Cibacron Blue and proteomics: the mystery of the platoon missing in action |
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Authors: | Di Girolamo Francesco Righetti Pier Giorgio D'Amato Alfonsina Chung Maxey C M |
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Affiliation: | Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Via Mancinelli 7, 20131 Milano, Italy. |
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Abstract: | The use of Cibacron Blue columns (HiTrapBlue) in proteome analysis for removal of plasma albumin, for facilitating biomarker discovery, has not borne any fruit. In fact, the visibility of low-abundance proteins was obscured. It is here reported that, upon albumin sequestering from plasma, there is adsorption, via hydrophobic interaction, of a substantial number of plasma proteins, which are lost for subsequent analysis if the blue resin is eluted via an ion shock (2 M NaCl) or with a somewhat more robust eluant (5 M urea, 2 M thiourea, 2% CHAPS, 2% sulphobetain 3-10) as recommended by manufacturers. Such treatments, in fact, release at most 25 to 30 unique gene products, including albumin. If, however, the Affigel-Blue resin, after elution with either of the two above eluants, is further eluted with boiling 4% SDS in 25 mM DTT, all the missing proteins (amounting to at least 112 unique species) are desorbed and biomarker analysis can be conducted in a correct way. It is also suggested that such blue-resin treatment could be coupled to ProteoMiner adsorption, this coupled treatment further enhancing the chances of success for discovery of low-abundance proteins. |
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