T in the 2DE, or using a high throughput technique, as

T in the 2DE, or using a high throughput technique, as liquid chromatography coupled with tandem Rosiglitazone mass spectrometry (LC-MS/MS).PolysaccharidesSimilarly to what occurs with the proteins, little information is available on the polysaccharides of yECM, other than the presence of a few glycoproteins in S. cerevisiae colonies, and the identity of some components of C. albicans biofilms [12,14,20]. The sugar fraction of yECM was therefore collected and analysed, for which the procedures widely used to extract the high Eukaryotes ECM were adjusted [31-33]. The samples wereFaria-Oliveira et al. BMC Microbiology 2014, 14:244 http://www.biomedcentral.com/1471-2180/14/Page 4 ofFigure 2 SDS-PAGE and 2DE separation of yECM proteins. (A) SDS-PAGE of the ECM proteins from S. cerevisiae and C. albicans. The methodology was capable of recover the extracellular proteins from two yeast species and detect the differences in the ECM composition. (B) 2DE of the S. cerevisiae strain ECM proteins. The 2DE allowed the identification of proteins present in the yECM (see Methods).subjected to the action of the broad range protease papain, eliminating the proteins and releasing polysaccharides that might be attached to them. This reaction was performed overnight to warrant complete digestion. After protein separation by centrifugation, the samples containing solubilized polysaccharides were treated with ethanol overnight at 4 . Ethanol, disrupts the hydration of the polysaccharide and the charged ions, and when combined with low temperatures, maximizes the precipitation of both neutral and acidic polysaccharides. The utilisation of ethanol in this step is also beneficial because it is easily removable by evaporation. The precipitated polysaccharides can be dissolved, without difficulty, in deionized water or other required solution for the subsequent analysis. The S. cerevisiae and C. albicans yECM polysaccharides recovered by this procedure were evaluated using 1,3diaminopropane acetate agarose electrophoresis (DAE) (Figure 3A). This technique allows the separation of compounds with different degrees of chemical substitution, and is commonly used to separate sulphated polysaccharides [34]. S. cerevisiae presented two differently migrating compounds, whereas using C. albicans only one compound was detected. This suggests that the yECM composition is different from species to species. Importantly, the methodology proved capable of detecting those differences, showing its appropriateness for the study of yECM.S. cerevisiae ECM polysaccharides were further analysed by fast performance liquid chromatography (FPLC) (Figure 3B). PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/16989806 Identically to DAE, this technique also separates polysaccharides through the total charge-to-mass ratio. In this way, fractions of S. cerevisiae were collected and tested for (1) total sugars through reaction using sulphuric acid:phenol reaction [35], (2) uronic acids, through carbazole method [36], and (3) possible chemical substitution, analysing the metachromatic shift of 1,9-dimethylmethylene blue (DMMB) [37] (Figure 3B). In total accordance with the DAE results, the S. cerevisiae FPLC profile shows two major peaks (dark circles), indicating the existence of at least two different polysaccharide species. The presence of some kind of uronic acid in their composition (white circles), is suggested by carbazole staining, and chemical substitution by the presence of metachromasia (black line). Uronic acids are very common in extracellular polysac.