TY - JOUR
T1 - "Proteomineering" or not? The debate on biomarker discovery in sera continues
AU - Di Girolamo, Francesco
AU - Boschetti, Egisto
AU - Chung, Maxey C M
AU - Guadagni, Fiorella
AU - Righetti, Pier Giorgio
PY - 2011/5/1
Y1 - 2011/5/1
N2 - 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 150. mM 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 25. mM 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 4. M 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.
AB - 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 150. mM 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 25. mM 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 4. M 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.
KW - Biomarkers
KW - Combinatorial peptide ligand libraries
KW - Low-abundance proteome
KW - Sera
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U2 - 10.1016/j.jprot.2011.01.013
DO - 10.1016/j.jprot.2011.01.013
M3 - Article
C2 - 21295168
AN - SCOPUS:79953293314
VL - 74
SP - 589
EP - 594
JO - Journal of Proteomics
JF - Journal of Proteomics
SN - 1874-3919
IS - 5
ER -