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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:
muwang@indiana.edu.
Tel: (317) 278-0296. Fax: (317) 278-9739.
Accepted
28 March, 2008 |
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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. |
