title: "Proteomineering" or not? The debate on biomarker discovery in sera continues.
abstract: The performance of sera pre-treatments for biomarker discovery has been recently assessed as very poor not only for immuno-subtraction, in turn evaluated as a tool unable to look deep into the low-abundance proteome (LAP) and thus incapable to lead to any novel biomarker discovery (J Proteome Res 2010;9:4982-4991), but also for combinatorial peptide ligand libraries (CPLL) (Proteomics 2010;10:1416-1425). The performance of both tools has been given as enabling to barely detect a meagre 25% more as compared to control, untreated sera. Meanwhile, other studies indicated the extreme effectiveness of peptide libraries to enlarge the knowledge of proteome compositions. In this contradictory situation we are here re-evaluating some protocol aspects and report that indeed CPLL is an excellent tool, able to dig really deep into the low-abundance proteome. The problem is that in those reports under-optimized capture and elution protocols had been adopted. With the protocols here reported, namely (a) abandoning the step of adding 150mM salt to the sample; (b) capture at three different pH values (pH 4.0, 7.0 and 9.3) and (c), most importantly, eluting from CPLL beads in 4% boiling SDS in the presence of 25mM DTT, we can largely expand the windows of visibility. In particular, it is here shown that a common elution protocol adopted in several reports, in 4M urea and 1% CHAP, barely elutes about 15% of the captured species. Nevertheless if the CPLL beads thus treated are further eluted with boiling SDS-DTT, an additional 80% is recovered.
authors.affiliation: Multidisciplinary InterInstitutional BioBank (BioBIM), Dept. Laboratory Medicine & Advanced Biotechnologies, IRCCS San Raffaele, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Di Girolamo
authors.forename: Egisto
authors.initials: E
authors.lastname: Boschetti
authors.forename: Maxey C M
authors.initials: MC
authors.lastname: Chung
authors.forename: Fiorella
authors.initials: F
authors.lastname: Guadagni
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Righetti
mesh_terms: D015415:Biomarkers / Q000032:analysis / Q000097:blood; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D020543:Proteome