Identifying transient protein-protein interactions in EphB2 signaling by Blue Native PAGE and Mass Spectrometry

Kimmel Center for Biology and Medicine at the Skirball Institute, New York, NY, USA.
Proteomics (Impact Factor: 3.81). 12/2011; 11(23):4514-28. DOI: 10.1002/pmic.201000819
Source: PubMed


Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling.


Available from: Costel Darie, Jan 04, 2014
  • Source
    • "Proteomics is an emerging field that focuses on the study of proteins. The workhorse of proteomic research is largely mass spectrometry (MS), which can be used to provide unbiased assessment of the protein components of a biological sample [1] [2] [3] [4] [5] [6] [7] [8] [9]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Biomarkers are greatly needed in the fields of neurology and psychiatry, to provide objective and earlier diagnoses of CNS conditions. Proteomics and other omic mass-spectrometry-based technologies are tools currently being utilized in much recent CNS research. Saliva is an interesting alternative biomaterial for the proteomic study of CNS disorders, with several advantages. Collection is non-invasive and saliva has many proteins. It is easier to collect than blood and can be collected by professionals without formal medical training. For psychiatric and neurological patients, supplying a saliva sample is less anxiety-provoking than providing a blood sample, and is less embarrassing than producing a urine specimen. The use of saliva as a biomaterial has been researched for the diagnosis of and greater understanding of several CNS conditions, including neurodegenerative diseases, autism and depression. Salivary biomarkers could be used to rule out non-psychiatric conditions that are often mistaken for psychiatric/neurological conditions, such as fibromyalgia, and potentially to assess cognitive ability in individuals with compromised brain function. As mass spectrometry and omics technology advances, the sensitivity and utility of assessing CNS conditions using distal human biomaterials such as saliva is becoming increasingly possible. This article is protected by copyright. All rights reserved.
    PROTEOMICS - CLINICAL APPLICATIONS 01/2015; DOI:10.1002/prca.201400153 · 2.96 Impact Factor
  • Source
    • "The samples were analyzed by LC-MS/MS using a NanoAcuity UPLC (Micromass/Waters, Milford, MA) coupled to a Q-TOF Micro MS (Micromass/Waters, Milford, MA) [22], [25], [26]. The peptides were loaded onto a 100 µm×10 mm nanoAquity BEH130 C18 1.7 µm UPLC column (Waters, Milford, MA) and eluted over a 150 min gradient of 2–80% organic solvent (ACN containing 0.1% FA) at a flow rate of 400 nL/min. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.
    PLoS ONE 08/2013; 8(8):e71859. DOI:10.1371/journal.pone.0071859 · 3.23 Impact Factor
  • Source
    • "The resulting peptide mixture was analyzed by reverse phase liquid chromatography (LC) and MS (LC–MS/MS) using a NanoAquity UPLC coupled directly to a Q-Tof Premier MS or Q-Tof Micro MS (Waters, Milford, MA). The procedure used was previously described [3] [7] [22] [23]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.
    Biochimica et Biophysica Acta 04/2013; 1834(8). DOI:10.1016/j.bbapap.2013.04.022 · 4.66 Impact Factor
Show more