Plasma proteomics for biomarker discovery: A study in blue

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.