Development And Glycomic Application Of A Series Of Novel Methods For Release,Purification And High-throughput LC-MS Analysis Of Glycoprotein Glycans

As the third type of biological macromolecules except nucleic acid and protein,sugar chains have proved to be a partner in a series of important biological processes.Thus,more and more attention has been paid to glycomics,which is focused on studies of the correlations between the structure and function of glycans located on proteins.In recent years,rapid progress has been made in many research areas of glycomics,such as the release of glycans from glycoproteins and glycan purification,derivatization,structure elucidation,quantification and structure database construction,providing essential methodological tools for glycomic studies.However,now glycomics is being faced with some difficulties in several basic and key technologies,which are listed in the following.First,serious peeling degradation occurs during the nonreductive release of O-glycans from glycoproteins by ?-elimination.Second,serious peeling degradation occurs during the nonreductive release of core?1,3-fucosylated N-glycans from glycoproteins byalkaline hydrolysis.Third,the glycan purification methods by affinity adsorption are unsuitable for glycan quantification,though they are easy to operate.Fourth,there isn't a universal and reliable method for high-resolution separation and high-throughput analysis by online liquid chromatography coupled with mass spectrometry(LC-MS).Fifth,various glycan structure databases are troubled with data complexity,poor retrieval reliability and inconvenient applicability.All those problems have hindered glycomics from development.On this basis,this study is focused on the design and development of a series of systematic methods for the release,purification,high-resolution separation by high-performance liquid chromatography(HPLC),high-throughput LC-MS analysis and structure database construction of glycoprotein glycans,expecting a resolution of those methodological problems in glycomic studies and a provision of some convenient,reliable and universal new methodological tools for glycomic research.The achievements are summarized in the following.1.The concept and mechanisms of the one-pot reaction of O-glycoproteins have been first proposed and explained in detail,and its essence in theory has been revealed as the simultaneous accomplishment of three reaction processes in an identical system,including the release of O-glycans from glycoproteins by?-elimination,the derivatization of released glycans with 1-phenul-3-methyl-5-pyrazolone(PMP),and the derivatization of O-glycosylation sites with PMP.The one-pot reaction has been first confirmed by experiments on porcine stomach mucin.The optimization strategy has been designed and the optimization process has been finished.As a result,three sets of one-pot reaction conditions were successfully developed.These reaction conditions have been evaluated as follows:the peeling reaction yield and O-glycan relative yield of the NaOH-PMP one-pot method are 1.1%and 100%,respectively;the peeling reaction yield and O-glycan relative yield of the NH3 H2O-PMP one-pot method are 1.2%and 112%,respectively;the peeling reaction yield and O-glycan relative yield of the DMA-PMP one-pot method are 3.3%and 54.9%,respectively;in N-glycan yield,the NH3·H2O-PMP one-pot method is rather high and correspondent with the DMA-PMP one-pot method,while the NaOH-PMP one-pot method is very low;the three methods all can't cause either desialylation or conspicuous degradation of peptides and need 100 ng glycoprotein sample at least;the three methods all can cause a derivatization of O-glycosylation sites with PMP.The newly developed NaOH-PMP one-pot method has been applied to the release and analysis of O-glycans of bovine submaxillary mucin,chicken and duck ovomucin,as well as human milk and seminal plasma,demonstrating the excellent stability,reliability and applicability to various complex biological samples.2.The novel one-pot reaction of N-glycoproteins has been first designed and accomplished,and its essence has been revealed as the simultaneous accomplishment of two reaction processes in an identical system,including the release of core?1,3-fucosylated N-glycans from glycoproteins by alkaline hydrolysis,and the derivatization of released glycans with 1-phenul-3-methyl-5-pyrazolone(PMP).Based on the already established NH3 H2O-PMP one-pot method of O-glycoproteins,the optimal reaction time has proved to be 48 h after a reaction condition optimization process,and the peeling yield of core al,3-fucosylated N-glycans during release keeps below 5%.The newly developed one-pot method for N-glycoproteins has been successfully applied to several samples derived from plants and lower animals for the release and analysis of core ?1,3-fucosylated N-glycans,demonstrating the applicability of this method to complex biological samples for N-glycan release.3.A novel method for the quantitative enrichment and purification of reducing glycans has been first developed based on sulfonyl hydrazine-functionalized polystyrene(SHPS)material,and a set of optimal reaction conditions have been obtained.The dynamic linear range of the method for the quantification of reducing glycans has been revealed to be between 0.04 mM and 20 mM;the recovery of reducing glycans has been revealed to be about 63.9%;the coefficient of variability(CV)of the method for glycan quantification has been revealed to be below 3.3%.The newly developed procedure has been successfully applied to a series of samples for glycan purification and analysis,including ribonuclease B,chicken egg albumin,several milk samples,human plasma and fetal bovine serum,demonstrating the excellent applicability of the method to glycomic studies of various biological samples.4.The universal method for the separation of glycans as bis-PMP derivatives by hydrophilic interaction liquid chromatography(HILIC)and reverse-phase high performance liquid chromatography(RP-HPLC)has been first developed,and the optimal elution conditions have been obtained.The relative standard deviation(RSD)values of the retention time of glycans as bis-PMP derivatives during HILIC and RP-HPLC separation have been revealed to be 0.76%and 0.31%,respectively.The limit of detection(LOD)of the both method has been revealed to be ca.5 pmol.The O-glycans as bis-PMP derivatives released from porcine stomach mucin,bovine submaxilary mucin,and bovine fetuin has been well separated.It has been found that the bis-PMP derivatives of glycans are separated in HILIC mainly by molecular weights,while in RPHPLC by linkages and three-dimensional structures,providing theoretical basis for the construction of glycan structure database.The new method has been successfully applied to the separation and analysis of the bis-PMP derivatives of O-glycans released from bovine serum and frog egg-jelly coat,demonstrating the excellent of the method in the analysis of bis-PMP derivatives of O-glycans derived from complex biological samples.5.The novel concept and method have been first designed and proposed for the construction of O-glycan structure database using ordered real number consist of the HILIC relative retention time GU value and the RP-HPLC relative retention time OMCU value.A set of chromatographic conditions have been obtained based on stepwise optimization,for the high-resolution separation by combined use of HILIC and RP-HPLC.The O-glycans released from swallow nest mucin by NaOH-PMP one-pot method have been successfully separated and analyzed by combined use of high-throughput online HILIC-MS/MS and RP-HPLC-MS/MS.As a result,19 O-glycan structures,including 3 pairs of glycan isomers,has been successfully separated and identified.An O-glycan structure mini-database has been successfully constructed based on the GU value and the corresponding OMCU value of each O-glycan structure,and a swallow nest mucin O-glycome information database has been successfully developed based on the GU value,the corresponding OMCU value and the occupancy ratio of each O-glycan structure,providing a set of new research methods for investigations in biological O-glycomes6.Qualitative and quantitative O-glycome comparison has been made between hepatocellular carcinoma tissue(HCT)and adjacent normal liver tissue(ANT),as well as hepatoma serum and normal serum,based on the newly established NaOH-PMP one-pot method and combined use of the high-throughput online HILIC-MS/MS and RP-HPLC-MS/MS.As a result,it has been found that all of the four types of samples contains 3 O-glycan structures,including T antigen,ST antigen with mono-N-neuaminic acid,and ST antigen with bis-N-neuaminic acid.There is no conspicuous difference in abundance of all of the 3 O-glycan structures between hepatoma serum and normal serum.Similarly,there is also no conspicuous difference in abundance of the both sialylated O-glycan structures between HCT and ANT.However,a conspicuous difference in the abundance of T antigen has been found between HCT and ANT(p<0.05).Therefore,the T antigen can be further investigated as a candidate O-glycan biomarker of hepatocellular carcinoma.This research has demonstrated further the reliability,systematicness and applicability of the newly developed method series in the glycomic studies of complex biological samples,and can also provides a technological reference for the further screening of glycan biomarkers of hepatocellular carcinoma and more diseases.