Optimizing protein recovery yield from serum samples treated with beads technology.

Proteomics studies are often complicated by the wide dynamic range of the biologicalfluids, 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 themost-abundant proteins. Unfortunately, the probability of codepletion is a noteworthydrawback associated with these strategies. The ProteoMinerTM (PM) technology is anovel approach, consisting of a combinatorial library of hexapeptide ligands coupled tobeads, that allows the capture of all species present in a proteome, but at much reducedprotein concentration differences, simultaneously enhancing the concentration of themost dilute species. In this study, we evaluated the compatibility of the PM kit’s elutionreagent with 2-DE analysis, comparing five different purification methods on serumsamples eluted from the beads: the ‘‘ReadyPrep 2-D Clean-up kit’’ and precipitation withorganic solvents, as acetone/methanol, TCA/acetone, ACN, and chloroform/methanol.Considering protein recovery yield (quantity) and 2-DE spot pattern (quality), precipitationwith ACN offered the most promising approach, showing the best spot resolution inall regions of the pH gradient and the greatest number of protein spots visualized on 2-Dgels.