Phosphokinase antibody arrays on dendron-coated surface

PLoS One. 2014 May 6;9(5):e96456. doi: 10.1371/journal.pone.0096456. eCollection 2014.

Abstract

Monitoring protein phosphorylation at the cellular level is important to understand the intracellular signaling. Among the phosphoproteomics methods, phosphokinase antibody arrays have emerged as preferred tools to measure well-characterized phosphorylation in the intracellular signaling. Here, we present a dendron-coated phosphokinase antibody array (DPA) in which the antibodies are immobilized on a dendron-coated glass slide. Self-assembly of conically shaped dendrons well-controlled in size and structure resulted in precisely controlled lateral spacing between the immobilized phosphosite-specific antibodies, leading to minimized steric hindrance and improved antigen-antibody binding kinetics. These features increased sensitivity, selectivity, and reproducibility in measured amounts of protein phosphorylation. To demonstrate the utility of the DPA, we generated the phosphorylation profiles of brain tissue samples obtained from Alzheimer's disease (AD) model mice. The analysis of the profiles revealed signaling pathways deregulated during the course of AD progression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alzheimer Disease / immunology
  • Alzheimer Disease / pathology
  • Animals
  • Anthracenes / chemistry*
  • Antibodies, Immobilized / immunology*
  • Antigen-Antibody Reactions / immunology*
  • Brain / immunology
  • Brain / pathology
  • Dendrimers / chemistry
  • Disease Models, Animal
  • Humans
  • Immunoassay / methods
  • Male
  • Mice
  • Mice, Transgenic / immunology
  • Phosphorylation / immunology*
  • Phosphotransferases / immunology*
  • Signal Transduction / immunology

Substances

  • Anthracenes
  • Antibodies, Immobilized
  • Dendrimers
  • dendron
  • Phosphotransferases

Grant support

This study was supported by Proteogenomics Project, NRF-2013-003102, NRF-2013M3C7A1069644, and the Institute for Basic Science (CA1308) grants from Korean MEST, POSCO research fund (Project NO. 2013Y008), and 10182KFDA992 and 12182KFDA651 grants from Korean FDA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.