Research Of Proteins Related HCC In Plasma And Serum By Comparative Proteomics

Hepatocellular carcinoma (HCC) is the second cancer killer in China and accounts for 53% of all HCC deaths worldwide. Despite substantial progresses made in HCC clinical and basic research, the overall prognosis of HCC remains dismal because of the late diagnosis and low resection rate. Although alpha fetoprotein (AFP) remains the best HCC biomarker, the specificity and sensitivity of which are still not satisfied. Therefore, screening novel HCC biomarkers are still needed.Proteomics is becoming the hot field of biological study in the functional genomic era .The scope of proteomics is to study the expression profile,funtion and cross reactivity of all cell, tissue or organism proteins under specific circumstances. Because of the high throughput and resolving power, proteomics techniques are becoming strong tools for contemporary medical research.The challenge of clinical proteomic studies is to link protein expression profiles to specific disease phenotypes and to find out relevant biomarkers in order to develop diagnostic tools . There are an estimated 10,000 proteins that may be commonly present in plamsa/serum, which are present across an extraordinary dynamic range of concentration that is likely to span more than 10 orders of magnitude, so it is very difficult to get purposes by only one separation technique. In this research ,we adopted two techniques as two-dimensional electrophoresis-mass spectrometer (2-DE-MS) and reversed-phase high performance liquid chromatogrpahy -mass spectrometer (RP-HPLC-MS) to identificate of plamsa/serum protein(s) related Hepatocellular Carcinoma, respectively. And the satisfactory results were obtained.PartⅠSample handing of plasma/serum with HCCThe blood samples were collected in accordance with Human Proteome Organization Plasma Proteome Project. After depleting of the six plasma proteins of highest abundance by the MARS, the remaining low- and medium-abundance plasma proteins were isolated and concentrated.PartⅡIdentification of plasma/serum proteins related to HCC by 2-DEThe two-dimensional gel electrophoresis(2-DE) of plasma/serum was established, and all of the factors of 2-DE was regulated and perfected, setting up a stable technique of 2-DE of plasma/serum. Result: the depleting of the high-abundance proteins in the blood raised the number and resolution of the proteins of the 2-DE picture; the resolution of IPG by 4-7 in pH is higher than that of IPG by 3-10 in pH; urea 9M, sulfocarbamide 2M, CHAPS 2%, ampholine 1∶1(pH4~6, pH5~7) being the sample buffer, are more favourable to the separation of plasma/serum proteins; 300μg proteins are the best sample amount for the experiment; the plasma/serum proteins could be perfectly separated out with 10% density of gel and the given condition of electrophoresis (5μA 30 min, 30μA 2h, 20μA to attend 6h); the lower abundance proteins in the target one could also be relatively detected.By making use of the established 2-DE, the plasma/serum protein group between the healthy persons and patients with HCC could easely be separated and identified. After comparing, analyzing and identifying by the software PDQuest(7.4,0), the enzymolysised production of differential expression proteins were analysised by MALDI-TOF-MS, we can obtain the PMF data, which can be used to identify proteins through MS-Fit Swissport data bank. Result: In the plasma of the HCC, 20 differential proteins have been identified, among which 18 types of proteins express up-regulation and 2 down- regulation. They are RING finger protein 34,Wilms' tumor 1-associating protein,CD147 antigen,Tumor necrosis factor-inducible protein TSG-14,PRAD1 oncogene,Melanoma-associated antigen 12,Cell growth regulator with RING finger domain 1,Vacuolar ATP synthase subunit d,IgM-associated peptide,Zinc finger protein 306,Fibrinogen gamma chain precursor , MRG-binding protein , B and T lymphocyte-associated protein,Mitogen-activated protein kinase 14 , C-reactive protein precursor ,1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase ,Regulating synaptic membrane exocytosis protein 4,IgG Fc receptor II-c,Follistatin precursor,FADD protein, respectively. In 10 samples, the average of identification is 52% towards 20 general differential proteins; the average of the repeated identification of each protein in the 10 samples is 52%. In the serum of the cancer patients, 23 differential proteins have been identified, among which 17 proteins express up-regulation and 6 down-regulation. They are Serpin B10 , Keratin ,Alpha-1-antitrypsin,Protein kinase NYD-SP9,Tumor necrosis factor receptor superfamily member 19 precursor,Histone-binding protein RBBP4 , HNF-4a coactivator , Galectin-2 , Metastsis suppressor protein 1,Heat shock 70 kDa protein 8,Zinc finger protein 202,Serum albumin precursor,Apolipoprotein A-IV,Melanoma-associated antigen C3,Myc box dependent-interacting protein 1,Zinc finger CCHC domain-containing protein 5,Histone deacetylase 1 (HD1),FADD protein,26S proteasome regulatory subunit p28,CD82 antigen,Autophagy protein 5 respectively. In 8 samples, the average of the identification to the 23 general differential proteins is 54.59%, the average repeated identification of each protein in the 8 samples is 53.80%. 2 types of differential proteins have altogether been identified in the plasma and serum of the cancer patients.PartⅢIdentification of plasma/serum differential proteins by HPLC/MSTo obtain the differential proteins by the method of RP-HPLC in separating and analyzing the sample of plasma after depleting of the six plasma proteins of highest abundance, and to identify the relative molecular mass by Maxtrix assisted laser deorption/ionization time of flight mass spectrometry. Result: There exist 3 groups differential proteins in the RP-HPLC chromatogram of the plasma between the healthy persons and cancer patients, the proteins with the molecue amounted between 15kD~50kD expresses higher among the healthy persons, while the the molecue amounted between 50kD~90kD expresses higher among the patients with liver cancer. The experiement result shows that the HPLC-MS technigue could be used as the tool to screen the proteins related to blood diseases, which can strongly assist the 2-DE technigue in screening the proteins related blood diseases. The thorough separation, identification towards the proteins and biological funtion of the related proteins are under further study.