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

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R.G. Trischuk, B.S. Schilling, M. Wisniewski and L.V. Gusta
tides from complex protein mixtures (whole cell) that have been first separated in the
first dimension by a strong cation exchange column followed by reverse phase chromatography
(RPC) in the second dimension. Throughout the process RPC fractions containing
peptides with different chemical characteristics are collected and fed directly
into a MS. Due to the direct link of the PRC to the MS, this system provides a very
efficient high throughput method of protein isolation. As a result it is possible to detect
and quantify low abundance proteins that would be difficult via 2D-PAGE. Although
MudPIT provides a more efficient method for the mass identification of proteins compared
to 2D-PAGE, it also possesses limitations. The first limitation is that this process
of protein identification and quantification is sequence dependent (van Wijk, 2001).
Labeling of proteins for ICAT ion exchange requires a cysteine residue, which is not
universally present in all proteins (e.g. myoglobin). As a result a certain quantity of
peptides are not labeled and are therefore missed. The opposite effect can occur in
peptides that have large numbers of cysteine residues present in their sequence, as
proteins possessing too many labels are difficult to quantify (Blackstock and Mann,
2000). Another major limiting factor of MudPIT is that it is not capable of differentiating
between modified forms of the same protein (e.g. post translational modifications)(Gygi
et al., 1999). In instances where post translational modifications have been made to a
protein, there is now way to visualize this with MudPIT as is possible in gel electrophoresis
To date there are few published results on high throughput proteomic analysis
on plant material that has been exposed to low temperature. In one study 2D-PAGE
was utilized to isolated nuclear proteins from the nuclei of Arabidopsis that were exposed
to low temperature (Bae et al., 2003). Out of a total of 184 proteins identified in the
nuclear proteome, 54 were found to increase a minimum of two-fold in response to the
cold. Of these 54 proteins a few notables included a 60s ribosomal protein, a transcriptional
regulator, a DEAD box RNA helicase and an HSP-70. In another study, utilizing
2D-PAGE, a wide spread proteomic analysis of Brassica napus has revealed that approximately
100 proteins accumulate or disappear at some point in the cold acclimation
process in a winter and a spring variety of canola (Trischuk and Gusta, unpublished
results). Some of the more interesting proteins identified included carbohydrate metabolism
enzymes, free radical scavenging enzymes as well as many proteins that possessed
homology to late embryogenesis abundant (LEA) proteins which have been
associated with the development of freezing tolerance. Another interesting discovery
from this research is that the set of proteins that accumulate upon initial exposure to LT
(LT 50
-7°C) are very different than those that are present once the plants have become
fully acclimated (LT 50
-15°C).
Despite the lack of large scale proteomics data on cold treated plant material,
a great deal of work on proteins has been accomplished. To date thousands of publications
have been written, listing a tremendous variety of proteins that are induced by LT
or involved in the cold acclimation process. The majority of the proteins identified fall
into a few distinct classes based on function. The first major class are the LEA-D11