Study Of Glycoproteome And Its Glycan In The Hepatocellular Carcinoma

The International Agency for Research on Cancer (IARC) indicated that Hepatocellular Carcinoma (HCC) was one of the top five prevalent primary malignant tumors. It showed that malignant carcinoma has been the most common cause of disease death and the HCC has been the second most common cause of cancer death in China. The diagnosis of HCC is usually achieved at a late stage, which results in the high mortality. A rapid and sensitive diagnosis method of the HCC would be particularly necessary. The study of the glycoproteomics can reveal the functions of the glycoproteins and explore the molecular mechanism of the HCC, and also devebpe a method for clinical diagnosis.For the first part:The epoxy-activated magnetic particles were synthesized by modification of the Fe3O4fluids using silaning agent.The method to preparation the lectin-magnetic particle conjugates was established using the home-made epoxy-activated magnetic particle. The epoxy-activated magnetic particles were characterized as uniform superparamagnetism material with3.50μm diameter,69.55emu/g saturation magnetic moments, and96.29m2/g specific surface area. For the biological activated lectin-magnetic particle conjugates preparation (LMPCs), the binding buffer, binding time, binding capacity, and surface coverage were optimized. It was estimated that the each gram of the magnetic particles could be saturated with186.31±5.16mg Concanavalin A (Con A).For the second part:A simple, rapid and high-throughput method was established for the N-linked glycoprotein isolation using lectin-magnetic particle conjugates (N-glyco-LMPCs). Meantime, combined with the mass spec tro me try could implement the identification and characterization the glycoprteins and glycans in one flow. At first, the strategy was involved in the isolation and identification the glycoprotein RNase B from model protein. The peptide spectra of the RNase B were identified by the MALDI-MS. The microheterogeneity of the glycan on the RNase B was characterized as five glycoform. Additional, the Con A-magnetic particle conjugate-based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and HCC case sera. Followed the isolated glycoproteins and their JV-linked glycans were identified by LC-ESI-MS/MS and MALDI-TOF/TOF-MS, respectively.93glycoproteins from the healthy donors and85glycoproteins from the HCC cases were identified. There were34different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34).28glycans from the healthy donors and30glycans from the HCC cases were detected and there were22different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Moreover, the lectin blotting, western blotting and lectin/glyco-antibody microarrays were applied to definitely elucidate the change of the expressions of selective protein and their glycosylatio n, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.For the third part:The glycoproteins of the normal donors and HCC cases sera were isolated by the single-and multi-lectin-magnetic particle conjugates and identified by the high-accuracy ESI-Q-TOF-MS, respectively. The exponentially modified protein abundance index (emPAI) method was applied in the absolute quantification of the glycoproteia371N-linked glycoproteins were identified from the two serum pools. From the results, it showed that the binding specificity of the identified glycoproteins to the four lectins (Con A, Lens Culinaris Agglutinin (LCA), Aleuria Aurantia Lectin (AAL), Wheat Germ Agglutinin (WGA)) and the glycan structures of the identified glycoproteins were tentative estimated. With the stringent criteria emPAI quantification, it indicated that315(84.9%) glycoproteins were no significant difference in the two serum pools. However,49(13.2%) glycoproteins were measured up-regulation and27(7.3%) were down-regulation in the HCC cases. The up-regulation proteins included the verified HCC biomarkers AFP and the potential biomarker Component C6, Ig mu chain C, β-2-glycoprotein, G-protein coupled receptor, and so on.For the fourth part:We applied the filter aided sample preparation-based N-linked glycopeptides enrichment (N-glyco-FASP) from the healthy donors and HCC cases sera.144N-glycosylation sites were mapped on74proteins in the two serum pools using high-accuracy mass spectrometry. With lectin enrichment, glycopeptides were28%of total peptides, indicating an enrichment factor of nearly60-fold. Combined the N-glyco-LMPCs and the N-glyco-FASP,451proteins were identified in the two serum pools, which305and298proteins were identified from the normal donors and HCC cases sera, respectively. After the analysis of the functional of the proteins by the Blast2Go, It indicated that the mainly molecular function of the protein was binding, catalytic activity, enzyme regulator activity. The proteins mainly located at the cell, the extracellular region, as well as organelle. The biological processes mediated by the proteins were cellular processes, response to stimulus, biological regulation and metabolic process. After the functional cluster analysis of the proteins only identified in the HCC, it showed that the proteins were secreted glycoproteins with signal peptide.For the fifth part:We developed a filter aided sample preparation-based total glycoprotein N-linked glycans enrichment (N-glycan-FASP-T) and lectin binding glycoprotein N-linked glycans enrichment (N-glycan-FASP-L) from the normal donors and HCC cases sera, respectively. The enriched N-linked glycans were characterized by the MALDI-TOF/TOF-MS to obtain the glycan spectra and structures.26glycans were annotated from the total glycoproteins using N-glycan-FASP-T in the two serum pools, which were centralized in the small molecular weight Additional,24glycans were annotated from the lectin binding glycoproteins using N-glycan-FASP-L in the two serum pools, which distributed more homogeneous and wider. The glycans between the two serum pools were mostly same using the two methods, which9glycans were identified both in the healthy donors and HCC cases sera using N-glycan-FASP-T, and also9glycans for the N-glycan-FASP-L. It showed that the characterized glycans only in the HCC were fucosylated oligosaccharide whatever by qualification (6/16) or by quantification (4/5). The two strategies for the glycan isolation and characterization displayed preference and could be mutual complementation in the study of the glycomics in HCC.