title: Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics.
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.
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal. ffariaoliveira@bio.uminho.pt.
authors.forename: Fábio
authors.initials: F
authors.lastname: Faria-Oliveira
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal. joana.scarvalho13@gmail.com.
authors.forename: Joana
authors.initials: J
authors.lastname: Carvalho
authors.affiliation: Institute of Medical Biochemistry, Laboratory of Glycoconjugates Biochemistry and Cellular Biology, Federal University of Rio de Janeiro/ Polo de Macaé, Macaé, Brazil. celso_belmiro@yahoo.com.br.
authors.forename: Celso L R
authors.initials: CL
authors.lastname: Belmiro
authors.affiliation: Unidad de Proteómica, Universidad Complutense de Madrid - Parque Científico de Madrid UCM-PCM), Madrid, Spain. montserrat.martinez@fpcm.es.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Martinez-Gomariz
authors.affiliation: Unidad de Proteómica, Universidad Complutense de Madrid - Parque Científico de Madrid UCM-PCM), Madrid, Spain. mlhernae@farm.ucm.es.
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Hernaez
authors.affiliation: Institute of Medical Biochemistry, Laboratory of Glycoconjugates Biochemistry and Cellular Biology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. mpavao@hucff.ufrj.br.
authors.forename: Mauro
authors.initials: M
authors.lastname: Pavão
authors.affiliation: Unidad de Proteómica, Universidad Complutense de Madrid - Parque Científico de Madrid UCM-PCM), Madrid, Spain. conchagil@farm.ucm.es.|Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain. conchagil@farm.ucm.es.
authors.forename: Concha
authors.initials: C
authors.lastname: Gil
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal. clucas@bio.uminho.pt.
authors.forename: Cândida
authors.initials: C
authors.lastname: Lucas
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Braga, Portugal. celiamjf@gmail.com.
authors.forename: Célia
authors.initials: C
authors.lastname: Ferreira
mesh_terms: D002176:Candida albicans / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods; D005109:Extracellular Matrix / Q000737:chemistry; D005656:Fungal Proteins / Q000032:analysis* / Q000302:isolation & purification*; D054794:Glycomics / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides