ISSN 2756-3707
International Journal of Histology and Cytology ISSN 2447-9535 Vol. 4 (5), pp. 318-328, May, 2017. © International Scholars Journals
Full Length Research Paper
Identification of FANCA interacting proteins in mammalian cells using tandem affinity purification and mass spectrometry
Sarah L. Conner and Mu Wang*
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
*Corresponding author. E-mail: [email protected]. Tel: (317) 278-0296. Fax: (317) 278-9739.
Accepted 04 September, 2016
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 gene fanca 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.