Identification and confirmation of biomarkers using an integrated platform for quantitative analysis of glycoproteins and their glycosylations

Abstract

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. However, accurate diagnosis can be difficult as most of the patients who develop this tumor have symptoms similar to those caused by longstanding liver disease. Herein we developed an integrated platform to discover the glycoprotein biomarkers in early HCC. At first, lectin arrays were applied to investigate the differences in glycan structures on serum glycoproteins from HCC and cirrhosis patients. The intensity for AAL and LCA was significantly higher in HCC, indicating an elevation of fucosylation level. Then serum from 10 HCC samples and 10 cirrhosis samples were used to screen the altered fucosylated proteins by a combination of Exactag labeling, lectin extraction and LC-MS/MS. Finally, 27 HCC and 27 cirrhosis serum samples were used for lectin-antibody arrays to confirm the change of these fucosylated proteins. C3, CE, HRG, CD14 and HGF were found to be biomarker candidates for distinguishing early HCC from cirrhosis, with a sensitivity of 72% and specificity of 79%. Our work gives insight to the detection of early HCC, and the application of this comprehensive strategy has the potential to facilitate biomarker discovery on a large scale.

Figure 1

Workflow showing the integrated strategy for the discovery of serum glycoprotein biomarkers in early hepatocellular carcinoma (HCC).

Figure 2

Lectin Microarray. (a) Sample randomization on the slides. H represents early hepatocellular carcinoma; C represents cirrhosis. (b) Response of 16 different lectins to glycoproteins in depleted serum from HCC and cirrhosis patients. AAL and LCA showed significantly different response to HCC and cirrhosis. (c) Summary of AAL and LCA response. Error bars indicate S.E. from 14 HCC and 14 cirrhosis patients; * p<0.05; **p<0.01. (d) ROC curves of sensitivity and specificity for AAL and LCA. (e) Scatter plot of fucosylation level in different serum samples detected by AAL and LCA.

Figure 3

LC-MS/MS analysis. (a) A representive nano LC-MS/MS base peak chromatogram, showing the detection of peptide ions across 120 min gradient separation (90 min shown here). (b) MS/MS sequencing data of a peptide from histidine-rich glycoprotein identified in the AAL extracted fractions.

Figure 4

Fucosylated protein alteration confirmed by Antibody Microarray. (a) AAL-assisted antibody array of 26 selected proteins in the serum from HCC and cirrhosis patients. Proteins which showed significantly different response to AAL between HCC and cirrhosis were indicated by red rectangle (p<0.05). (b) Comparison of response intensity of C3, CE, HRG, CD14 and HGF to AAL in HCC and cirrhosis. Each spot represents one serum sample, error bars indicate the standard deviation from 27 HCC and 27 cirrhosis patients. (c) ROC curves for C3, CE, HRG, CD14 and HGF.

Figure 5

Statistical analysis of antibody microarray. (a) Heatmap based on the signal intensity of antibody microarray. Each column indicates a selected protein tested in the antibody array experiment, and each row indicates an individual serum sample from HCC or cirrhosis patients. The intensity increased from blue color to red color. (b) Scatter plot in log 2 scale and a regression line of histidine-rich glycorprotein (HRG) response to AAL between every pair of two independent experiments with the same sample set. (c) Combined ROC curve of C3, CE, HRG, CD14 and HGF. (d) Principle component analysis. Shown is the response intensity of 26 selected serum proteins from 27 HCC and 27 cirrhosis patients onto the two leading principal components.

Figure 6

Western blotting analysis of C3, CE, HRG, CD14, HGF, VDB and AFM. Depleted serum samples from 10 HCC patients (left lane) and 10 cirrhosis patients (right lane) were pooled, respectively. Five micrograms of proteins were separated by a 4%-12.5% gel. The expression level of C3, CE, HRG CD14 and HGF were increased in the serum from HCC, while the control proteins VDB and AFM showed no significant difference between HCC and cirrhosis.