spectrometry has the advantages of high sensitivity,high throughput and anti-impurity interference,which has been successfully applied to the study of serum glycomics.However,the analytical methods based on bio-mass spectrometry still have a lot of limitation,such as glycan chain enrichment,purification,labeling.In this paper,based on the advantages of bio-mass spectrometry,the N-glycans in guinea pig serum was systematically studied for the first time under two derivatization techniques,moreover,several analytical methods were compared.Furthermore,the optimized analytical method was applied to the study in N-glycan’s change of the colorectal cancer’s serum and the N-glycan’s change of IgG in human serum which influenced by environmental pollutants.The details are as follows:As an important model organism,guinea pigs are often used in immunology,toxicology and pharmacology.The analysis of N-glycan in guinea pig serum is conductive to further understanding its physiological function.In order to enhance the mass spectrum response of N-glycans,we derive N-glycans from guinea pig serum by permethylation and methylamination respectively,and then identify 31 kinds of permethylated N-glycans and 43 kinds of methylaminated N-glycans by MALDI-MS.O-acetylation of sialic acid in serum of guinea pigs was first discovered.The results showed that mild methylamination did not destroy the structure of O-acetylated sialic acid,which was more suitable for the analysis of sialic acid glycans.Meanwhile,54 kinds of methylaminated N-glycans and more than 200 kinds of corresponding methylaminated N-glycan isomers were identified based on nanoLC-ESI-MS,furthermore,their structures were confirmed by MS/MS.It was found that O-acetylation not only affected the number of N-glycan isomers,but also changed the hydrophobicity of N-glycan to some extent.The results showed that although nanoLC-ESI-MS has more advantages in identifying the structure of N-glycan,MALDI-MS is more suitable for large-scale serum samples because of its simple operation and shot analysis.Colorectal cancer is one of the most common digestive tract malignant tumors with the highest morbidity and mortality in the world.Accordingly,the clinical diagnosis of colorectal cancer has become particularly important.In this chapter,the MALDI-MS method established in the previous chapter was used to analyze the serum N-glycans between healthy people and patients with colorectal cancer,and 64 kinds of human serum N-glycans’ structure were identified accurately.After the obtained mass spectrometry data was normalized,it was confirmed that three N-glycans with core fucose structure are closely related to colorectal cancer which could be used as good colorectal cancer markers by the t-test and the receiver operating characteristic curve.Besides,in the study of the relationship between the classification of cancer stages and the change of N-glycans,it was found that the change of N-glycans in colorectal cancer’s serum was not obvious in stage I,while the changes of N-glycans in colorectal cancer’s serum were relatively significant in stage II,III and IV,which confirmed that the early stage of colorectal cancer is not easy to be diagnosed from the perspective of glycomics.The results indicate that the application of MALDI-MS in screening N-glycans markers can not only accurately identify the structure of N-glycans and obtain the corresponding disease markers,but also understand the mechanism of disease development.Perfluorooctane sulfonate(PFOS)and perfluorooctanoic acid(PFOA)are two common organic pollutants widely existing in the natural environment which could cause adverse immune reactions in human body,such as the decrease of thymocytes and spleen cells,the increase of liver weight and the increase of peroxisome.Immunoglobulin(IgG)is the main executor of the immune system.Glycosylation on IgG is closely related to the occurrence and development of many diseases.Therefore,this chapter will further study the immunotoxicity of PFOS and PFOA from the perspective of glycosylation on IgG.Combined methylamination derivatization with MALDI-MS,a comparative study was conducted on the N-glycan changes in serum extracted from 190 serum samples,including 95 adult and 95 adolescent groups,and then 28 kinds of N-glycans’ structure derived from IgG were identified.With the change of PFOS and PFOA concentration in serum,the mass spectrometry data of corresponding samples were analyzed statistically.It was found that 11 kinds of N-glycans with significant changes along with the changes of PFOS and PFOA concentration in adult group,while 12 kinds of N-glycans with significant changes along with the changes of PFOS and PFOA concentration in children group.In terms of characteristics of N-glycans,the changes of mannosylation,fucosylation and sialification are the main manifestations in adult group,while the changes of fucosylation and sialification are the main manifestations in children group.Therefore,the existence of PFOS and PFOA is closely related to IgG glycosylation.This chapter reveals that PFOS and POFA have different effects on different populations from the perspective of glycomics for the first time.On the basis of the discovery of IgG N-glycan structure which is significantly correlated with the changes of PFOS and POFA concentrations,the relationship between PFOS and POFA and the response of human immune system can be interpreted to some extent.In summary,mass spectrometry was applied to the systematic study of serum N-glycans in vivo.The complete information of serum N-glycans in guinea pigs was obtained for the first time,meanwhile,potential N-glycan markers in colorectal cancer were found.Furthermore,the glycomics research method was applied to analyze environmental pollution and health problems for the first time which could provide a new way to further understand the mechanism of environmental pollution.The development of this paper will provide technical means and theoretical basis for clinical disease diagnosis,drug development and public health in the future which has broad application prospects.