Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC)

Abstract

A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.

Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

“…[15][16][17][18][19][20][21][22][23][24] Ikeguchi and Nakamura reported phosphopeptide enrichment using titania. 15 Other metal oxides, such as zirconia 23 and aluminium hydroxide, 22 have also been employed. Larsen et al utilized aromatic substituted carboxylic acids, such as 2,5-dihydroxybenzonic acid (DHB) and phthalic acid, as competitive chelating reagents to reduce the binding of non-phosphopeptides to titania.…”

Highly efficient and selective enrichment of phosphopeptides using porous anodic alumina membrane for MALDI-TOF MS analysis

“…The utility of zirconium dioxide microtips for phosphopeptide isolation before mass spectrometric analysis has also been demonstrated [28]. The metal hydroxide Al(OH) 3 , for phosphopeptide and phosphoprotein bindings, has also proved to be effective and more selective than commercial phosphoproteinenrichment kits [29].…”

Advances in purification and separation of posttranslationally modified proteins

“…However, the similarity of purification results obtained using these kits to those obtained using IMAC/MOAC matrices (e.g. [76]) suggests that they are also probably based on IMAC/MOAC technology. Chromatofocusing and ion exchange chromatography are options that can be used to selectively enrich acidic proteins from a sample.…”

“…Aluminum hydroxide (Al(OH) 3 ) matrices have also been used for MOAC phosphoprotein enrichment. These matrices are relatively inexpensive and provide comparable yields to those obtained using commercial phosphoprotein purification kits [76]. The numbers of IMAC-and MOAC-based methods are constantly increasing.…”