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: 7.25). 10/2010; 9(10):2162-72. DOI: 10.1074/mcp.M110.000091
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Current sample preparation protocols for quantitative phosphoproteome analysis are tedious and time-consuming. Here, a facile in-situ sample processing approach (iSPA) is developed by using the macroporous Ti(IV)-IMAC microspheres as the preparation "beds", where all sample preparation procedures including the enrichment of phosphoproteins, tryptic digestion of proteins, enrichment and isotope labeling of phosphopeptides are performed in-situ sequentially. As a result of the in-situ processing design and the seamless procedures, extra steps for desalting and buffer exchanging, which are always required in conventional approaches, are avoided and the sample loss and contamination could be greatly reduced. Thus better sensitivity and accuracy for the quantitative analysis of phosphoproteome were obtained. This strategy was further applied to differential phosphoproteome analysis of human liver tissues with or without hepatocellular carcinoma (HCC). Totally 8548 phosphorylation sites were confidently quantified from three replicate analysis of 0.5 mg of human liver protein extracts.
    Journal of Proteome Research 07/2014; 13(9). DOI:10.1021/pr500454g · 5.00 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regulation of synaptic plasticity and homeostasis. At the molecular level, MeCP2 is described as a repressor capable of inhibiting gene transcription through chromatin compaction. Indeed, it interacts with several chromatin remodeling factors, such as HDAC-containing complexes and ATRX. Other studies have inferred that MeCP2 functions also as an activator; a role in regulating mRNA splicing and in modulating protein synthesis has also been proposed. Further, MeCP2 avidly binds both 5-methyl- and 5-hydroxymethyl-cytosine. Recent evidence suggests that it is the highly disorganized structure of MeCP2, together with its post-translational modifications (PTMs) that generate and regulate this functional versatility. Indeed, several reports have demonstrated that differential phosphorylation of MeCP2 is a key mechanism by which the methyl binding protein modulates its affinity for its partners, gene expression and cellular adaptations to stimuli and neuronal plasticity. As logic consequence, generation of phospho-defective Mecp2 knock-in mice has permitted associating alterations in neuronal morphology, circuit formation, and mouse behavioral phenotypes with specific phosphorylation events. MeCP2 undergoes various other PTMs, including acetylation, ubiquitination and sumoylation, whose functional roles remain largely unexplored. These results, together with the genome-wide distribution of MeCP2 and its capability to substitute histone H1, recall the complex regulation of histones and suggest the relevance of quickly gaining a deeper comprehension of MeCP2 PTMs, the respective writers and readers and the consequent functional outcomes.
    Frontiers in Cellular Neuroscience 08/2014; 8:236. DOI:10.3389/fncel.2014.00236 · 4.18 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.
    Molecular &amp Cellular Proteomics 07/2014; DOI:10.1074/mcp.O114.038588 · 7.25 Impact Factor