In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 10/2010; 9(10):2162-72. DOI: 10.1074/mcp.M110.000091
Source: PubMed


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.

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    • "LC-MS/MS analysis of a peptide mixture can detect and characterize diverse types of PTMs, such as phosphorylation (Witze et al., 2007). Together with the improvement in phosphopeptide enrichment methods (Neville et al., 1997; Pinkse et al., 2004; Iliuk et al., 2010), mass spectrometry based large scale phosphorylation profiling has largely replaced metabolic labeling with radioisotopes and Edman sequencing to identify endogenous phosphoproteins and sites of phosphorylation (Cañas et al., 2006). Furthermore, a variety of quantitative methods for LC-MS/ MS analysis have been successfully applied to quantify phosphorylation change on specific sites. "
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    ABSTRACT: Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.
    04/2013; 8(2). DOI:10.1007/s11515-013-1257-z
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    • "Recently, an alternative affinity technique, PolyMAC, was introduced for phosphopeptide enrichment (Iliuk et al. 2010). The phosphopeptide-capturing compound was composed of a soluble dendrimer, to which two types of side group were bound (Fig. 6). "
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    ABSTRACT: Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies. In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.
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