Titanium Oxide-Based Phosphopeptide Enrichment from Arabidopsis Seedlings

Protein phosphorylation is one of the most widely studied posttranslational modifications, and its role in signal transduction has gained particular attention. The relatively low abundance of the phosphorylated form of proteins makes identification by mass spectrometry challenging in the absence of selective enrichment. Titanium oxide-based enrichment of phosphopeptides in the presence of acidic modifiers is highly selective and makes it technically possible to detect thousands of phosphopeptides in a single sample by liquid chromatography-mass spectrometry. In this chapter, we describe a detailed protocol for the selective enrichment of microsomal and cytosolic phosphopeptides from Arabidopsis seedlings. The resulting phosphopeptide fractions enable routine identification of several thousands of phosphopeptide spectra per sample by mass spectrometry.