NDLI: Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics

Content Provider	Springer Nature : BioMed Central
Author	Faria-Oliveira, Fábio Carvalho, Joana Belmiro, Celso LR Martinez-Gomariz, Montserrat Hernaez, Maria Luisa Pavão, Mauro Gil, Concha Lucas, Cândida Ferreira, Célia
Abstract	Background 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 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.
Related Links	https://bmcmicrobiol.biomedcentral.com/counter/pdf/10.1186/s12866-014-0244-0.pdf
Ending Page	9
Page Count	9
Starting Page	1
File Format	HTM / HTML
ISSN	14712180
DOI	10.1186/s12866-014-0244-0
Journal	BMC Microbiology
Issue Number	1
Volume Number	14
Language	English
Publisher	BioMed Central
Publisher Date	2014-10-25
Access Restriction	Open
Subject Keyword	Microbiology Biological Microscopy Mycology Parasitology Virology Life Sciences Extracellular Polymeric Substance Uronic Acid Ammonium Bicarbonate Triose Phosphate Isomerase Chemical Substitution
Content Type	Text
Resource Type	Article
Subject	Microbiology Microbiology (medical)
Journal Impact Factor	4/2023
5-Year Journal Impact Factor	4.6/2023

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