Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns

Abstract

Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO 2 microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO 2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO 2 . We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented. Molecular & Cellular Proteomics 4: 873-886, 2005.Phosphorylation is among the most widespread post-translational modifications in nature, and it has been estimated that more than 30% of the proteins in a given mammalian cell at some point during their expression are phosphorylated (1). Phosphorylation and dephosphorylation of proteins regulates a large number of biological processes such as signal transduction (2), molecular recognition and interaction, and other cellular events. A fundamental understanding of these biological processes at the molecular level thus requires a characterization of the phosphorylated sites in the proteins. It is therefore essential to develop sensitive and selective methods for this task.A wide variety of methods are known for characterization of phosphorylated proteins. The most widely used have been peptide sequencing using Edman degradation combined with 32 P labeling. This method is well established and very robust but has several limitations. For example, in Edman degradation the peptides have to be separated before the analysis using liquid chromatography. This decreases the overall sensitivity and increases analysis time, and it is therefore not well suited for analysis of complex samples.Recently a number of MS-based strategies have been developed that are relatively sensitive and in many cases easier to perform than Edman degradation with respect to handling complex mixtures (e.g. Ref.3). The increased sensitivity is especially needed for low stoichiometric phosphorylation. However, presently ...

Optimizing TiO2-Based Phosphopeptide Enrichment for Automated Multidimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry

“…The most thoroughly studied condition in these reports has been the acid content of the loading buffer. For TiO 2 -based enrichment, it has been shown that increases in the concentration of a particular acid or replacing an acid with one of a lower pH in the loading buffer reduces non-specific binding of unphosphorylated peptides, while enhancing the detection efficiency of phospopeptides 16,18 . The same trend has been reported with the use of IMAC and ZrO 2 as well 15, 19 . Additionally, Kokubu et al showed that increasing the concentration of acetonitrile in a loading buffer enhances the enrichment of phosphopeptides using IMAC 19 .…”

Selective Enrichment and Fractionation of Phosphopeptides from Peptide Mixtures by Isoelectric Focusing after Methyl Esterification

“…Within the past five years, many highly specific enrichment methods have been developed, such as immobilized metal affinity chromatography (IMAC) after methyl esterification of peptide mixtures 1 and improved TiO 2 , ZrO 2 and Al(OH) 3 chromatography 2-4 . These methods have greatly reduced nonspecific binding of non-phosphorylated acidic peptides.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…New metal ions, e.g. titanium [100] and zirconium [101], have been successfully employed for phosphopeptide enrichment, and additional peptide fragmentation methods, e.g. electron-capture dissociation (ECD) [102] and electron-transfer dissociation (ETD) [103], have been used for better MS analysis of phosphopeptides.…”