Journal: Journal of Extracellular Biology

Article Title: Proteomic Profiling of Bacterial Extracellular Vesicles for Exploring Ovarian Cancer Biomarkers

doi: 10.1002/jex2.70073

Figure Lengend Snippet: Characterisation and mass spectrometry analysis of patient ascites EV. (A) Schematics of EV collection from ascites samples. (B) EVs were recovered from the ascites samples of 10 patients with ovarian cancer and five patients without cancer by size exclusion (SEC). Size distribution obtained by NTAs for isolated EVs derived from representative samples of ascites samples. (C) Transmission electron microscopy analysis of isolated cancer and non‐cancer ascites EVs. Scale bar = 100 nm. (D) Immunoblot analyses for CD9, CD81 and GRP94 of EV samples of cancer and non‐cancer ascites samples. Uncropped Western blotting data are shown in Figure . (E) Schematics of mass spectrometry analysis of patient ascites EV with human reference. (F) MS data with human reference was obtained for cancer and non‐cancer ascites EV. According to protein content–based EV characterisation from MISEV2023, EV‐associated proteins were categorised into 1a to 5b. Each protein is identified by its gene symbol. The data were converted to a log10 scale. (G) The heat map shows the protein expression obtained from the same MS data as in Figure (total = 2284 proteins). The data were converted to log2 fold change (log2FC). The details of the protein names are given in the Supplementary Information. (H) PCA of the MS data obtained in Figure . (I) Volcano plot analysis between Cancer and non‐cancer samples of MS data obtained in Figure . The p values for each protein were calculated using the Wald test in Limma.

Article Snippet: After blocking with Blocking One (Nacalai tesque) for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: anti‐CD9 Antibody, clone MM2/57 CBL162 (Merck, Darmstadt, Germany), CD81 Antibody (B‐11) sc‐166029 (Santa Cruz Biotechnology, Dallas, TX, USA) and GRP94 Antibody (H‐10) sc‐393402 (Santa Cruz Biotechnology), which were diluted 1:100 in 10 % Blocking One/Tris‐buffered saline with 0.1% Tween 20 (TBST).

Techniques: Mass Spectrometry, Isolation, Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing