The Research Of Comparative Serological Proteome In Evolving Process Of Hepatocarcinogenesis And N-polysaccharide Characteristic Analysis Of Haptoglobin

Hepatocellular carcinoma(HCC)is the second cancer killer in China. Hepatocarcinogenensis is a complex process involving multiple changes in gene expression and usually occurs at early stages.It is known that development of HCC is linked to cirrhosis and the chronic hepatitis caused by hepatitis B virus (HBV)is a predominant reason for cirrhosis.The changes on ingredient of serum reflected the organism process of the physiological and pathological conditions.It is useful for serum samples to monitor patients to screen the biomarker or evaluate the efficacy of treatment regimens because serum can be readily obtained from patients.Although current methods for the diagnosis of HCC rely on serological markers such as alpha fetoprotein(AFP)and certain liver enzymes,the specificity and sensitivity of which are still not satisfied, therefore,screening the key proteins in each stage of hepatocarcinogenensis are still needed.It is common knowledge that approximately 50%of proteins in malignant tumors are glycosylated and that glycans play crucial roles in various biological events.There is still no any systematic glycomic method to screen aberrantly glycosylated proteins.It is common that a-1,6 fucose(core fucose) exists in many glyproteins,whose aberrantly glycosylation relates to the development and diagnosis of chronic hepatitis and HCC. Here we have used two-dimensional electrophoresis(2-DE)for serum proteomics,followed by matrix assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF-MS/MS),to screen different proteins and to approach the rule of the different proteins in evolving process of hepatocarinogenesis.Then we validated some potential biomarkers.At the same time,we have used MP technique coupled with two-dimensional electrophoresis (2-DE),followed by lectin blot and precipitation.And we built up serum glycoprotein profiles of HCC,liver cirrhosis,inactive carrier and healthy controls by using the former approach;we found some aberrantly a1-6 fucosylated glycoproteins with different express by using the latter approach, which related to the development of HCC,such as hp-βchain.All in all,we detected the change on hp-βprotein expression levels,and oligosaccharide structures in these different sera samples to discover whether these alterations played important roles in evolving process of HCC.Part 1 Comparison and optimization of proteomic methods for human serumObjective:To establish and optimize a two-dimensional polyacrylamide gel electrophoresis(2-DE)technique for serum proteomics.Methods:Through modifying the sample preparation,such as depleting high abundance protein,optimizing rehydration buffer and comparing different methods of protein staining,we enhanced the resolution for 2-DE serum proteomics. Results:Comparing Coomassie Brilliant Blue and SYPRO-Ruby fluorescent dye staining,the average spots in the gels of pH 3-10 IPG strip for 2-DE profiles were respectively 324±25(n=3)和785±30(n=3);and the average spots in the gel for neat serum and high abundance protein(albumin and IgG)depletion serum were 501±25(n=3)and 813±22(n=3)separately.Depletion of high-abundance protein combined with SYPRO Ruby fluorescent dye staining could enhance low abundance protein detection sensitivity.Conclusions:Established and optimized 2-DE technique of serum protein can be effectively applied in serum proteomics of diseases.Part 2 The research of comparative serological proteome in evolving process of hepatocarcinogenesisObjective:To establish high resolution and reproducibility of a two-dimensional polyacrylamide gel electrophoresis(2-DE)technique for serum proteomics,and then to look for different proteins in evolving process of hepatocarcinogenesis to approach the rule of their processes.Method:We examined sera from 27 Hepatocellular carcinoma patients,10 liver cirrhosis patients,23 chronic HBV inactive Carrier patients and 25 healthy controls.After pretreatment including sonication,albumin and immunoglobulin (IgG)depletion and desalting,sera were subjected to 2-DE and then stained with SYPRO Ruby.After image acquisition and analysis,protein spots were excised by Genetix gelPix Spot Cutter and identified by MALDI-TOF-MS.Result:After sera pretreatment,more protein spots(853 vs.513)were detected on reference gel because of the depletion of album in and IgG.By comparing the 2-DE sera profiles of HCC,liver cirrhosis,inactive carrier,healthy controls,the average number of each group spots was 850±10(mean±SD),755±37,825±29 and 784±39(n=3),respectively.From optimized 2-DE gel images of the four groups,Image master 6.0 software selected 96 protein spots that showed a more than 2-fold difference in intensity between the two mixtures with statistical significance and identified by MALDI-TOF-MS to be twenty-five proteins. Selecting healthy controls as standard,we found these changeable patterns as follows:①12 proteins show different expression among more than 2 groups: these proteins were unremitting down-expressed from healthy controls to liver cirrhosis,otherwise,over-expressed in HCC,such as Hp,hp2-alpha, preprohaptoglobin,SP40 and so on;②on the contrary,2 proteins were unremitting over-expressed from healthy controls to liver cirrhosis,while down-expressed in HCC.These proteins were both Ig mu heavy chain disease protein and Immunoglobulin J chain;③And 4 proteins were over-expressed in inactive carrier compared with normal liver tissue,then down-regulated in liver cirrhosis and over-expressed in HCC finally,such as Chain A,Human Serum, Transferrin,similar to human albumin and son on;④only one proteins show different expression only between 2 groups of the samples:this unnamed protein product over-expressed in liver cirrhotic compared with in inactive carrier or HCC,and had almost no difference in expression level among other samples.7 different spots were all one kind of protein among above 30 different spots, belonged to haptoglobin family.And this kind of protein was the most significant patterns in four groups. Conclusions:At last,after the depletion of high-abundance proteins,we required the better images of 2-DE sera profiles.At the same time,we found a group of different proteins in evolving process of hepatocarcinogenesis by the method of serum proteomics and revealed apparently changeable patterns related to dynamics process of HCC.haptoglobin over-expressed significantly from liver cirrhosis to HCC.Thus,this new discovered biomarkers might be an aid in the diagnosis of liver cirrhosis which resulted from the occurrence and development of HCC,though further validation is needed.Part 3 Verification on Differential Expression of haptoglobin and Its N-polysaccharide characteristic analysisObjective:To further validate haptoglobin expression level and approach the oligosaccharide structures of HP.Method:sera from 30 healthy volunteers,30 hepatitis B virus(HBV)infected patients,30 liver cirrhosis patients and 30 HCC patients were used for validation study.We examined sera total haptoglobin in the same four groups by immunoturbidimetry and western blot(each group 13 sera).At the same time, we used MP technique coupled with two-dimensional electrophoresis(2-DE), followed by lectin blot and precipitation;Then,we used western blot to confirm the 2-DE results that showed the expressions of haptoglobin,too.At the same time,we have used multiplexed proteomics(MP,Glycoprotein stain Emerald 488 and Total protein stain SYPRO Ruby)coupled with two dimensional electrophoresis(2-DE),followed by lectin blot in several different kinds of lectin and precipitation to inference glycoproteins which related to the development of HCC with different structures.Result:The results from immunoturbidimetry and western blot for members in haptoglobin family(Hp,hp2-alpha and preprohaptoglobin),confirm to the 2-DE results that showed the expressions of haptoglobin family in four groups of human sera.And we built up serum glycoprotein profiles of HCC,liver cirrhosis, inactive carrier,healthy controls through the Image master 6.0 software to calculate vol%of hp,we found the extents of glycosylation of hp were same as the protein express levels of hp except the preprohaptoglobin;AAL,LCA,DSA, Con A,PHA-E and RCA-I were employed to observe the alternations in their N-glycan structures.Compared to HBV,Hp had a higher affinity for AAL,and RCA-I in both LC and HCC,while Con A and PHA-E almost no affinity in LC. At the same time,it was found that the binding of DSA to hp was not obviously different between N,HBV and LC,and lower affinity in HCC,compared to above three groups.Conclusions:we validated the change on hp-βprotein expression levels,and observed the alternations in their N-glycan structures in these different serum samples to discover whether these alterations played important roles in evolving process of HCC.Haptoglobin was proved to have core-fucosylated type high mannose-core,triantennary type and terminal GalNAc type sugar chain structure. Fucosylated haptoglobin was increased in the serum in evolving process of HCC. Conclusion1.We established and optimized a two-dimensional polyacrylamide gel electrophoresis(2-DE)technique for serum proteomics through sample preparation,staining comparison and so on.2.We established 2-DE profile from Hepatocellular carcinoma patients,liver cirrhosis patients,chronic HBV inactive Carrier patients and healthy controls, and identified twenty-five proteins by MALDI-TOF-MS and found four changeable patterns,one of the most significant alternation patterns proteins in four groups was haptoglobin family.3.We used western blot to confirm the 2-DE results that showed the expressions of haptoglobin family in four groups of human sera.4.We built up serum glycoprotein profiles of HCC,liver cirrhosis,inactive carrier and healthy controls by using MP technique coupled with two-dimensional electrophoresis(2-DE);Except some aberrantly fucosylated glycoproteins with different expression,we found other sugar chain alteration which related to the development of HCC by lectin blot and precipitation.Novelty of this project1.25 different proteins were identified among hepatocellular carcinoma patients, liver cirrhosis patients,chronic HBV inactive Carrier patients and healthy controls using serum proteomics techniques.To our best knowledge,four changeable patterns in evolving process of hepatocarcinogenesis were firstly reported in four groups.2.What haptoglobin play a role in four groups was firstly validated by our works.3.our works showed hyproglycosylated HP related to HCC occurrence and development.These results indicated inference of haptoglobin structure of sugar chain may affect the process of hepatocarcinogenesis.The potential application of this work1.2-DE based serum proteome analysis can be useful in detecting proteins expression alteration and screening for new biomarkers in amount of disease.2.Haptoglobin might be an aid in the diagnosis of liver cirrhosis which resulted from the carcinogenesis and development of HCC.It was good to research about glycoprotein profile and elucidate the molecular mechanism of HCC occurrence and development through providing the inference of haptoglobin's structure of sugar chain,especially,some aberrantly glycosylated proteins.