Glycoproteome-wide Identifications of Novel GALNT14 Substrates Using Tandem Mass Spectrometry
| Autor: | Lin, Yu-Hua, 林郁樺 |
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| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 The glycosyltransferase GALNT14 was recently shown to mediate the oncogenesis and treatment responses of multiple cancers, including hepatocellular carcinoma, cholangiocarcinoma, colon cancer, esophageal cancer, neuroblastoma and breast cancer. Particularly, the genotype of GALNT14 was tightly associated to the therapeutic response of hepatocellular carcinoma in an accumulation of more than 900 patients treated in Taiwan. However, the substrates of GALNT14 are not entirely known, except the death receptor (DR) 5 which mediates the extrinsic apoptosis signaling of cancer cells. We aim to investigate novel substrates of GALNT14 using a lectin enrich glycoproteome-wide screening method combined with high performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS). The GALNT14 genomic DNA was cloned to plasmids which were then transfected to the hepatocellular carcinoma cell line including Huh7, J7 and Mahlavu, for generating stable GALNT14 overexpression cell lines. Their corresponded control cell lines were also established with only the plasmid backbone without the GALNT14 DNA. Glycoproteins of the three cell lines were captured using self-made lectin affinity column, which was packed with Peanut Agglutin lectin (PNA) or Vicia Villosa Lectin (VVA). The captured proteins were following by trypsin digestion and high performance liquid chromatography – tandem mass spectrometric analyses. In summary, 305 glycoproteins were identified in GALNT14 overexpressed cells but not in the control cells. They are found to be involved in ribosome pathway and clearly shown to be associated with hepatocellular carcinoma. Therefore, this study successfully demonstrated the relation between GALNT14 and cancers, and could pave the way for a deeper understanding of carcinogenesis studies. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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