"Frontmatter". In: Plant Genomics and Proteomics

Arabidopsis whole genome oligonucleotide array to see whether any “un-
expected” transcripts were found and then to go back and look for their 
distribution and possible function in Arabidopsis. 
An expression profile is a snapshot of the mRNA populations at a given
moment in time. However, it gives no information about the stability of the
RNA or about the translation rates of any given message. Therefore, when-
ever expression profiling is undertaken in a plant tissue it may be necessary
to identify the protein components to maximize the information that can be
gleaned from the expression profiles. The protein information will confirm
the translation of the messages into proteins, as well as potentially identify-
ing those proteins that are subsequently modified, whether for the purpose
of their activation, inactivation, or degradation.
P
ROTEOMICS
The advent of high-throughput technologies has facilitated a more holistic
approach to the study of gene expression, allowing the coordinated charac-
terization of many genes simultaneously, compared with previous studies
that looked at genes or proteins individually (Anderson et al., 2000; Dutt and
Lee, 2000; Lopez, 2000). Proteomics is the systematic analysis of “all” the
individual proteins within a cell or tissue populations at a given time. These
analyses should result in the characterization of all proteins simultaneously,
as well as identifying their interactions. The characterizations should include
the sequences and cellular localization as well as the identification of any
posttranslational modifications and splice variants. The way in which pro-
teins interact within the cell is additional information that will be essential
for our understanding of cell functions. However, as with all the other high-
throughput methodologies, data handling and analysis become critical
issues (Patterson, 2001).
A proteomics experiment essentially consists of four steps:
1 2 0
6. F
U N C T I O N A L
G
E N O M I C S

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∑ Sample preparation
∑ Protein separation 
∑ Identification 
∑ Functional analysis 
High-throughput proteomics techniques used to characterize the protein
complement include:
∑ Two-dimensional gel electrophoresis (2-DE) 
∑ Image analysis
∑ Protein microsequencing
∑ Mass spectrometry
A sample flow of a proteomics experiment is shown in Figures 6.6 and
6.7. These figures illustrate the isolation of the proteins followed by their sep-
aration on 2-DE electrophoresis and the subsequent analysis of the individ-
ual separated proteins. 
The choice of the proteins to be further characterized usually depends
on the question being asked. For example, whenever comparisons are being
made between tissues or treatments the 2-DE results are usually subject to
image analysis and the differences in protein abundance of the various con-
stituents identified (Jacobs et al., 2000). The protein spots can be character-
ized on the basis of peptide mass fingerprints by matrix-assisted laser
desorption ionization-time of flight (MALDI-TOF) and by partial amino acid
sequencing. The information generated is sufficient for protein identification
when coupled with database searches (Tabb et al., 2002; Lin et al., 2003). Ini-
tially, the protein identification was most effective in those cases in which
large EST collections or genome sequences were available, so that the protein
could be identified directly from the mass of the digest-produced peptides.
However, the introduction of methods for the de novo peptide sequencing
has meant that proteomics investigations can now be carried out effectively
on proteins from species for which the nucleic acid sequence databases are
insufficient. The use of both RNA and protein expression characterization
has become an increasingly powerful combination for understanding the
relationship between external perturbations and gene expression, as well as
for identifying gene regulatory regions within the genome. Major technical
challenges in plant proteomics will include the quantitative isolation of pro-
teins for all compartments of the cell, the analysis of low-abundance pro-
teins, the absolute quantification of expressed proteins, and the mapping of
posttranslational modifications.
The disruption of the cell followed by the extraction of all of the proteins
only allows the characterization of the individual protein molecules. Fre-
quently, however, the information that is vitally important is how particular
P
R O T E O M I C S
1 2 1

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1 2 2
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size
pH
Mass Spectrometry
Determine mass
or
Determine amino
acid sequence
Compare with
existing databases
to find matches and
identify proteins
Extract proteins
from the gel. Split
into peptiode
fragments
Low abundance high
Sample 1
Sample 2
FIGURE 6.6.
Pr
oteomics experimental flow
. Isolation of pr
oteins followed by their separation by 2-D gel
electr
ophor
esis. The pattern of the pr
oteins is compar
ed, and those of inter
est ar
e excised, fragmented, and
separated by mass spectr
ometry
. The amino acid composition or sequence is determined (depending on the