Immobilized Iron(III) Metal Affinity Chromatography for the Separation of Phosphorylated Macromolecules: Ligands and Applications

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

“…However, thorough washing of the column before use and a judicious selection of binding ligands can minimize these problems. For example, IDA is a tridentate ligand, whereas NTA is a tetradentate ligand; hence, the latter binds more strongly to the metal ion to better prevent leaching (Hochuli, Dobeli, & Schacher, 1987; Holmes & Schiller, 1997).…”

Investigation of the use of immobilised metal affinity chromatography for the on-line sample clean up and pre-concentration of nucleotides prior to their determination by ion pair liquid chromatography-electrospray mass spectrometry: a pilot study

“…Several models are used to explain the differential retaining behaviour of metal ions in IMAC. The primary action of binding is based on the formation of co-ordination bonds between the immobilised metal ion and electron donating groups delivered by the compounds of interest [41][42][43]. The different selectivity exerted by various metal ions towards different kinds of nucleophiles is generally explained by the Hard and Soft Acids and Bases (HSAB) principle of Pearson [44].…”

“…The applied test mixtures were made of cytosine, cytidine, CMP, CDP, CTP, adenine, adenosine, AMP, ADP and ATP dissolved in 0.1% HOAc and each at a concentration of 1 × 10 −5 M. Elution, based on competing hydroxyl binding [42], was realised by changing the mobile phase to aqueous NH 4 OH (pH = 10), 2 min after injection. The effluent of the column both during the sample loading-and eluting step was monitored using full scan ES(−)MS. An evaluation of the extracted ion chromatograms and the corresponding MS-spectra of the compounds under investigation showed that the nucleobases and nucleosides were not retained at all and that they eluted with the void volume of the column.…”

“…Thus a 0.1% (w/v) NH4OAc pH = 10 ( In order to find a solution free phosphate was introduced and tested on its ability to elute nucleotides trapped on a Fe 3+ -loaded IMAC-column. Unlike acetate ions, inorganic phosphate has two possible binding sites and therefore it is believed to be inherently more competitive than acetate [42,45]. One could expect the free phosphate to have a similar affinity towards immobilised iron(III) as the phosphate groups present in nucleotides.…”

Mass spectrometric identification of Rab23 phosphorylation as a response to challenge by cytidine 3′,5′‐cyclic monophosphate in mouse brain

“…A series of incubations of mouse brain tissue with ATP as phosphate donor were carried out in the presence of cAMP, cGMP, cCMP or a non‐cyclic nucleotide control. The use of immobilized metal affinity chromatography (IMAC)28 with immobilized Fe(III) to chelate phosphate groups linked to threonine, tyrosine or serine in proteins,29, 30 followed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐ToFMS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS), allowed the identification of a protein phosphorylated in response to cCMP but not to cAMP and cGMP. After testing a number of different protocols in trial experiments the strategy depicted in Fig.…”