| Cellular membrane proteins and cell derived exosomes play important roles in our biological system.However,the extraction of membrane proteins remains as a challenge,due to innate hydrophobicity,dynamic discrepancy,and restrain effect of membrane lipids.The development of new enrichment strategies will contribute to their functional research,particularly in cancer research.Exosomes are major players in signaling transduction.They have an intimate relationship to the occurrence,development,and metastasis of cancer,and might be useful for diagnosis and therapy of cancers.Quantitative comparison of changes in membrane proteome as well as exosomes proteome of cancer group and control group can help to discover biomarkers for cancer diagnosis and explore the molecular mechanisms of carcinogenesis and cancer metastasis.The findings of this dissertation are as follows.Firstly,carbon nanomaterial based new membrane and membrane-associated proteins extraction and enrichment method were developed.Nanomaterials with high specific surface area have competency of hydrophobic-hydrophobic lipid interactions.We found that both graphene and graphene oxide dissolved in solubilization buffer are solid matrix for extraction of membrane proteins and greatly improved enrichment efficiency.LCMS/MS analysis further revealed that graphene(50-200 nm)and graphene oxide(50-200 nm)can enrich more kinds of membrane proteins than a commercially available kit.Graphene was further applied to the enrichment of membrane proteins from normal cells as well as cancer cells and 1079 and 872 proteins were identified,respectively,among which 56.5% and 60.5% were membrane proteins.In particular,241 proteins were significantly regulated in cancer cells.The gene expression of candidate proteins was verified by qRT-PCR,and the four proteins were then verified by immunoassay.These data collectively demonstrate that graphene has great potential to improve membrane protein extractions and thus can serve for downstream cancer proteomics.Secondly,serum exosomes were isolated and used for the analysis of exosomes proteome of hepatocellular carcinoma.Serum exosomes were isolated from the serum of patients with hepatocellular carcinoma,liver cirrhosis,and healthy controls,and from the culture media of hepatocellular carcinoma cells(HepG2)and normal hepatocytes(LO2).These isolated exosomes were confirmed by size distribution measurement,morphological analysis,and surface biomarker test.There were 61 and 63 proteins differentially expressed in serum exosomes between patients with cirrhosis and patients with hepatocellular carcinoma(P < 0.05).Immunoassay further confirmed hepatocellular carcinoma-related protein candidates,including thrombin-sensitive protein-1(THSB1),fibrin-1(FBLN1),and fibrinogen gamma chain(FGG),which could differentiate healthy controls from hepatocellular carcinoma or cirrhosis.Our results confirmed that cancerrelated proteins are present in exosomes of patient’s serum and can be used to detect hepatocellular carcinoma and liver cirrhosis.In summary,we developed a novel bio-separation method for membrane proteins and conducted cellular membrane proteomics research,which resulted in the discovery of key proteins related to lung cancer.The newly developed membrane proteins extraction method has the advantages of simplicity,high efficiency,and high selectivity as compared to kit method,which is expected to be widely used in biomedical research.Furthermore,we revealed that liver cancer related proteins present in serum exosomes.Serum exosomal proteins can be used as candidates for the detection and diagnostics of liver diseases. |
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