title: Plasma proteomics for biomarker discovery: a study in blue.
abstract: 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.
authors.affiliation: Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Milano, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Di Girolamo
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Righetti
mesh_terms: D000042:Absorption; D015415:Biomarkers / Q000032:analysis; D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004396:Coloring Agents / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D040901:Proteomics / Q000379:methods*; D014227:Triazines / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D004396:Coloring Agents; D014227:Triazines; C012842:Cibacron Blue F 3GA