RT Journal Article
T1 Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics.
A1 Faria Oliveira, Fábio
A1 Carvalho, Joana
A1 Belmiro, Celso LR
A1 Martinez Gomariz, Montserrat
A1 Hernaez, Maria Luisa
A1 Pavão, Mauro
A1 Gil, Concha
A1 Lucas, Cândida
A1 Ferreira, Célia
AB BACKGROUNDIn 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.RESULTSThis 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 chemical methods were also used to test for uronic acids and sulphation.CONCLUSIONSThe 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.
PB BioMed Central
SN 1471-2180
YR 2014
FD 2014
LK https://hdl.handle.net/20.500.14352/34911
UL https://hdl.handle.net/20.500.14352/34911
LA eng
NO Fundação para a Ciência e a Tecnologia
NO Marie Curie Initial Training Network GLYCOPHARM
NO FCT/MEC through Portuguese funds (PIDDAC) -
DS Docta Complutense
RD 6 oct 2024