title: Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis.
abstract: Since red blood cells (RBCs) lack nuclei and organelles, cell membrane is their main load-bearing component and, according to a dynamic interaction with the cytoskeleton compartment, plays a pivotal role in their functioning. Even if erythrocyte membranes are available in large quantities, the low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional 2D gel-based proteomic approaches. So, in order to increase the efficiency of RBC membrane proteome identification, here we took advantage of a simple and reproducible membrane sub-fractionation method coupled to Multidimensional Protein Identification Technology (MudPIT). In addition, the adoption of a stringent RBC filtration strategy from the whole blood, permitted to remove exhaustively contaminants, such as platelets and white blood cells, and to identify a total of 275 proteins in the three RBC membrane fractions collected and analysed. Finally, by means of software for the elaboration of the great quantity of data obtained and programs for statistical analysis and protein classification, it was possible to determine the validity of the entire system workflow and to assign the proper sub-cellular localization and function for the greatest number of the identified proteins.
authors.affiliation: Proteomics and Metabolomics Unit, Institute for Biomedical Technologies-CNR, Via Fratelli Cervi 93, I-20090 Segrate, Milan, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: De Palma
authors.forename: Antonella
authors.initials: A
authors.lastname: Roveri
authors.forename: Mattia
authors.initials: M
authors.lastname: Zaccarin
authors.forename: Louise
authors.initials: L
authors.lastname: Benazzi
authors.forename: Simone
authors.initials: S
authors.lastname: Daminelli
authors.forename: Giorgia
authors.initials: G
authors.lastname: Pantano
authors.forename: Mauro
authors.initials: M
authors.lastname: Buttarello
authors.forename: Fulvio
authors.initials: F
authors.lastname: Ursini
authors.forename: Massimo
authors.initials: M
authors.lastname: Gion
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Mauri
mesh_terms: D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D015999:Multivariate Analysis; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012984:Software
chemical_list: D008565:Membrane Proteins