PNNL-13501 - Pacific Northwest National Laboratory

Figure 2. Hybridization of a cy3-labeled control RNA to a
microarray with 1920 cDNA fragment to reveal immobilized
polymerase chain reaction product
analysis of peptide fragments that are representative of the
complete protein sequence. To improve these methods,
we developed a combination of protein digestion and gel
extraction procedures that provide >95% coverage of a
test protein. These procedures should be suitable for
determining protein modifications with a high degree of
confidence.
Protein Arrays
A major goal of this task is the development of protein
arrays that will allow for the rapid identification and
characterization of a large number of proteins involved in
cell signaling pathways important in the genesis of
ovarian cancer. In order to accomplish this, we are
developing arrays of antibodies that will selectively bind
cell-signaling proteins. Bound proteins will be evaluated
using sophisticated mass spectrometric methods. This
coupling of protein array technology with advanced mass
spectrometric detection provides a high-throughput
approach and a means to identify and characterize
biologically important changes in the proteome
(Figure 4).
We have developed the initial methodologies necessary
for the attachment of antibodies to a solid support that are
compatible with mass spectrometry. Currently two
different attachment chemistries have been employed. In
both cases, a self-assembled monolayer is generated on
gold, a surface that is preferred for MALDI-MS. The
antibody is then covalently attached to the self-assembled
monolayer. The first self-assembled monolayer we tried,
dithiobis(succinimidyl propionate), is an amine-reactive,
50 FY 2000 Laboratory Directed Research and Development Annual Report
homobifunctional cross-linker that covalently attaches the
antibodies to the gold surface. We have also achieved
good protein attachment using an alkanethiolated selfassembled
monolayer. For this attachment strategy, a
zero-length carbodiimide linker is employed to covalently
link the terminal carboxylic acid group of the selfassembled
monolayer with a primary amine from the
protein. Additionally, we developed the necessary
methods to analyze the different attachment chemistries
both to monitor the attachment of the self-assembled
monolayer and antibody and to address the functionality
of the immobilized antibody. We are currently using
these methods to optimize and extend our attachment
strategies.
Summary and Conclusions
Technologies to characterize the relationship between
signal transduction, environmental exposures, and human
diseases are being implemented. These include
• cDNA microarrays
• antibodies to capture specific proteins
• protein characterization by mass spectrometry
• protein identification using bioinformatics programs.
References
Hobson et al. 2000. “Signal transduction mechanisms
linking increased extracellular calcium to proliferation in
ovarian surface epithelial cells.” Exp. Cell Res. 258:1-11.
McNeil et al. 1998. “Functional calcium-sensing
receptor expression in ovarian surface epithelial cells.”
Amer. J. Obstet. Gynec. 178:305-313.
Wong KK, RS Cheng, and SC Mok. “Identification of
differentially over expressed genes in ovarian cancer cells
by MICROMAX cDNA microarray.” BioTechniques (in
press).
Publications and Presentations
Wong KK, RS Cheng, and SC Mok. “Identification of
differentially over expressed genes in ovarian cancer cells
by MICROMAX cDNA microarray.” BioTechniques (in
press).
Zangar RC, KL Wahl, CE Petersen, RD Rodland, DL
Springer. 2000. “Protein primary structure determined
by protease digestion and MALDI-MS peptide analysis.”
To be presented at Hanford Symposium, Richland,
Washington.