Abstract

Tandem affinity purification (TAP) allows for the isolation of protein complexes under close-to-physiological conditions for subsequent protein identification by mass spectrometry. Although TAP has been successfully applied to yeast system, there are only a few reports in mammalian cells. In this study, the genefanca was cloned into the commercially available TAP construct from Stratagene and transiently transfected into human embryonic kidney cells (Hek, 293). The FANCA interacting proteins were TAP purified and subsequently identified by tandem mass spectrometry under both mitomycin C (MMC) treated and non-treated conditions. Several novel protein-protein interactions were identified by liquid chromatography (LC) tandem mass spectrometry (MS) under both conditions. The interaction between FANCA and Huntingtin (HTT), which was induced by MMC treatment, was also confirmed by western blot analysis. Although more studies need to be done to determine the biological implications of these interactions, this study provides a useful method for understanding protein functions through identification of protein-protein interactions.

Key words: Tandem affinity purification, fanconi anemia, proteomics, mass spectrometry.