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Proteomics and Protein Analysis Core B

Proteomics and Protein Analysis Core B

Core Director:  Mamuka
Kvaratskhelia, PhD
, The Ohio State University

The Specific Aims of Core B are:

The Aim 1. To perform
MS-based protein footprinting experiments.

This
technology will enable identification of contact amino acids in the context of
large biologically relevant nucleoprotein and protein-protein complexes. This
information will help individual projects to rationally design site directed
mutants and test the functional importance of the interacting interfaces.

 Aim 2. To develop novel
applications of the EnSpire multimode plate reader.

This will allow for
biochemical characterization of protein-nucleic acid and protein-protein interactions
using homogeneous time-resolved fluorescence energy transfer (HTRF), AlphaLISA,
and AlphaScreen (ALPHA) based technologies. A rapid and sensitive
mix-and-measure format will allow us to monitor formation of virtually any
biological complexes from small peptide or oligonucleotide interactions to
complexes as large as 200 nm in size. Furthermore, these approaches will allow
us to determine binding affinities for low or high affinity protein-protein and
protein-nucleic acid interactions.

Aim 3. To provide a column
chromatography service using AKTA-FPLC.

Core B will
assist individual PPG projects to isolate low abundance native protein-protein
complexes from mammalian cells. Furthermore, we will purify milligram
quantities of poorly soluble recombinant proteins from E. coli for their
subsequent biochemical and biophysical characterization. These protein
preparations will also be provided to Core
C
to generate antibodies.

 Aim 4. To conduct MS-based
proteomics experiments to identify interacting partners and protein posttranslational
modifications.

We
have established very productive and highly cost effective collaboration with
the proteomic core of OSUCCC to assist individual PPG projects with proteomic
studies. Core B personnel have
combined expertise in virology and protein chemistry, which enable us to
optimally design the proteomic experiments and prepare high quality samples.
Furthermore, Core B will devote
significant effort helping individual PPG projects to interpret the proteomic
results and optimally select the hits for subsequent validation. These skills
and efforts are highly complementary to the LC-MS/MS capabilities available at
the proteomic core of OSUCCC and are essential to effectively accomplish the
aims of the individual PPG projects.

 Taken together, Core B will provide a wide range of
protein analysis services. Importantly, these services will be utilized by all
four PPG projects.

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