FR AB - Science Reference

P101-M
Implementing surface plasmon resonance biosensors in
drug discovery.
D.G. Myszka; Univ. of Utah, 50 N. Medical Dr./School of Medicine Rm
4A417, Salt Lake City, UT 84132
Recent improvements in instrument hardware, experimental design, and
data processing make it possible to utilize surface plasmon resonance (SPR)
biosensor technology in the discovery and development of small-molecule
drugs. The key features of SPR biosensors, real-time binding analysis and lack
of labeling requirements, make this technology suitable for a wide range of
applications. Current instruments have a throughput of �100–400 assays per
day, providing a complement to high-throughput screening. The ability to
collect kinetic data on compounds binding to therapeutic targets yields new
information for lead optimization. Small-molecule analysis and emerging
applications in the areas of ADME and proteomics have SPR biosensors
poised to play a significant role in the pharmaceutical industry.
P103-S
Mapping of protein-protein interactions using mass spectrometry.
D. Figeys, H. Duewel; MDS-Ocata, Toronto, 480 University Avenue
Suite 401, Toronto, Ontario M5G 1V2, Canada
Functional proteomics is becoming the next generation of large-scale proteomic
approaches. It is based on the concept that the function of a protein
is defined by its interactions. Therefore, large-scale approaches for the mapping
of protein-protein interactions at the cellular level will be essential to
the comprehensive understanding of the interactome, i.e. the protein-protein
interactions related to a proteome. Here we will present an approach for the
large-scale screening of the interactome using mass spectrometry. This technology
identifies proteins involved in specific protein-protein interactions
using protein identification by mass spectrometry. We will also discuss the
incorporation in the process of novel technology, such as the ICATtm technology,
for the rapid and relative quantitation of proteins by mass spectrometry.
POSTER ABSTRACTS
ABRF 2001 ABSTRACTS
P102-T
Study of the protein-DNA interaction responsible of the carbon
catabolite repression in Lactobacillus casei.
C.D. Esteban1, K. Mahr2, G. Pérez-Martínez1, W. Hillen2, F. Titgemeyer2; 1Agrochem. and Food Technol. Inst., Burjassot, Spain, Apartado de
correos 73, Burjassot, Valencia 46100 Spain, 2Erlangen-Nürnberg Univ.
In the industrially relevant lactic acid bacterium Lactobacillus casei the preferential
utilization of carbon sources is controlled by the mechanism of carbon
catabolite repression (CCR). As in other low-G�C gram-positive bacteria,
CCR takes place through the binding of the transcriptional repressor,
CcpA, to an operator called cre (catabolite responsive element). CcpA binding
to cre sequences is enhanced by its correpressor HPr-ser46-P.
It was our aim to characterize this protein-DNA interaction by Surface Plasmon
Resonance (SPR). For this purpose CcpA was overexpressed and purified
both with an N-terminal poly-histidine tag and without this tag. HPr was
overexpressed, purified and in vitro phosphorylated. A synthetic biotinylated
double stranded oligonucleotide containing the cre sequence present in the
promoter of the lac operon of L. casei was immobilized on a streptavidin sensor
chip. SPR experiments were performed flowing a range of CcpA (with
and without his tag) concentrations over the chip in the presence and
absence of saturating amounts of HPr-ser46-P. The experimental parameters
for data acquisition were optimized and equilibrium and semiquantitative
kinetic analysis allowed the calculation of KD and estimation of kinetic association
and dissociation rate constants for protein-DNA interaction.
The experimental parameters for the study of this protein-DNA interaction by
SPR have been established. Binding of unmodified CcpA to the cre sequence
was characterized by equilibrium and kinetic rate constants. The presence of
the his tag was shown not to interfere (results with and without his tag are
comparable). In conclusion, we have developed a powerful system to accurately
characterize the intermolecular relationships of histidine tagged CcpA
that could allow efficiently characterizing mutations in CcpA in the near
future.
P104-M
Affinity chromatography and mass spectrometry in dissecting
EGFr signaling interdicted by the quinazoline EGFr inhibitor
OSI-774.
J.D. Haley1, A. Thelemann1, H. Pan1, D. Fenyo2; 1OSI Pharmaceut. Inc.,
106 Charles Lindbergh Blvd., Uniondale, NY 11553, 2Proteometrics LLC
The blockade of EGFr signaling by the quinazoline tyrosine kinase inhibitor
OSI-774 was investigated by direct affinity chromatography, reverse-phase
chromatography and mass finger-printing. Phosphotyrosine complexes from
OSI-774 and control treated HN5 squamous carcinoma cells were prepared
by both Triton X-100 and RIPA lysis. Protein fractionation by SDS-PAGE was
compared with capillary HPLC. Protein fractions were proteolytically digested
with either trypsin, GluC or LysC, desalted by microC18 reverse phase tips
and subjected to matrix assisted laser desorption- time of flight mass spectrometry.
C4 chromatography greatly improved the signal strength and resolution
of the peptide spectra obtained, when compared to direct MALDI MS
of digested immunoaffinity fractions.
Eighty-eight spectra were evaluated from five HN5 phosphotyrosine protein
complex chromatography separations. Mass analysis was performed using a
PerSeptive DE-Pro mass spectrometer using a-cyano-4-hydroxycinnamic acid
or dihydrobenzoic acid matrices. Data were analyzed using RADARS, a SQL
compliant database search engine allowing comparison across experiments
of proteins identified under different experimental conditions (e.g. trypsin vs.
GluC). The predominant protein identified was the epidermal growth factor
receptor (EGFr) which was found in the major C4 HPLC protein fraction.
Phosphorylation on both P1 and P2 C-terminal tyrosines was readily observed
by mass spectrometry. A large number of well known and less defined
proteins which either (1) contain phosphotyrosine or (2) form stable complexes
with targets proteins were identified from multiple experiments. The
Triton X100 lysis and affinity capture methodology allows the identification
of known and novel protein complexes not detected by gel-based techniques.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 213