Purification And Identification Of The Glycoproteins From Sera Of HCC Patients

Background:Hepatocarcinoma(HCC) is one of the most common malignant tumors in the clinical, and the mortality is high. It has become a disease with a heavy menace to human health and life, so we should not overlook the hazard. In the human body, there are more than half of the proteins being glycosylated. The glycosylation not only plays a vital role in protein structure, stability and orientation, but also participates in some significant biological processes such as cell adhesion, apoptosis, receptor activation, signal transduction, endocytosis and so on. With some researches, it is showed that the aberrant glycosylation of proteins has a close relationship with cancer and other diseases, and some glycoproteins can be considered to be potential biomarkers of HCC. Lectins can specifically recognize sugar moieties of the glycoprotein and reversibly bind to them by non-covalent bond. Before we identify and analyze glycoproteins by mass spectrometry, lectins can be used to isolate them from serum to lessen the interference from high abundance non-glycoproteins.Method:In our experiment, Aleuria Aurantia Lectin(AAL), Wheat Germ Agglutinin (WGA)and Concanavalin A(Con A) were coupled with epoxy-coated magnetic particles. Then, AAL-magnetic particle conjugates, WGA-magnetic particle conjugates and Con A-magnetic particle conjugates were utilized to isolate glycoproteins from45healthy volunteers mixed serum and45HCC patients mixed serum, respectively. Finally, we employed LC-MS/MS to identify and analyze these glycoproteins.Result:(1) A total of32unique glycoproteins binding with AAL were identified from healthy volunteers sera.23proteins have been annotated as glycoproteins in the Unitprot.9proteins were predicted to be N-glycoproteins by NetNGlyc software, and3proteins were identified as glycoproteins in the9proteins by our experiment. A total of35unique glycoproteins binding with AAL were identified from HCC patients sera.24proteins have been annotated as glycoproteins in the Unitprot.11proteins were predicted to be N-glycoproteins by NetNGlyc software, and1protein was identified as a glycoprotein in the11proteins by our experiment. There were15glycoproteins in both kind of sera. After quantifying the15glycoproteins with emPAI label-free method, it is showed that7glycoproteins were markedly higher and1glycoprotein was markedly lower in HCC petients sera than in healthy volunteers sera.20glycoproteins were only identified in HCC patients sera, in which we discovered3up-regulated glycoproteins.(2) A total of81unique glycoproteins binding with WGA were identified from healthy volunteers sera.62proteins have been annotated as glycoproteins in the Unitprot.19proteins were predicted to be N-glycoproteins by NetNGlyc software, and4proteins were identified as glycoproteins in the19proteins by our experiment. A total of78unique glycoproteins binding with WGA were identified from HCC patients sera.58proteins have been annotated as glycoproteins in the Unitprot.20proteins were predicted to be N-glycoproteins by NetNGlyc software, and3proteins were identified as glycoproteins in the20proteins by our experiment.There were53glycoproteins in both kind of sera. After quantifying the53glycoproteins with emPAI label-free method, it is showed that4glycoproteins were markedly higher and8glycoproteins were markedly lower in HCC petients sera than in healthy volunteers sera.25glycoproteins were only identified in HCC patients sera, in which we discovered1up-regulated glycoprotein.(3) A total of32unique glycoproteins binding with Con A were identified from healthy volunteers sera.26proteins have been annotated as glycoproteins in the Unitprot.6proteins were predicted to be N-glycoproteins by NetNGlyc software. A total of28unique glycoproteins binding with Con A were identified from HCC patients sera.22proteins have been annotated as glycoproteins in the Unitprot.6proteins were predicted to be N-glycoprotein by NetNGlyc software, and1protein was identified as a glycoprotein in the6proteins by our experiment. There were25glycoproteins in both sera. After quantifying the25glycoproteins with emPAI label-free method, it is showed that1glycoprotein was markedly higher and4glycoproteins were markedly lower in HCC petients sera than in healthy volunteers sera.3glycoproteins were only identified in HCC patients sera, and all of them were up-regulated glycoproteins.(4) In healthy volunteers sera and HCC patients sera, there were24glycoproteins isolated only by AAL,76glycoproteins isolated only by WGA and14glycoproteins isolated only by Con A. In the glycoproteins isolated by the three types of lectins, there were11same glycoproteins isolated only by AAL and WGA,4same glycoproteins isolated only by WGA and Con A,2same glycoproteins isolated only by AAL and Con A and14same glycoproteins isolated by the three kinds of lectins.Conclusion:AAL-magnetic particle conjugates, WGA-magnetic particle conjugates and Con A-magnetic particle conjugates can be employed to isolate and purify unique glycoproteins from sera.18glycoproteins can be regarded as potential biomarkers for HCC. With searching for some literatures, it is reported that3glycoproteins of the18identified glycoproteins could be considered to be biomarkers for HCC.