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J Biomol Tech. 2007 February; 18(1): 24–25.
PMCID: PMC2291957

P72-T Using Titanium Dioxide IMAC for Enrichment of Phosphopeptides Prior to Tandem Mass Spectrometry


Protein phosphorylation plays a significant role in regulating cellular processes such as signal transduction, cell division, cell motility, apoptosis, metabolism, differentiation, gene regulation, and carcinogenesis. Typically, there are 10–20% of proteins which are phosphorylated. Due to the low level of phosphoproteins in the presence of overwhelming amounts of non-phosphorylated proteins, as well as those proteins’ wide dynamic variation over time, identification of phosphopeptides is still a formidable task. In addition, phosphopeptides often have poor ionization efficiency in MS analysis. Thus, a highly sensitive detection method plus phosphopeptide enrichment is extremely important for a successful phosphopeptide identification. Currently, immobilized metal affinity chromatography (IMAC) is the method of choice for enriching phosphopeptides from complex biological samples. Typically, nickel, iron, and gallium–based IMAC shows significant binding of non-phosphorylated peptides that have multiple acidic residues. Forest White et al. used a kind of chemistry to put methyl esters onto those acidic groups (D and E) to solve the problem of nonspecific binding to the IMAC beads. However, this approach brings in a lot of side reactions to that chemistry, and raises issues of how complete the modifications are. Recently, several papers and posters have been published demonstrating the unique ability of titanium dioxide and zirconium dioxide to selectively retain phosphopeptides contained in complex biological mixtures. In this application, a TiO2-based IMAC method was successfully developed to enrich phosphopeptides and adapted to a complex biological sample, Saccharomyces. Trapping phosphopeptides are demonstrated via the analysis protein CaO19_4593 (gi|68466366), a family of GTPase-activating proteins which contains multiple kinase-binding domains.

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