Physiology and Molecular Biology of Stress ... - KHAM PHA MOI

Functional Genomics of Stress Tolerance
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ions according to their masses. MALDI-TOF (matrix-assisted laser desorption/ionization-time
of flight) is one such source that generates peptide fingerprints resulting from
digestion of protein with proteases, trypsin being the most commonly used protease.
The mass spectrum of eluted peptide mixture results in ‘peptide mass fingerprints’
which can be compared with the predicted sequences from genomes or cDNA available
in databases (Henzel et al., 1993). MALDI-TOF identification procedure may not be
appropriate in cases where full-length genes are not sequenced. Another source, electro
spray ionization (ESI) provides more information via fragmentation of individual peptides
(Pandey and Mann, 2000). The peptide ions are fragmented by collision to isolate
one species at a time producing different amino and carboxy terminal fragments. Peptide
masses are compared to available databases and analyzed. This gives more specific
information than MALDI fingerprinting. It not only provides information for protein
sequence databases but also for nucleotide sequences such as EST databases (Kuster
et al., 2001). In Arabidopsis, nuclear proteome has been comprehensively characterized
in response to cold stress using 2D gel electrophoresis and MALDI-TOF-MS (Bae et
al., 2003). Out of 184 protein spots, 40 proteins were induced and 14 repressed in
response to cold stress by more than two folds. Many of them were already known to
be involved in stress, including heat-shock proteins, transcription factors (AtMYB2
and OBF4), DNA binding proteins (DRT102 and Dr1), catalytic enzymes, syntaxin,
calmodulin, and germin-like proteins. Similar study of proteome analysis in wheat grains
following heat stress identified a total of 37 proteins which were significantly changed
by heat treatments (Majoul et al., 2003). Costa et al. (1998) in a proteomic study of
drought stress in maritime pine quantified about 1000 proteins and found 38 were stress
responsive. Similarly, in rice, out of >1000 quantified proteins from stressed and well
watered rice leaves, 42 proteins were found responsive to stress (Salekdeh et al., 2002).
Eight proteins showing reversible behavior in leaves under drought stress were identified
by tryptic fingerprinting with MALDI-TOF MS and partially sequenced with ESI-
Q-TOF MS/MS. There are several examples where proteins associated with various
processes affecting response to stress conditions have been identified, e.g. maize
(Riccardi et al., 1998) and wild watermelon (Kawasaki et al., 2000).
Protein chips are particularly useful for assessing and analyzing protein diversity
on a large scale including hydrophobic and large proteins that are difficult to
visualize by 2D method. This approach is based on use of arrays for proteome analysis.
The surface of protein chips can be immobilized with antibodies or recombinant proteins
into an array format (Lueking et al., 1999). The protein chip is probed with a sample
of interest. Different fluorescently labeled cell lysates can be added and proteins bound
to chip surface are detected by a change in colour. The bound proteins can be eluted
and identified by mass spectrometry. Alternatively, instead of cell lysates, phage cDNA
display library can be added to the chip. Phage display method expresses the protein of
interest fused to coat protein of bacteriophage (Li, 2000). Immobilized proteins of the
chip can capture proteins displayed on phage particles. The cDNA of interest expressing
the desired protein can be sequenced to know about the interacting proteins. In