ABSTRACT
Protocols to identify lipopeptide biosurfactant extracts contained in complex residual streams are very important, as fermented agri-food matrices are potential sources of these valuable compounds. For instance, corn steep liquor (CSL), a secondary stream of the corn wet-milling industry, is composed of a mixture of microbial metabolites, produced during the corn steeping process, and other natural metabolites released from corn, that can interfere with the purification and analysis of lipopeptides. Electrophoresis could be an interesting technique for the purification and further characterization of lipopeptide biosurfactant extracts contained in secondary residual streams like CSL, but there is little existing literature about it. It is necessary to consider that lipopeptide biosurfactants, like Surfactin, usually are substances that are poorly soluble in water at acidic or neutral pH, forming micelles what can inhibit their separation by electrophoresis. In this work, two lipopeptide biosurfactant extracts obtained directly from CSL, after liquid-liquid extraction with chloroform or ethyl acetate, were purified by applying a second liquid extraction with ethanol. Following that, ethanolic biosurfactant extracts were subjected to electrophoresis under different conditions. Lipopeptides on Tricine-SDS-PAGE (polyacrylamide gels) were better visualized and identified by fluorescence using SYPRO Ruby dye than using Coomassie blue dye. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of lipopeptide isoforms separated by electrophoresis revealed the presence of masses at 1,044, 1,058, and 1,074 m/z, concluding that Tricine-SDS-PAGE electrophoresis combined with MALDI-TOF-MS could be a useful tool for purifying and identifying lipopeptides in complex matrices.
ABSTRACT
We studied the specific changes of the secreted protein clusterin and its cytoplasmic precursor regarding colorectal tumorigenesis, using in vitro differentiation of Caco-2 cells. In tumor-like stage, we observed an overexpression of both precursor and secreted clusterin, corroborated in the cell line SW-480. Noticeably, SW-620 cells (from a tumoral node, thus with metastatic capacity) did not show overexpression of either precursor or secreted clusterin, suggesting a downregulation related to local metastasis. We further investigated clusterin in serum, finding a significant increase in colorectal cancer patients, with 81% sensitivity, 79% specificity, and an area under the ROC curve of 0.85.
