In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble...

The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro- and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling.