Sequential Phosphopeptide Enrichment for Phosphoproteome Analysis of Filamentous Fungi: A Test Case Using Magnaporthe...

A number of challenges have to be overcome to identify a complete complement of phosphorylated proteins, the phosphoproteome, from cells and tissues. Phosphorylated proteins are typically of low abundance and moreover, the proportion of phosphorylated sites on a given protein is generally low. The challenge is further compounded when the tissue from which protein can be recovered is limited. Global phosphoproteomics primarily relies on efficient enrichment methods for phosphopeptides involving affinity binding coupled with analysis by fast high-resolution mass spectrometry (MS) and subsequent identification using various software packages. Here, we describe an effective protocol for phosphopeptide enrichment using an Iron-IMAC resin in combination with titanium dioxide (TiO₂) beads from trypsin digested protein samples of the filamentous fungus Magnaporthe oryzae. Representative protocols for LC-MS/MS analysis and phosphopeptide identification are also described.