omation mbers - Society for Laboratory Automation and Screening

4:30 pm Wednesday, February 4 Proteomics – Technology 1 Room B1
Philip E. Dawson
The Scripps Research Institute
10550 N. Torrey Pines Road
La Jolla, California 92037
dawson@scripps.edu
Probing Multicomponent Protein Assemblies Using Site-directed Attachment of
Fluorophores and Crosslinking Agents
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Co-Author(s)
John H. Griffin
Jose A. Fernandez
Subramanian Yegneswaran
Enzymes in the blood coagulation pathway are typically composed of a sequence specific peptide cleaving
active site. However, much of the specificity and regulation of these enzymes is dictated by several protein
binding surfaces (exosites) that mediate specific protein:protein interactions. For example, thrombin is specific
for cleavage at Arg residues but gains specificity for cleavage of fibrinogen through interactions in exosite-1.
Fibrinogen cleavage can be blocked by interaction with thrombomodulin that competes for binding to exosite-1.
Similarly, the anitcoagulant leech peptide hirudin binds to the catalytic site and exosite-1 of thrombin. In order
to better understand the relationship between active site and exosite binding between these serine proteases,
we are developing methods to introduce a site specific, covalent label at either the active site or exosites in
a manner that blocks binding and introduces a fluorophore and a chemical crosslinking agent. We have used
this approach to unambiguously label the active site of the serine protease factor Xa with fluorescein and a
benzophenone crosslinking agent. This enabled us to monitor the interaction of this protein with other components
of the prothrombinase complex, an assembly of proteins that activates prothrombin to thrombin. Specifically, the
binding of the substrate prothrombin to factor Xa was directly measured by both fluorescence anisotropy and by
chemical crosslinking. In addition, this binding was increased 50-fold by the addition of the cofactor factor Va. We
are applying this approach to the structural and functional characterization of the large multiprotein assemblies
involved in blood coagulation.
5:00 pm Wednesday, February 4 Proteomics – Technology 1 Room B1
Travis Boone
ACLARA BioSciences, Inc.
1288 Pear Avenue
Mountain View, California 94043
tboone@aclara.com
Co-Author(s)
Tina Tian, Po-Ying Chan-Hui, Yuping Tan,
Yining Shi,
Hossein Salimi-Moosavi, Kathryn Stephens,
Lilly Chen, Sharat Singh
A Systems Biology Approach to Tracking Protein/Gene Expression and Interactions in
Oncology and Toxicology Using the eTag Assay System
The eTag Assay System enables the precise, simultaneous quantitation of tens of cellular pathway or clinical
biomarkers across thousands of samples, providing high-value information about the expression and interaction
of proteins and genes within cells. The eTag Assay System is a homogeneous method to analyze proteins and/
or mRNAs directly from tissue samples, whole cells, or cell lysates. The multiplexing aspect of the technology is
derived from the use of eTag reporters, which are fluorescent, biologically compatible labels that have distinct
electrophoretic mobilities and can be separated from one another using capillary electrophoresis. The eTag
reporters can be conjugated to a wide variety of biological probes including oligonucleotides, antibodies, proteins
and peptides and are released in reaction as a consequence of a target-specific binding event. Mixtures of
released eTag reporters are efficiently separated and sensitively detected using commercial capillary array DNA
sequencing systems and accurately identified and quantified using proprietary, user-friendly software. The eTag
Assay System has been employed in a systems biology approach to study gene and protein expression, cell
signaling and pathway activation, and protein-protein interactions in oncology and toxicology applications. Results
will be presented tracking receptor dimerization and signaling pathway phosphoproteins in breast cancer cell
lines, as well as identifying markers in tissue samples. Data demonstrating simultaneous protein/gene profiling for
toxicology screens on rat hepatocytes, across a set of drug compounds, will also be shown.
PODIUM ABSTRACTS