title: Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast.
abstract: Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Porto Conte Ricerche, Tramariglio, Alghero, Italy.
authors.forename: Maria Filippa
authors.initials: MF
authors.lastname: Addis
authors.affiliation: Porto Conte Ricerche, Tramariglio, Alghero, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tanca
authors.affiliation: Dipartimento di Agraria, Università di Sassari, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Landolfo
authors.affiliation: Porto Conte Ricerche, Tramariglio, Alghero, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Abbondio
authors.affiliation: Dipartimento di Agraria, Università di Sassari, Italy.
authors.forename: Raffaela
authors.initials: R
authors.lastname: Cutzu
authors.affiliation: Porto Conte Ricerche, Tramariglio, Alghero, Italy.
authors.forename: Grazia
authors.initials: G
authors.lastname: Biosa
authors.affiliation: Porto Conte Ricerche, Tramariglio, Alghero, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pagnozzi
authors.affiliation: Porto Conte Ricerche, Tramariglio, Alghero, Italy.|Dipartimento di Scienze Biomediche, Università di Sassari, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
authors.affiliation: Dipartimento di Agraria, Università di Sassari, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Mannazzu
mesh_terms: D001709:Biotechnology; D002338:Carotenoids / Q000096:biosynthesis; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D063990:Gene Ontology; D040901:Proteomics / Q000379:methods; D012248:Rhodotorula / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D005656:Fungal Proteins; D002338:Carotenoids
keywords: 2D-DIGE; carotenoid; protein extraction; red yeast; shotgun proteomics