Discovery of a Hidden Proinflammatory Signaling Proteome Using a Large-Scale, Targeted Antibody Microarray Platform

Dynamic post-translational processes regulate protein expression in eukaryotic cells. However, the processes are difficult to assess on a proteomic scale because protein levels actually reflect the sum of individual biosynthesis and degradation rates. These rates are presently hidden from the conventional proteomic technologies. We present here a novel and dynamic, antibody microarray-based time-resolved approach to simultaneously measure not only the total protein changes but also the rates of biosynthesis of low abundance proteins in the proteome of lung epithelial cells. In this chapter, we describe the feasibility of this technique by investigating the complete proteomic kinetics of 507 low abundance proteins in cultured cystic fibrosis (CF) lung epithelial cells using ³⁵[S] methionine or ³²[P] and the consequences of repair by gene therapy with [wildtype] CFTR. This novel antibody microarray-based technology identifies relevant, hidden proteins whose regulation by the CF genotype would never have been detected by simple measurements of total proteomic masses.