Functional analysis of the nuclear proteome of human A549 alveolar epithelial cells by HPLC‐high resolution 2‐D gel electrophoresis

Abstract

The airway epithelial cell plays a central role in coordinating airway inflammatory responses, where significant changes in the proteome occur in response to infectious stimuli. To further understand the spectrum of proteins and the biological processes they control, we have initially determined the nuclear proteome of human type II-like alveolar epithelial cells (A549) using a sequential method of organellar enrichment followed by HPLC prefractionation prior to 2-DE-based protein identification using MALDI-TOF MS. This approach yielded 719 high-confidence identifications, 433 mapping to unique gene identifiers. Expert classification showed that these proteins controlled chromatin remodeling, protein refolding, cytoskeletal structure, membrane function, metabolic processes, mitochondrial function, RNA binding, protein synthesis, signaling, and transcription factor activities. The proteins were mapped to gene ontology classifications, where metabolism and catalytic activity functions were significantly enriched, representing 43 and 32% of the protein set, respectively. Pathways analysis indicated a protein network affecting tumor necrosis factor-nuclear factor-kappaB signaling pathway interacting with intermediate cytoskeletal filaments. Forty-five proteins of unknown function were subjected to domain analysis and inferred to have additional nuclear functions controlling purine nucleotide metabolism and protein-protein interactions. This database represents the most comprehensive data set of mammalian nuclear proteins and will serve as a foundation for further discovery.