Establishment Of A Glycopeptide Microarray Method For The Rapid Analysis Of Glycan-binding Protein And Its Application

The interaction of protein and glycan on cells surface promote the adhesion between cells,and plays a role in vertebrate embryonic development.This kind of protein interaction with sugar is called glycan-binding protein.Glycan microarray fixed glycan on nitrocellulose membrane or slides,with high flux,systemic,sensitive detection interactions between glycan and glycan-binding proteins.But,kinds of glycans which can fix to microarray is limited.Therefore,it is important for the immobilization of glycans and the development of glycan microarray to have more glycans isolated from the nature or artificially synthesized.Use trypsin enzyme to digest the protein treated by IAM and iodoacetamide in turn.Use lectin to extract glycopeptide containing acertain glycan.Fix the glycopeptide to the epoxidation-modified glass by cycloaddition reaction between glycopeptide N-end and epoxy group.Fix the Cy3 tagged sample protein and glycopeptide microarrayar for incubation,scan the fluorescent signal value of the microarrayar after incubation,analyze the changes of glycan-binding proteins.The result showed the fluorescent intensity reached the highest level when spotting buffer was 0.1 M Na2CO3,pH9.3.The best spotting humidity is 45%?The best microarrayar drying temperature is 37?,3 h.The best blocking buffer is phosphate buffer solution containing 1%BSA.The best incubation buffer is phosphate buffer solution containing 1%BSA,0.58%NaCl,0.05%Tween-20.The peptide micromarry was used to study glycan-binding protein related to liver cancer,the expression difference of glycan-bing protein between four stages of development of liver cancer(normal,hepatitis B,cirrhosis and liver cancer)in different samples(serim,saliva)were carried out.There are four kinds of glycan-binding protein identificated by different glycan have been changed in saliva:the level of GBPS from saliva of hepatitis,liver cirrhosis and liver cancer patient identificated by Terminal in GalNAc and Gal and Galal-3(Fucal-2)Gal have been raised than which from healthy volunteers' saliva.The level of GBPS from saliva of liver cancer patient identificated by Bisecting GlcNAc,biantennary complex-type N-glycan with outer Gal have been rasied than healthy volunteer.The level of GBPS from liver cancer patient identificated by(GlcNAc)n,high mannose-type N-glycans have been lower than liver cirrhosis patient.There are nine kinds of glycan-binding protein identificated by different glycan have been changed in serum:the level of GBPS from serum of hepatitis B,liver cirrhosis and liver cancer patients identificated by Gal?-1,4GlcNAc(type II),Galpl-3GlcNAc(type I)and Bisecting GlcNAc,bi-antennary N-glycans,tri-and tetra-antennary complex-type N-glycandifferent have been rasied than healthy volunteers.The level of GBPS from hepatitis B patient identificated by High-Mannose,Manal-3Wan,Man?l-6Man,Man5-GlcNAc2-Asn ? terminating in GalNAc?/?1-3/6Gal have been lower than healthy volunteers.The level of GBPS from serum of liver cirrhosis and liver cancer patient identificated by GlcNAc and agalactosylated tri/tetra antennary glycans have been rasied than healthy volunteers.The level of GBPS from serum of hepatitis B,liver cirrhosis and liver cancer patient identified by Terminal in GalNAc and Gal and ?-Gal,?-GalNAc,Gal?-1,3Gal,Gal?-1,6Glc have been lower than healthy volunteers.The level of GBPS from serum of hepatitis B,liver cirrhosis and liver cancer patient identified by Gal?1-3(Fucal-2)Gal have been lower than healthy volunteers.The level of GBPS from liver cirrhosis patient identified by Multivalent Sia and(GlcNAc)n have been rasied than healthy volunteers and hepatitis B patient.Bulied the glycopeptied-grain compound with the glycopeptides cantaining Gala 1-3(Fucal-2)Gal to extract GBPS from saliva and serum of healthy volunteers,hepatitis B,liver cirrhosis and liver cancer patients for LC-MS/MS analyzes.There are 34 of 39 GBPS from saliva and 31 of 33 GBPS from serum of healthy volunteers have been annotated.There are 35 of 40 GBPS from saliva and 54 of 67 GBPS from serum of liver cancer patients have been annotated.