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Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC)
Author(s) -
Wolschin Florian,
Wienkoop Stefanie,
Weckwerth Wolfram
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200402049
Subject(s) - affinity chromatography , phosphorylation , chromatography , proteomics , chemistry , protein phosphorylation , biochemistry , ion chromatography , biology , enzyme , protein kinase a , gene
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 α‐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.