Application of On-Line nanoLC-IT-TOF in the Identification of Serum β-Catenin Complex in Mice Scald Model

Abstract

Severe burn shock remains an unresolved clinical problem with an urgent need to explore novel therapeutic treatments. Intracellular β-catenin, through interaction with other proteins, has been reported to be able to regulate the size of cutaneous wounds. Higher expression of β-catenin is associated with larger sized wounds. However, the identification of serum β-catenin complex is difficult and has been rarely reported. The exploitation of more binding partners can contribute to uncovering the exact mechanisms behind serum β-catenin mediated biological effects. Here, we describe a method that consists of immunoprecipitation, SDS-PAGE, in-gel digestion, and nanoLC coupled to LCMS-IT-TOF for the investigation of serum β-catenin complex in mice scald model. Among selected gel bands obtained from the protein gels, a total of 31 peptides were identified and sequenced with high statistical significance (p<0.01). Three proteins (alpha-2-marcoglobulin, serine protease inhibitor A3K, and serine protease inhibitor A1A) were identified and validated with high reliability and high reproducibility. It was inferred that these proteins might interact with serum β-catenin, which could affect the wound healing resulting from burn shock. Our study demonstrated that the on-line coupling of nano-LC with a LCMS-IT-TOF mass spectrometer was capable of sensitive and automated characterization of the serum β-catenin complex in mice scald model. © 2012 Huang et al.