Plasma proteomics for biomarker discovery: A study in blue

Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Milano, Italy.
Electrophoresis (Impact Factor: 3.03). 12/2011; 32(24):3638-44. DOI: 10.1002/elps.201100307
Source: PubMed


The performance of Cibacron Blue dye (HiTrapBlue or Affigel Blue) in depleting albumin from plasma, as a pre-treatment for biomarker searching in the low-abundance proteome, is here assessed. It is shown that (i) co-depletion of non-albumin species is an ever-present hazard; (ii) the only proper eluant able to release quantitatively the proteins bound to the dye is boiling 4% SDS-25 mM DTT, an ion shock (2 M NaCl) being quite ineffective in releasing the low-abundance species tightly bound to the dye moiety; (iii) the mechanism of dye-protein interaction, after an initial ion-ion docking, is a robust hydrophobic interaction, which progressively augments at lower and lower pH values; (iv) at pH 2.2 in the presence of 0.1% TFA, the blue resin behaves, for all practical purposes, just as a reverse-phase chromatography column, since all residual proteins present in plasma are completely harvested. However Cibacron Blue technology should not necessarily be discarded: As long as also the plasma fraction adsorbed is properly released and analyzed, together with the flow through, one should be able to perform a viable analysis of the low-abundance proteome.

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    • "After incubation with a settled bed volume of 100 lL Blue Sepharose 6 Fast Flow beads for 1 h at 20 °C with mild agitation, the beads were sedimented by centrifugation at 2000 g for 2 min and the supernatants were transferred to new tubes. Bound proteins were recovered after three washes with 1.0 mL incubation buffer by boiling with 100 lL 4% SDS and 25 mM DTT (Di Girolamo and Righetti, 2011). Albumin depletion by extraction with CB-Sepharose was tested with human, bovine and porcine samples under various conditions, including variation of the pH (see Appendix A: Supplementary Fig. 2), salinity and the polarity of the buffer (data not shown). "
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