PNNL-13501 - Pacific Northwest National Laboratory

cellular processing (such as post-translational modifications).
Protein modifications, such as phosphorylation,
can modulate protein function and are essential to the
understanding of regulatory mechanisms. Changes in the
proteome occur, for example, when environmental
conditions change, upon exposure to different chemicals
and ionizing radiation, during the cell cycle, with the
onset of disease states and in the “normal” aging process.
The biological processes involved in cell signaling,
transcription, regulation, responses to stresses, etc., are
elements of the complex linkages that determine system
properties. The capability to measure such changes in the
proteome with good precision is important for
understanding these complex systems and the functions of
their individual components.
At present, no rapid and sensitive technique exists for
large-scale proteome studies. The study of proteins on a
one-at-a-time basis, as conventionally practiced, provides
an implausibly slow route to understanding cellular
processes dictated by the expected 50,000 to 100,000
gene products expected from the human genome, and the
additional 10- to 100-fold complexity introduced by their
various modified forms. Beyond such considerations, it is
generally accepted that such reductionist approaches are
unlikely to provide insights into systems-level properties.
The closest comparable technology, two-dimensional
polyacrylamide gel electrophoresis (2-D PAGE), is a
slow, labor intensive, and cumbersome technology.
The most common mechanism by which cells propagate
signals via protein pathways and networks is
phosphorylation. Studies estimate that as many as onethird
of all cellular proteins derived from mammalian
cells are phosphorylated. Cellular processes ranging from
differentiation, development, cell division, peptide
hormone response, and protein kinase activation are all
regulated via phosphorylation. The large number of
phosphorylated proteins requires methods that are able to
rapidly identify proteins that undergo phosphorylation.
The predominant method of studying protein
phosphorylation is to label the proteins using radioactive
phosphorous-32-labeled inorganic phosphate. The use of
32P (inorganic) to label proteins does not lend itself to
high-throughput proteome-wide analysis due to the
problems associated with the handling of radioactive
compounds and the radioactive contamination of
instrumentation. In addition, while immuno- and metalaffinity
columns have been used to enrich mixtures for
phosphopeptides, these strategies result in the isolation of
many non-phosphorylated peptides through non-specific
interactions complicating the analysis by introducing
uncertainty about the nature of each peptide.
Approach
One objective of this project is to demonstrate the
detection and identification of large numbers of proteins
from yeast (or other organisms for which a full genomic
sequence is available) and new stable isotope labeling
methods to provide precise levels of expression of all
proteins for eukaryotic proteomes. This approach will
provide a systems-level view, with greatly improved
sensitivity and high-precision quantitation of protein
expression in cells, and the basis for new understandings
in cell signaling and protein function. The information
obtained would also provide essential input for modeling
studies aimed at simulations of cells. In the second year
of this project, we will continue development of the new
proteome expression measurement technology described
below and undertake the initial developments necessary
for its extension and application to mammalian tissues.
We will use these approaches to demonstrate new
methods for 1) the proteome-wide identification and
quantitation of phosphorylated proteins (highly relevant to
signal transduction), and 2) for the determination of
protein-protein interactions. The project efforts will also
include the visualization tools necessary to display and
interpret the extremely large and complicated data sets, so
as to enable the study and understanding of complex
networks and pathways associated with cellular responses
at the systems level. An essential part of the data analysis
tools to be developed will be those necessary for mining
the data to extract information on protein modifications,
unexpected modes of protein expression (recoding) and
sequence frame shifts and other biological insights.
Our Laboratory is presently developing new experimental
capabilities that provide major advances for proteomics.
This new technology is based upon capillary
electrophoresis separations in conjunction with direct
mass spectrometric analysis and thus, avoids the laborintensive
process of individual spot excision and analysis
as well as the time-consuming gel-based separation.
Unlike 2-D PAGE separations that yield protein “spots”
that must still be individually analyzed, our approach
provides simultaneous information on expression level,
protein identification, and protein modifications.
However, new challenges for effective data analysis arise
due to the quantity of information from each proteome
measurement. Data analysis and effective use of the
information presently constitutes an enormous bottleneck
to the use of the “flood of data” that can now be
generated. A complete two-dimensional capillary
electrophoresis Fourier-transform ion cycloton resonance
proteome analysis is projected to include a total of
perhaps 1,000 mass spectra having generally high
complexity, and amounting to approximately 1 to 10
Biosciences and Biotechnology 61