The performance of sera pre-treatment for biomarker searching via combinatorial peptide ligand libraries (CPLL) has recently been challenged (Proteomics 2010, 10, 1416-1425) and stated to allow discovery of only medium to high-abundance proteins. We have thus investigated four elution protocols, as published in recent reports: (i) in 4M urea+1% CHAPS; (ii) in 4M urea+1% CHAPS+5% acetic acid; (iii) in 8M urea+2% CHAPS+5% acetic acid; (iv) in boiling 4% SDS+25mM DTT. One milliliter of serum, in all cases, was captured with 50μL of CPLL beads, which were then eluted with the four eluants described above. In the first three cases, after the first elution, the beads were re-eluted with cocktail (iv), known to offer maximal release of proteins adsorbed by the CPLL ligands. Eluant (i) released only ca. 20% of the species adsorbed, eluant (ii) ca. 60%, eluant (iii) ca. 80%. Thus, the poor performance of the CPLL methodology, as reported in (i) is not due to any fault of the capture technique, but simply to the adoption of a very poor elution protocol. Even those using eluants (ii) and (iii) should know that a substantial fraction of the captured species still remains bound to the beads and is thus not available to biomarker discovery. Once more, eluant (iv) is recognized as the only one able to offer optimal recovery from the CPLL baits.
- Combinatorial peptide ligand libraries
- Elution protocols
- Low-abundance proteome
ASJC Scopus subject areas
- Clinical Biochemistry