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 & 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.

Full-text preview

Available from:
    • "Recently, advances have been made in developing phosphopeptide enrichment strategies using metal oxide affinity chromatography (MOAC) [15]. A new technique designated polymer-based metal ion affinity capture with titanium dioxide-functionalized soluble nanopolymers (PolyMAC-Ti) was developed and proved highly selective for enrichment of phosphorylated peptides [15] [16]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: Global analysis of stimulus-dependent changes in the neutrophil phosphoproteome will improve the understanding of neutrophil signal transduction and function in diverse disease settings. However, gel-free phosphoproteomics of neutrophils in clinical studies is hampered by limited sample amounts and requires protein extract stability, efficient tryptic digestion and sensitive phosphopeptide enrichment in a protease-rich environment. For development of an appropriate workflow, we assessed neutrophil protein stability in urea-based lysis buffers and determined feasibility of gel-free phosphoproteomic analyses using polymer-based metal ion affinity capture (PolyMAC). Methods: Western blotting, phosphopeptide enrichment and mass spectrometric analyses of samples of neutrophils were performed. Results: Degradation of proteins in neutrophil extracts was observed after preparation with an urea-containing lysis buffer and could be prevented by addition of highly concentrated protease inhibitors. Subsequent tryptic digestion and PolyMAC-based phosphopeptide enrichment proved efficient with accordingly prepared neutrophil samples. Applying the new workflow, formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was detected after gel-free and gel-based phosphoproteomic analyses as proof of principle from 20 ml of whole blood. Furthermore, phosphorylation of other ERK1/2 pathway-associated proteins was monitored. Conclusion: We provide a workflow for efficient, gel-free phosphoproteome analyses with small-sized neutrophil samples, suitable for application in clinical studies.
    No preview · Article · Oct 2015 · Clinica chimica acta; international journal of clinical chemistry
  • Source
    • "Similarly, defective clearance of SGs leads to the pathological accumulation of RNP particles and underlies the pathology of amyotrophic lateral sclerosis, Huntington's disease, frontotemporal lobar degeneration and AD (Harris and Rubinsztein, 2011; Buchan et al., 2013; Koppers et al., 2012; King et al., 2012; Vanderweyde et al., 2012; Waelter et al., 2001; Goggin et al., 2008; Liu-Yesucevitz et al., 2010; Neumann et al., 2006; Menzies et al., 2015). Large scale proteomic screens from our laboratory have identified multiple SG-associated proteins as binding partners and substrates for SYK (Iliuk et al., 2010; Xue et al., 2012; Galan et al., 2011). We found that SYK was recruited to SGs in MCF7 cells when exposed to sodium arsenite or proteasome inhibitors (Krisenko et al., 2015). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1–42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells.
    Full-text · Article · Oct 2015 · EBioMedicine
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Apr 2013
Show more