Analysis of thrombin‐antithrombin complex formation using microchip electrophoresis and mass spectrometry

Nielsen, JB., et al. Analysis of thrombin‐antithrombin complex formation using microchip electrophoresis and mass spectrometry, Electrophoresis. 10.1002/elps.201900235

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin‐antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.We used the data from the GENT database and immunohistochemistry staining to assess the AGTR1 expression in epithelial ovarian cancer (EOC) patients and to assess its role in cancer progression. Colony formation assay, 3D culture assay, and transwell assays were used to analyze the effect of ANGII on the MCS formation and cell migration. The signaling pathways of AGTR1 and transactivation of epidermal growth factor receptor (EGFR) transactivation were investigated by the western blotting analysis. Xenograft models were used to determine the role of AGTR1 in ovarian cancer metastasis. ANGII release from ovarian cancer cells and ANGII levels in the EOC ascites fluid were measured by immunoassay. A shotgun proteomic approach was used to explore the detail molecular mechanism. Modulation of lipid desaturation and endoplasmic reticulum stress were verified by the in vitro and in vivo functional assays.

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