Analysis Of Low Abundance Diferential Proteome In Plasma From Hepatocellular Carcinomas Patients

Pramary hepatocellular carcinoma (HCC) is one of the most prevailing and lethal malignancies in the world. At present, in Asia, there is a very large number of patients suffering from hepatocellular carcinoma (HCC), in particular, hepatitis B and hepatitis C virus infection related to the HCC Suffering. HCC is highly invasive, poor prognosis, so early diagnosis and treatment of liver cancer is of particular importance. AFP (alpha-fetoprotein, AFP) on the clinical diagnosis of HCC is the most common tumor markers, but low sensitivity (70%), lower specificity (64%), a separate application on HCC diagnosis easily lead to misdiagnosis and missed diagnosis. As a result, from the plasma / serum to discover new biomarkers for early diagnosis of HCC is the indicator of a major research hot spots.In the present work,we applied multi-dimensional chromatography tandem mass spectrometry as analysis strategy.Samples of human plasma were depleted six most abundance proteins,using an immunoaffinity chromatography Agilent MARS Spin Cartridge,then were separated under the prefered conditions by Reversed-phase LC.Over-expressed differential fraction of low abundance proteins were collected and tryspined, tryspined peptides were enriched and fractioned and indentified,using HPLC-CHIP/MS.The aim of this study is to screen a viable techology route for discovering novel biomarkers of HCC from the plasma.First of all,to elect and handle sample . In strict accordance with the criteria of clinical screening patients with hepatocellular carcinoma, hepatocellular carcinoma patients were selected and obtained first-hand clinical data. And in accordance with the reference specimen standard applied in HUPO (Human Proteome Organization) preliminary studies selected healthy volunteers as the control group.Plasma were collected and handled according to criteria applied in pilo-phase study of HUPO.The second, removal of high abundance proteins of plasma .The removal of high abundance proteins of plasma is one of the most important bottleneck. In this work, immunoaffinity chromatography MARS (Agilent Mutiple Affinity Remove System) Spin Cartridge extensively applied HUPO PPP were applied to effective removal six major high abundance proteins (albumin, Haptoglobin , Immunoglobulin A, immunoglobulin G, transferrin,α-1-antitrypsin), and the flow fractions of low-abundance plasma proteome has been concentrated by Amicon Ultra-4 (3 kDa) concentration device after one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) to verify the removal of high-abundance plasma proteins from the control group.Bradford assay of quantitative results of crude plasma,the flow-through fraction and the bound fraction indicate the removal ratio of total proteins mass is 85%,consistently reproducible. The SDS-PAGE of 4%-12% is further authenticated the removal efficiency which manifested the MARS column enriched the low-abundance proteins and immunodelepted the six high-abundance proteins, which provided ground for the successive research.The third, the prefractionation of low-abundance proteome with RP-HPLC. Low-abundance plasma proteome were prefractionated by Reversed phase high performance liquid chromatography(RP-HPLC) taking into account the hydrophobic protein biomarkers in the importance of research on basis of optimized RP chromatography parameters,the sample pre-treatment,and delay time of manually collecting chromatography peak. Then the diferential chromatography peak were collected precisely and frozen-dried.Indicating that diferential peak B of over-expressed and diferetial peak C of low-expressed are consistent in immuodepletion plasma of 5 HCC,dierential peak A of over expressed is only emerged in No.1 HCCThe last, HPLC-CHIP/MS analysis .The over-expressed differential peak B and the special over-expressed peak A of low-abundance proteins of immunodepletion plasma in No.1 HCC were analyzed with HPLC-CHIP/MS.The peptide mixture was enriched and fractionated with HPLC-CHIP(Agilent 1100 Series HPLC systems),CHIP included a enrich column of Zorbax 300SB-C18(5 mm×300μm,5μm) and a separate column of Zorbax 300SB-C18(43 mm×75μm, 3.5μm).then identified by linear ion trap mass spectrometry, The data of MS and MS/MS were analyzed with Spectrum Mill MS Proteomeics Workbench (Rev A.03.03.078) against the database of UniProtKB/SWISS-PORT,Homo Sapiens (Human),renewed every two weeks. about 27 kinds of proteins were indentfied.23 kinds of protein are related to vary diseases or tumors, Among them,IMP3, ARNT2 and GRIP1 may become potential biomarkers of diagnosis of HCC .