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

Freezing Stress
141
(Barnett et al., 1980; Key et al., 1981). Unlike the heat shock response, CA material
continues to accumulate ‘housekeeping’ proteins (Marmiroli et al., 1986; Yacoob and
Filion, 1986, 1996a; Guy and Haskell, 1987; Gilmour et al., 1988; Guy et al., 1988; Perras
and Sarhan, 1989), as CA is a process that requires energy and continued metabolism
(Guy and Haskell, 1987).
With the discovery of new molecular techniques and the improvement of
existing techniques, changes in gene expression were commonly quantified via the
detection of mRNA levels. Recent evidence has shown that transcript expression data
does not always correlate with protein accumulation patterns (Whitelegge, 2002). In
order to accurately identify and quantify changes with in a cell or organism it is now
believed that one must surpass the transcript and look directly at the protein level. The
systematic analysis of proteins present within the plant at any given period is referred
to as proteomics (van Wijk, 2001). Proteomics is currently the leading technology for
the high throughput analysis of protein accumulation patterns. Applications of
proteomic analysis include the identification of proteins that accumulate within a cell or
organism, the analysis of post translational modification of proteins and the interaction
of proteins within a living system (protein-protein interactions)(Whitelegge, 2002). The
mass identification of proteins, commonly referred to as the ‘Brute Force’ method most
commonly occurs via two dimensional electrophoresis (2DE), which uses immobilized
pH gradient strips to separate proteins in the first dimension (based on pI) and SDS-
PAGE in the second dimension to separate proteins based on molecular weight (MW)
(Blackstock and Mann, 2000). Following separation, gels are typically analyzed with
the assistance of computer programs for changes in accumulation patterns, and differentially
accumulated proteins are robotically isolated from the gel. Isolated proteins are
then digested (typically via trypsin) into peptide fragments and the masses are determined
by LC MS/MS (Blackstock and Mann, 2000). Once masses data are obtained,
they are compared to databases and subsequently identified. This method of protein
identification has only recently become efficient, as a result of the large quantities of
sequence data generated by early generation genomics programs (Rowley et al., 2000).
As with all highly technical analysis, 2D-PAGE does possess limitations that involved
problems with dynamic resolution (Blackstock and Mann, 2000), quantification and
identification of low abundance and hydrophobic proteins (van Wijk, 2001). To reduce
the problems associated with 2DE, a new technique that involves differentially labeling
protein samples with fluorescent dyes has been developed (Amersham Biosciences).
This technique, referred to as DIGE, uses the same principles as 2D-PAGE except multiple
protein samples (labeled with different florescent dyes) are analyzed within one
gel. By analyzing all samples within the same gel, gel to gel variation is eliminated
resulting in increased quantification and dynamic range accuracy (Swatton et al., 2004).
A different high throughput method for protein isolation and quantification
requires the utilization of multidimensional separation methods, that are different but
complimentary to those used in 2D-PAGE (pI and MW). This method is referred to as
multi-dimensional protein identification technology (MudPIT) and it generates pep-