Mohamed, Emida (2013) Proteomic analysis of sera of women with normal pregnancy, hydatidiform mole and cancers using lectin-and gel-based approaches / Emida binti Mohamed. PhD thesis, University of Malaya.
Abstract
Previous study conducted by Abdul-Rahman et al. (2007) has highlighted the enhanced expression of a 35 kDa glycoprotein, termed lectin-detected protein 1 (LDP1), in sera of patients with endometrial adenocarcinoma (EACa). In this study, LDP1 was isolated from sera of patients with EACa using immobilised champedak-galactose-binding (CGB) lectin affinity chromatography. Subjecting LDP1 to mass spectrometry analysis, LDP1 was identified as the 35 kDa cleavage fragment of inter-α-trypsin-inhibitor heavy chain 4 (ITIH4f). Subsequent to its identification, expression of ITIH4f in sera of patients with breast carcinoma (BrCa; n=10), epithelial ovarian carcinoma (EOCa; n=10), germ cell ovarian carcinoma (GOCa; n=10), nasopharyngeal carcinoma (NPCa; n=13) and osteosarcoma (OsSa; n=10) was investigated using 2-DE and HRP-CGB lectin approach. When compared to control sera, expression of ITIH4f was enhanced in sera of patients with BrCa, EOCa and GOCa, but not in patients with NPCa and OsSa. The data of this study, when taken together with reports of previous researchers, appeared to link the over-expression of ITIH4f with cancers associated with estrogen dysregulation. As such, the expression of ITIH4f is also likely to be enhanced in other diseases associated with increased levels of estrogen. Hence, its expression in women with normal pregnancy (NP; n=20), hydatidiform mole (HM; n=20) and menopausal women under the hormone replacement therapy (HRT; n=10) were studied. When compared to controls (n=20), it was demonstrated that the expression of ITIH4f was over-expressed in women with NP and HM, but was not altered in women with HRT. Hence, the expression of ITIH4f was not exclusive to cancers but was also enhanced in normal and benign conditions that are associated with estrogen dysregulation. An attempt to establish the serum protein profiles of women with NP and HM was subsequently performed via 2-DE. Protein detection was either performed by silver staining or CGB lectin. Image analyses performed on profiles of pregnant women demonstrated that the expression of ITIH4f, α1-antichymotrypsin (ACT) and leucine-rich-glycoprotein (LRG) was significantly enhanced in the women with NP. However, ITIH4f was the sole serum glycoprotein which appeared to be altered in sera of women with HM. Comparative analysis performed on the silver-stained protein profiles demonstrated enhanced expression of ACT, LRG, clusterin (CLU) and zinc-α2- glycoprotein (ZAG) in sera of women with NP. In contrast, women with HM were characterized by the up-regulated expression of CLU and LRG. However, the LRG of women with HM appeared to be differentially glycosylated from that of women with NP and CLU in women with HM seemed to be more cationic, relative to than that of NP women. All proteins which showed differential expression were identified by mass spectrometry analysis. The aberrant expression of the identified proteins was subsequently validated using immunoblotting technique. Finally, an attempt to simultaneously detect the differential expression of the respective proteins was performed using multiplex immunoassay. A multiplex immunoassay was developed to evaluate the expression of eight proteins but the development was only successful for three proteins- anti-thrombin III (ATR), CLU and ceruloplasmin (CLP). Nonetheless, the assay was capable of confirming the up-regulated expression of CLU and the unaltered expression of ATR and CLP.
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