Highly Efficient Enrichment Of O-GalNAc Glycopeptides By Using Immobilized Metal Ion Affinity Chromatography

O-GalNAc glycosylation is one of the most important post-translational modifications in organisms,and its abnormal expression is closely related to many diseases.The comprehensive analysis of O-GalNAc glycosylation can provide important information for the development of biomarkers for tumor diagnosis and treatment response assessment.Different from N-glycosylation,O-glycosylation is still in a relatively preliminary stage due to lack of corresponding glycosidase and low abundance.This thesis is dedicated to developing a new method for efficiently enriching O-GalNAc glycosylated proteome.The full text is divided into four chapters.The first chapter is divided into four parts.The first part summarizes proteomics;the second part describes the pretreatment methods of protein post-translational modification based on mass spectrometry;the third part describes protein glycosylomics;the fourth part introduces the research content of this works.In the second chapter,a total of 691 non-redundant O-GalNAc glycosylated peptides corresponding to 141 glycosylated proteins were identified from 1 μL normal human serum by using Ti(Ⅳ)-IMAC.Compared with the hydrophilic interaction chromatography(HILIC)method,the Ti(Ⅳ)-IMAC material can be enriched to nearly 2 times of O-GalNAc glycosylated peptides from only 0.1 μL of human serum samples.Furthermore,high-p H reversed-phase liquid chromatography(RPLC)was used to fractionate O-GalNAc glycosylated peptides.Combined with2 D LC-MS/MS analysis,2,093 O-GalNAc glycosylated peptides could be identified from only7.2 μL human serum samples,corresponding to 271 glycosylated proteins.It is one of the largest databases of O-GalNAc glycosylated peptide,and it has realized the large-scale analysis of OGalNAc glycosylation in complex biological samples.In the third chapter,the Ti(Ⅳ)-IMAC integration strategy was applied to the analysis of abnormal O-GalNAc glycopeptides in serum of hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma(ICC).A total of 1,253 intact O-GalNAc glycopeptides were detected in the three groups of samples(n = 3)by using lable-free quantification(LFQ)for quantitative analysis of O-GalNAc glycopeptides.According to the P value(-log 10)and t test of peak area among the three groups of samples of the control group,HCC and ICC,a total of 596 intact OGalNAc glycosylated peptides were quantified.Among them,52 significantly changes O-GalNAc glycosylated peptides were obtained between HCC and control serum samples,and 31 significantly changes O-GalNAc glycosylated peptides were obtained between ICC and control serum samples,indicating the potential of this method in clinical research.In summary,this paper developed an efficient enrichment of complete O-GalNAc glycopeptides by Ti(Ⅳ)-IMAC based on hydrophilic and chelating affinity interactions.This method has the advantages of simple operation and mild enrichment conditions,which is suitable for the qualitative and quantitative analysis of O-GalNAc glycosylated peptides in complex biological samples at scale,and has broad application prospects in clinical medicine and other practical fields.