N-glycosylation Analysis Of Serum Haptoglobin β Chain In Patients Bearing Liver Diseases

Glycoconjugate (glycoprotein, glycolipids and proteoglycan) studies are another emerging field followed by protein and nucleic acid analysis, especially the multiple structures and functions of glycoprotein. Glycans play an important role in a variety of biological processes like cell adhesion, molecular trafficking and clearance, receptor activation and signal transduction. Further, alterations in cell surface glycan are associated with various physiological and pathological status including malignant transformation and metastasis. Recently, relevance between glycans and human disease has been a major foucus of glycobiologists. The LCA-reactive fraction of AFP (AFP-L3) has become a more specific glyco-biomarker for hepatocellular carcinoma (HCC). There are great advances in N-glycan analysis and many methods are available for determining covalent structures of glycans attached to glycoproteins. However, accurate characterization of glycoproteins with multiple glycosylation sites and assessment of the glycan macroheterogeneity (glycosylation site occupancy) and inicroheterogeneity (glycan structure) are needed for understanding the functions of glycans.Serum haptoglobin (Hp) is an acid glycoprotein in the sera and body fluids of human and many mammalian. Hp β chain contains four potential N-glycosylation sites. Changes in glycan structure of Hp β chain were reported in patients with breast cancer and pancreatic cancer. In this study, standard antibody affinity purification for Hp-p and a novel strategy’iTRAQ plus18O’were established, MALDI-QIT-TOF MS, LTQ Orbitrap XL ETD combined with lectin blot analysis were used to analyze macroheterogeneity and inicroheterogeneity of sera Hp-β obtained from healthy individuals and patients with hepatitis B virus infection (HBV), liver cirrhosis (LC) and HCC. Alterations in glycosylation of sera Hp β chain may become alternatives or complements to clinical diagnosis of cirrhosis and HCC Part One N-Glycosylation site occupancy analysis of serum Hp-β in patients bearing liver diseasesA novel strategy combining iTRAQ with18O stable isotope labeling followed by mass spectrometry (MS) analysis was established to identify N-glycosylation site and quantify N-glycosylation site occupancy of serum Hp-β.’iTRAQ plus18O’supplied effective measurements of glycan macroheterogeneity on the target glycoprotein.Hp-β was purified from healthy individuals and patients with HBV, LC and HCC. Non-glycosylated peptides and three glycopeptides NLFLN207HSEN211ATAK MVSHHN184LTTGATLINEQWLLTTAK and VVLHPN241YSQVDIGLIK were obtained by trypsin digestion. iTRAQ reagents were used to label non-glycosylated peptides and glycopeptides.18O was used to label N-glycosylation site of glycopeptides. After the modification of MS precursor ion isolation window, tagged peptides were identified by LC-MS/MS, both glycopeptides and non-glycopeptides were quantified simultaneously using ProteinPilotTM Software. The glycosite occupancy was calculated by dividing abundance ratio of glycopeptides by abundance ratio of protein. With four samples to be maximally analyzed in parallel, this workflow supports accurate quantification of glycopeptides in a site-specific fashion.In this part,13non-glycosylated peptides (conf>95%) were detected and the average of non-glycosylated peptides (conf>95%) reflects the expression levels of Hp-β, which is6.43:2.28:1:2.84(N:HBV:LC:HCC). A reduced protein expression level of Hp-β was observed in HBV, LC patients, and increased protein expression in HCC patients. The abundance ratio of Hp-β glycopeptides were7.29:2.49:1:2.76(N: HBV:LC:HCC),9.11:2.37:1:4.84(N:HBV:LC:HCC) and6.73:2.47:1:3.19(N: HBV:LC:HCC) for NLFLN207HSEN211ATAK, VVLHPN241YSQVDIGLIK and MVSHHN184LTTGATLINEQWLLTTAK respectively.The N-glycosylation site occupancy of NLFLN207HSEN211ATAK, VVLHPN241YSQVDIGLIK and MVSHHN184LTTGATLINEQWLLTTAK were1.13:1.10:1:0.97(N:HBV:LC:HCC),1.42:1.04:1:1.70(N:HBV:LC:HCC) and1.05:1.08:1:1.12(N:HBV:LC:HCC) respectively. The relative ratio of glycosylation site occupancy more than1.5-fold (up-regulation) or less than0.6-fold (down-regulation) was considered to be standard with statistic significance. Glycosylation site occupancy in VVLHPN241YSQVDIGLIK changed significantly in HCC patients compared with LC patients (ratio1.70) and HBV patients (ratio1.63). Glycosite occupancy of Asn241was observed to change significantly in HCC patients compared with LC and HBV patients. This novel approach supports the screening of the target glycoproteins as biomarkers in clinical application.Part Two N-glycan changes of serum Hp-β in patients bearing liver diseasesThe objective of this part is to analyze N-linked glycan alterations of serum Hp-β obtained from healthy individuals and patients with HBV, LC and HCC. Hp was purified from the sera and β chain was separated by SDS-PAGE. N-glycans of Hp-β were released by in-gel PNGase F digestion. The structural analysis of N-glycans was acquired by MALDI-QIT-TOF MS/MS, using DHB as matrix. AAL blot analysis was performed to demonstrate the validity of glycan changes.The main types detected from Hp-β were biantennary glycan, triantennary glycan and fucosylated glycan. Increase of fucosylation in Hp-β became the major significant feature distinguishing group of HCC and LC from group of N and HBV. The result of lectin blotting also indicated that fucosylation was increased in Hp-β of LC and HCC patients and this result was consistent with MS results.This work suggested MALDI mass spectrometry with CID took advantage of the controlled fragmentation of precursor ions and was a powerful tool to aid the structural dissection of glycosylation. The fucosylated glycans were increased in LC and HCC patients compared with HBV patients and healthy individuals. Thus, alterations in glycan structure of Hp-β may become the significant signatures of LC and HCC patients.Part Three Site-specific N-glycans changes of serum Hp-p in patients bearing liver diseasesThis part is aimed at analyzing site-specific N-glycans of serum Hp-P in healthy individuals and patients with HBV, LC and HCC. Hp was purified from the sera and β chain was separated by SDS-PAGE. Non-glycosylated peptides and three glycopeptides NLFLN207HSEN211ATAK, MVSHHN184LTTGATLINEQWLLTTAK and VVLHPN241YSQVDIGLIK were obtained by trypsin digestion. All the peptides were subjected to LC-ES1-HCD-MS/MS and this MS system allowed glycopeptide identification and structural determination, along with the relative quantification of glycans presented on each glycopeptides.Asn207and Asn211had7glycoforms including biantennary glycan, triantennary glycan and fucosylated glycan.5glycoforms including biantennary glycan, triantennary glycan and fucosylated glycan were presented at Asn184. Asn241had7glycoforms including biantennary glycan, triantennary glycan, tetraantennary glycan and fucosylated glycan. The quantification results revealed the fucosylated structure increased significantly at each glycopeptide in LC and HCC patients compared with HBV patients and normal subjects.Thus, analysis of intact glycopeptides using LC/MS could better assess the glycan variation at each N-glycosylation site. The glycan alterations of glycopeptide may be useful as the novel glyco-biomarkers for LC and HCC patients.Conclusions1. A novel strategy’iTRAQ plus18O’was established to quantify N-glycosylation site occupancy of serum Hp-β. Glycosylation site occupancy of Asn241was observed to change significantly in HCC patients compared with LC and HBV patients.2. The N-glycans and site-specific N-glycans of serum Hp-β1in healthy individuals and liver disease patients were analyzed by MS. The fucosylated glycans at each glycopeptide were increased significantly in LC and HCC patients compared with HBV patients and healthy individuals.3. Increase of Asn241occupancy and fucosylation at each glycopeptide in Hp-β became significant features distinguishing group of HCC from group of N, HBV and LC. It suggested Hp-β could act as the candidate glyco-biomarker and had the potential diagnostic value for HCC. The Potential Application of This Work1.’iTRAQ plus18O’is a powerful tool to quantify glycosylation site occupancy on the target glycoprotein in four samples simultaneously and supports to screen out the glyco-biomarkers derived from the target glycoproteins for further clinical application.2. Variations in glycosylation site occupancy and glycan structure of Hp-β may be useful as the glyco-biomarker for liver diseases.Novelty of Project1.’iTRAQ plus18O’was established firstly to quantify glycosylation site occupancy on the target glycoprotein with potential clinical application.2. Glycan macroheterogeneity and microheterogeneity of Hp-β were assessed by different analytical methods and the aberrant alternation of Hp-β glycosylation had the potential diagnostic value for liver diseases.