Purification And Identification Of The Glycoproteins Recognized By Lectin LCA From Sera Of HCC Patients

Background:Hepatocarcinoma is one of the five most common malignant tumors in the world according to the latest data from the International Agency for Research on Cancer. In 2008, there were 749 thousand hepatocarcinoma patients in the world,697 thousand of whom died during that year, while 371 thousand patients of 401 thousand patients died in China. Mortality from malignant tumors is the highest among the major diseases in China in 2009 while mortality caused by hepatocarcinoma is the second highest, according to the China Health Statistics Annuals. One of the reasons for high mortality of hepatocarcinoma is the delayed treatment for lack of efficient early diagnosis and confirmation methods for hepatocarcinoma. More than half of the proteins in the human body were glycosylated. The glycosylation not only affect protein structure, biological distribution, biological function, but also play an important role in signal transduction, cells recognition, cell differentiation. The results of the early research showed that the aberrant glycosylation protein had relationship with tumor and other diseases. Currently, the FDA approved 19 types of disease biomarkers are all glycoproteins. Lectins can recognized some specific structure of sugar, and be used to purified glycoproteins from serum to reduce the high abundance protein which restrain the identification of the glycoproteins. Two-dimensional electrophoresis and mass spectrometry were widely used in proteomics research. In this study, we use of lectin affinity to purify glycoprotein from serum and then the two-dimensional electrophoresis and mass spectrometry were used to analyze and identify the glycoproteomics. The methods can be used to identify potential markers of HCC.Methods:Our laboratory has established the lectin-magnetic particle conjugates technology platform, we used magnetic particles coupled with the Lens Culinaris Agglutinin to purify glycoproteins from 40 HCC patients and 40 healthy volunteers's mixed serum, individually. To find out the difference serum glycoprotein between the HCC patients and healthy volunteers, the two-dimensional electrophoresis was applied. The puridied glycoproteins were digested in solution and the peptide was determined by LC-MS/MS. The MS/MS date was annotated by the MASCOT and Swiss-Prot database. The identified glycoproteins were analysed by Gene ontology.Results:1. Application the two-dimensional electrophoresis to identify the difference glycoprotein between the HCC patients and healthy volunteers's,322±6were found from HCC patients and 245±8were found from healthy volunteers's mixed serum by LCA-magnetic particle conjugates. In the found glycoproteins,152±5 were matched,22 were significant differences.2. Digested by the Trypsin and PNGase F of the LCA purified glycoproteins were identified using LC-MS/MS.(1) Thirty-five glycoproteins purified from the HCC patients serum were identified.25 proteins have been published in the Swiss-Prot as glycoprotein,5 proteins were predicted by N-glycosylation prediction software NetNGlyc as N-glycosylation proteins. In the five predicted glycoprotein,3 glycopeptides were identified by the LC-MS/MS; 2 identified proteins were predicted as O-glycoproteins by N-glycosylation prediction software NetOGlyc; 2 glycoproteins were predicted as non-N-X-S/T sequon; 1 was identified as glycoproteins by LC-MS/MS.(2) Thirty-six glycoproteins purified from the healthy volunteer's serum were identified.26 proteins have been published in the Swiss-Prot as glycoprotein,5 proteins were predicted by N-glycosylation prediction software NetNGlyc as N-glycosylation proteins. In the five predicted N-glycoprotein,3 glycopeptides were identified by the LC-MS/MS; 2 identified proteins were predicted as O-glycoproteins by O-glycosylation prediction software NetOGlyc; 2 glycoproteins were predicted as non-N-X-S/T sequon; 1 was identified as glycoproteins by LC-MS/MS.(3) Twenty-six identified serum glycoproteins were present both in HCC patients and healthy volunteer's serum.9 glycoproteins were presented only in HCC patients,10 glycoproteins were presented only in healthy volunteer's serum.(4) Quantity of proteins identified in this study can be estimated by Exponentially Modified Protein Abundance Index (emPAI). C4b-binding protein alpha chain, Protein AMBP and Alpha-1-antitrypsin were more than 1.5 fold up-regulated while Apolipoprotein A-Ⅰ, Apolipoprotein A-Ⅱ, Inter-alpha-trypsin inhibitor heavy chain H4, Serum paraoxonase/arylesterase 1, Beta-2-glycoprotein 1 and IGL@ protein were less than 0.66 fold down-regulated.Conelusion: LCA magnetic particles are useful to isolate and purify glycoproteins from serums; 4 glycoproteins of the 12 identified glycoproteins which are potential biomarkers for hepatocarcinoma are conformed as biomarkers according to several scientific papers. 8 differential expression glycoproteins that are not published as potential biomarkers before are also recognized in this study.