title: Optimizing protein recovery yield from serum samples treated with beads technology.
abstract: Proteomics studies are often complicated by the wide dynamic range of the biological fluids, in which few highly abundant proteins obscure the signal of low abundant ones. To overcome this problem, several techniques have been developed on the basis of "depletion principles," namely immuno-subtraction with specific antibodies against the most-abundant proteins. Unfortunately, the probability of codepletion is a noteworthy drawback associated with these strategies. The ProteoMiner (PM) technology is a novel approach, consisting of a combinatorial library of hexapeptide ligands coupled to beads, that allows the capture of all species present in a proteome, but at much reduced protein concentration differences, simultaneously enhancing the concentration of the most dilute species. In this study, we evaluated the compatibility of the PM kit's elution reagent with 2-DE analysis, comparing five different purification methods on serum samples eluted from the beads: the "ReadyPrep 2-D Clean-up kit" and precipitation with organic solvents, as acetone/methanol, TCA/acetone, ACN, and chloroform/methanol. Considering protein recovery yield (quantity) and 2-DE spot pattern (quality), precipitation with ACN offered the most promising approach, showing the best spot resolution in all regions of the pH gradient and the greatest number of protein spots visualized on 2-D gels.
authors.affiliation: Department of Laboratory Medicine, Medical Faculty, University Hospital of Modena and Reggio Emilia, Modena, Italy. elisa.bellei@unimore.it
authors.forename: Elisa
authors.initials: E
authors.lastname: Bellei
authors.forename: Emanuela
authors.initials: E
authors.lastname: Monari
authors.forename: Stefania
authors.initials: S
authors.lastname: Bergamini
authors.forename: Tomris
authors.initials: T
authors.lastname: Ozben
authors.forename: Aldo
authors.initials: A
authors.lastname: Tomasi
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011232:Chemical Precipitation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D008863:Microspheres; D009930:Organic Chemicals / Q000737:chemistry; D019151:Peptide Library; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000097:Acetonitriles; D001798:Blood Proteins; D009930:Organic Chemicals; D019151:Peptide Library; C032159:acetonitrile