PNNL-13501 - Pacific Northwest National Laboratory

8. Initial demonstration of an off-line approach for twodimensional
capillary isoelectric focusing and
capillary high-performance liquid chromatography
separations combined with ESI-FTICR mass
spectrometry for the rapid and ultrasensitive analysis
of the array of proteins expressed by cells.
9. Demonstration of methods for the identification of
large numbers of proteins from genomic databases
based upon the use of accurate mass tags obtained
from the high mass measurement accuracy provided
by Fourier-transform ion cyclotron resonance.
10. Demonstration of methods for precise determination
of relative levels of protein expression for all proteins
from the same measurement used for identification.
The project also includes the development of visualization
tools for effectively displaying multiple, hyperdimensional,
feature-rich data sets to allow one to rapidly
derive meaning from the often small differences in
expressed proteomes that are the result of environmental
perturbations. Significant developments have been made
in many areas needed to visualize and present the data,
but constant development is needed to keep pace with the
ever growing field on bioinformatics. We have and are
continuing to develop the tools to link that information to
repositories of biological data for deriving information
related to the function(s) of specific proteins.
Developments to aid data analysis have included methods
for visualization of relative protein expression rates
(systems-wide differential or comparative displays of the
results from perturbed proteomes). Important also to
future efforts will be the automation of protein
identification and the linkage to web databases that will
provide information on the function of known proteins.
Thus, the linkages we envision will provide the basis for
gleaning biological function of new genes and for
providing greatly improved understanding of the complex
networks and pathways that constitute cellular systems.
Summary and Conclusions
The ability to make high-throughput measurements of
proteomes will have enormous and immediate impacts on
broad areas of biological and biomedical research. One of
the key proteomic technologies will be the capability to
study entire proteomes and to directly probe the effects of
environmental perturbations and disease states, providing
the basis for inference of function of individual proteins
as well as new systems-level understandings.
Publications
Belov ME, MV Gorshkov, HR Udseth, GA Anderson,
and RD Smith. 2000. “Zeptomole-sensitivity
electrospray ionization Fourier transform ion cyclotron
resonance mass spectrometry of proteins.” Anal. Chem.
72:2271-2279.
Bruce JE, GA Anderson, MD Brands, L Pasa-Tolic, and
RD Smith. 2000. “Obtaining more accurate FTICR mass
measurements without internal standards using multiply
charged ions.” J. Amer. Soc. Mass Spectrom. 11:416-421.
Conrads TP, GA Anderson, TD Veenstra, L Pasa-Tolic,
and RD Smith. 2000. “Utility of accurate mass tags for
proteome-wide protein identification.” Anal. Chem.
72:3349-3354.
Gao H, Y Shen, TD Veenstra, R Harkewicz, GA
Anderson, JE Bruce, L Pasa-Tolic, and RD Smith. 2000.
“Two dimensional electrophoretic/chromatographic
separations combined with electrospray ionization-FTICR
mass spectrometry for high throughput proteome
analysis.” J. Microcolumn Separations 12:383-390.
Masselon C, GA Anderson, R Harkewicz, JE Bruce,
L Pasa-Tolic, and RD Smith. 2000. “Accurate mass
multiplexed tandem mass spectrometry for highthroughput
polypeptide identification from mixtures.”
Anal. Chem. 72:1918-1924.
Shen Y and RD Smith. 2000. “High-resolution capillary
isoelectric focusing of proteins using highly hydrophilicsubstituted
cellulose-coated capillaries.” J. Microcolumn
Separations 12:135-141.
Shen Y, SJ Berger, GA Anderson, and RD Smith. 2000.
“High-efficiency capillary isoelectric focusing of
peptides.” Anal. Chem. 72:2154-2159.
Shen Y, SJ Berger, and RD Smith. “Capillary isoelectric
focusing of yeast cells.” Anal. Chem. (in press).
Zhang C-X, F Xiang, L Pasa-Tolic, GA Anderson,
TD Veenstra, and RD Smith. 2000. “Stepwise
mobilization of focused proteins in capillary isoelectric
focusing mass spectrometry.” Anal. Chem. 72:1462-1468.
Biosciences and Biotechnology 63