S to generate, extract, purify and fractionate yeast ECM for analytical use in Rosiglitazone proteomics and glycomicsF io Faria-Oliveira1, Joana Carvalho1, Celso LR Belmiro2, Montserrat Martinez-Gomariz3, Maria Luisa Hernaez3, Mauro Pav 4, Concha Gil3,5, C dida Lucas1 and C ia Ferreira1*AbstractBackground: In a multicellular organism, the extracellular matrix (ECM) provides a cell-supporting scaffold and helps maintaining the biophysical integrity of tissues and organs. At the same time it plays crucial roles in cellular communication and signalling, with implications in spatial organisation, motility and differentiation. Similarly, the presence of an ECM-like extracellular polymeric substance is known to support and protect bacterial and fungal multicellular aggregates, such as biofilms or colonies. However, the roles and composition of this microbial ECM are still poorly understood. Results: This work presents a protocol to produce S. cerevisiae and C. albicans ECM in an equally highly reproducible manner. Additionally, methodologies for the extraction and fractionation into protein and glycosidic analytical pure fractions were improved. These were subjected to analytical procedures, respectively SDS-PAGE, 2-DE, MALDI-TOF-MS and LC-MS/MS, and DAE and FPLC. Additional PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25751659 chemical methods were also used to test for uronic acids and sulphation. Conclusions: The methodologies hereby presented were equally efficiently applied to extract high amounts of ECM material from S. cerevisiae and C. albicans mats, therefore showing their robustness and reproducibility for yECM molecular and structural characterization. yECM from S. cerevisiae and C. albicans displayed a different proteome and glycoside fractions. S. cerevisiae yECM presented two well-defined polysaccharides with different mass/charge, and C. albicans ECM presented a single different one. The chemical methods further suggested the presence of uronic acids, and chemical modification, possibly through sulphate substitution. All taken, the procedures herein described present the first sensible and concise approach to the molecular and chemical characterisation of the yeast ECM, opening the way to the in-depth study of the microbe multicellular aggregates structure and life-style.Background In multicellular organisms, communication between cells is essential, and is profoundly influenced by the extracellular matrix (ECM) components. This scaffolding structure coordinates the biochemical reactions of the different types of cells within the tissues and organs [1]. The mammalian ECM is composed by a wide array of functional molecules, biochemically and biophysically diverse, including proteins, glycosaminoglycans and proteoglycans.* Correspondence: celiamjf@gmail.com 1 CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal Full list of author information is available at the end of the articleThe glycosaminoglycans (GAGs) are some of the most dominant ECM components, greatly influencing the cellular behaviour. These highly charged polysaccharides, frequently sulphated, can be found covalently attached to protein cores, forming proteoglycans (PGs), which regulate the GAG distribution and turnover [2]. A high molecular diversity arises from the different combinations of PGs protein cores with one or more types of GAGs chain. These are responsible for the wide variety of biological roles, including structural scaffolding, signalling and growth factor s.