New approaches to phosphoproteomics: Clinical scale characterization of the AKT/mTOR pathway targets.

Protein phosphorylation is the most common mechanism of regulating protein function. With an expanding phosphoproteome of known functional pathways, much interest now turns towards targeted quantitative analyses based on candidates earlier identified in discovery screens for detailed comparison of differential phosphorylation and linking with genomic evidence of mutagenesis. Even complex tissue samples can be probed selectively for phosphopeptides of interest by devoting instrument sensitivity and sampling speed to a subset of relevant targets for peptide quantification. Here, we screen for phosphoproteins in the AKT/mTOR pathway using a new targeted sample prep approach (SureQuant). This approach provides a simple, robust method to multiplex immunoprecipitation and mass spec sample prep for multiple phosphopeptides simultaneously: this particular kit is used to identify and quantify 30 unique peptides from 10 phosphorylated proteins and can be used in conjunction with genomic analyses. This panel covers significant proteins throughout the AKT-mTOR signaling pathway from human clinical samples. Using this approach, we enriched for ∼300 proteins and ∼1k peptides from both cell lysates and tissues in untargeted runs. Most of the 10 AKT/mTOR targets were successfully identified from frozen tissue and lysates. Identification of a couple of targets in FFPE samples is encouraging and an indication of sensitivity even in these challenging samples. Quantitation of these targets is being evaluated in ongoing efforts. With this technology, we identify peptide targets from both cell lysates and frozen tissues, and additionally demonstrate viability for some of these target phosphoproteins in FFPE tissue, laying the foundation for connecting these observations with genetic screening. This new tool adds a critical 'next step' for phosphoproteomics approaches previously limited to qualitative screening and is portable to analogous protein targets in other research areas.