Development of Glycoprotein Capture-Based Label-Free Method for the High-throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma

• DOI: 10.1074/mcp.m110.006445
• Publication Date: 2011-04-08T04:07:02Z
• Authors: Rui Chen; Yexiong Tan; Min Wang; Fangjun Wang; Zhenzhen Yao; Liwei Dong; Mingliang Ye; Hongyang Wang; Hanfa Zou
• Keywords: Adult; Male; 0303 health sciences; Carcinoma, Hepatocellular; Liver Neoplasms; Enzyme-Linked Immunosorbent Assay; Middle Aged; Reference Standards; Sensitivity and Specificity; Chromatography, Affinity; 3. Good health; 03 medical and health sciences; Insulin-Like Growth Factor Binding Protein 3; Molecular Diagnostic Techniques; Antigens, Neoplasm; Tandem Mass Spectrometry; Case-Control Studies; Biomarkers, Tumor; Humans; Female; Carrier Proteins; Early Detection of Cancer; Glycoproteins

A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.