Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography

A1 Journal article (refereed)

Internal Authors/Editors

Publication Details

List of Authors: Imanishi, Kochin, Eriksson
Publication year: 2007
Journal: Proteomics
Journal acronym: Proteomics
Volume number: 7
Issue number: 2
Start page: 174
End page: 176
ISSN: 1615-9853
eISSN: 1615-9861


While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.

Last updated on 2019-16-09 at 06:54