Glycoproteomic Analysis Of Aberrantly Fucosylated Glycoproteins In Sera Of Patients With Hepatocellular Carcinoma

[Objective]Hepatitis B virus (HBV) infection is high in China. Infection with HBV is the major etiology of hepatocellular carcinoma (HCC). The high mortality associated with HCC is partly due to unresponsiveness to treatment, with a low 5-year survival rate after diagnosis. As therapeutic options increase, early detection of HCC is important to improving the prognosis. Determination of AFP levels is often included as a serum marker of HCC. However, AFP can be produced under many circumstances, including other liver diseases, and is not present in all those with HCC. Therefore, the use of AFP as a primary screen for HCC has been questioned. Specifically, AFP becomes fucosylated and this alteration is called AFP-L3. AFP-L3 is a more specific marker of HCC than is the total AFP protein level. Indeed, AFP-L3, gained approval from the US FDA in 2005 as the only diagnostic assay for HCC.AFP-L3 discovery prompted us fucosylated glycoproteins associated with the occurrence and development of HCC. Glycosylation is the most important post-translational modification of proteins. In many diseases, the development process, the glycan level of protein glycosylation or glycan structures have changed. This has led to glycoproteins and their host cells dysfunction, and even a malignant phenotype. The fucosylation change is not limited to AFP in HCC. The researchers found other glycoproteins such as serotransferrin, Golgi protein 73 also become fucosylated with the development of HCC and a recent study has proposed that these glycoforms may be valuable biomarkers of HCC.Because of lens culinaris agglutinin (LCA) can specifically capture fucosylated glycoproteins, we will use proteomics technology based on LCA affinity separation attempt to look for other serum fucosylated glycoproteins in HCC and verify the potential of the diagnosis of HCC.[Methods]1 Application isolation and identification two-dimensional gel electrophoresis based on LCA affinity separation fucosylated glycoproteins associated with HCC:The HCC group, chronic liver disease group and the healthy control group of 10 cases of serum samples were mixed by LCA-enriched fucosylated glycoproteins. Through the two-dimensional electrophoresis separation of these proteins, then analyzed and compared to identify differences in protein spots by mass spectrometry identification of differences in protein spots. Finally detected by Western blot, several of which protein in the serum of each group of fucosylation conditions.2 Application CLINPROT system based on LCA affinity separation, the establishment of HCC-related fucosylation of serum protein fingerprinting model: Application of LCA-magnetic beads were enriched HCC group, liver cirrhosis group, chronic hepatitis group and the healthy control group serum samples fucosylated proteins. Time of flight mass spectrometry technology was used to detect these proteins. Collecting relevant data to establish groups of fucosylated proteins fingerprint model. Through software analysis, establish the diagnosis model and use it to blind test.3 Application CLINPROT system based on LCA affinity separation, the establishment of HCC-related fucosylation of serum peptide fingerprinting model: Application of LCA-magnetic beads were enriched HCC group, liver cirrhosis group, chronic hepatitis group and the healthy control group serum samples fucosylated proteins. Use trypsin digestion part of these proteins. Then time of flight mass spectrometry to detect the peptides after trypsin digestion. Collecting relevant data to establish groups of fucosylated peptides fingerprint models. Through software analysis, establish the diagnosis models and use them to blind test.[Results]1 Identification and analysis of differentially expressed proteins:Isolated and identified 22 differentially expressed proteins from serum of the HCC group, chronic liver disease group and the healthy control group. The extent of fucosylation ofα-1-B glycoprotein, kininogen 1, hemopexin, etc. in HCC group, chronic liver disease group and healthy group in turn lower; in contrast, apolipoprotein H, complement factor H-related protein 1, etc. in turn increase; keratin 9, keratin 10 etc. in HCC group is higher than chronic liver disease group and healthy group; anti-thrombin 3, histidine-rich glycoprotein,α-1-microglobulin precursors in healthy group is higher than HCC group and chronic liver disease group; complement C7, and ATP-dependent RNA helicase DDX41 in healthy group is lower than HCC group and chronic liver disease group; RNA-binding protein 12 in HCC group is lower than chronic liver disease group and healthy group; a-2-macroglobulin and parvalbumin alpha in chronic liver disease group is lower than HCC group and healthy group.By Western blot test,α-1-B glycoprotein(A1BG) and fucosylatedα-1-B glycoprotein (Fc-A1BG) expression in the serum of each group:A1BG and Fc-A1BG expression in the HCC group, chronic liver disease group and the healthy control group were not significantly different (P>0.05).By Western blot test, Apolipoprotein H(APOH) and fucosylated apolipoprotein H (Fc-APOH) expression in the serum of each group:APOH and Fc-APOH expression in the HCC group, chronic liver disease group and the healthy control group were significantly different (P<0.05). Fc-APOH expression more obvious differences among three groups of serum samples (1:1.56:3.83).2 Establishment and application of HCC-associated fucosylation of serum protein fingerprinting model:Four groups of serum protein fingerprint model were compared to establish a series of various classification model. Analysis model found that serum glycoprotein fucosylation abnormalities associated with the occurrence and development of HCC. At the same time, diagnosis of HCC classification model through blind testing, HCC samples could be distinguished from all serum samples, the liver disease samples and liver cirrhosis samples with a sensitivity/specificity of 74.07%/76.81%,81.48%/83.72% and 88.89%/86.36% respectively. Combined with the AFP test, the sensitivity/specificity increased to 81.48%/88.41%,88.89%/90.70% and 92.59%/95.45% respectively.3 Establishment and application of HCC-associated fucosylation of serum peptide fingerprinting model:Four groups of serum peptide fingerprint model were compared to establish a series of various classification model. Analysis model found that serum glycopeptide fucosylation abnormalities associated with the occurrence and development of HCC. At the same time, diagnosis of HCC classification model through blind testing, HCC samples could be distinguished from all serum samples, the liver disease samples and liver cirrhosis samples with a sensitivity/specificity of 70.37%/69.57%,77.78%/74.42% and 81.48%/81.82% respectively. Combined with the AFP test, the sensitivity/specificity increased to 77.78%/88.41%,85.19%/88.37% and 88.89%/90.91% respectively.[Conclusions]1 Serum glycoproteins fucosylation abnormalities associated with the occurrence and development of HCC, but also in the process of occurrence and development of HCC, different glycoproteins fucosylation unusual manifestation of different forms, including increased gradually and decreased gradually.2 Fucosylatedα-1-B glycoprotein is not suitable as a candidate diagnostic marker for HCC.3 Fucosylated apolipoprotein H can be used as a potential marker of HCC for early diagnosis.4. HCC-associated fucosylation of serum protein/peptide fingerprinting early diagnosis of HCC model has the potential for clinical application.