| In the integrated CIEF/CRPLC approach,
peptides are systematically resolved by their differences
in pI and hydrophobicity. This is evidenced by the correlation
of the peptide pI value versus the CIEF fraction number
(from acidic to basic pIs). By using pharmalyte 3-10,
we are able to identify the peptides over a wide pH range
of at least 3.8-10.2. The pI range analyzed in this study
is comparable to that reported using strong cation exchange
chromatography. However, the degree of pI overlapping
in CIEF fractions is drastically lower than that in strong
cation exchange chromatography using a salt gradient at
acidic pH. For example, the percentage of identified peptides
present in more than one CIEF fraction is around 10-25%
and significantly less than 40-80% obtained from multidimensional
LC using strong cation exchange coupled with reversed-phase
separations.
A total of 1156 unique proteins and 1866 unique peptides
are identified in the soluble fraction of yeast cell
lysates. By using only the soluble fraction, our studies
already demonstrate the capabilities of CIEF-based multidimensional
separation technology for identifying a larger number
of soluble yeast proteins than other techniques reported
in the literature. Furthermore, the distribution of
codon adaptation index value for identified yeast proteins
approximates to that predicted for the entire yeast
proteome. Most importantly, the amount of tryptic peptides
employed for performing yeast proteome analysis is only
around 960 ng which is two to three orders of magnitude
less than those utilized in the current non-gel based
proteome techniques. Instead of using significantly
higher amounts of sample materials and additional sample
fractionation procedures, the combination of electrokinetic
focusing with two highly resolving and orthogonal separation
mechanisms in an integrated platform significantly enhances
both the dynamic range and the sensitivity of MS toward
the proteome analysis.
The CIEF-based multidimensional separation/concentration
technology is further employed for the comprehensive
and ultrasensitive analysis of Drosophila proteomics
during cell death. Steroid hormones regulate the metabolism,
reproduction, and development of higher eukaryotes.
The regulation of these diverse biological phenomena
has been conserved in organisms that are as different
as insects and humans. Thus, Drosophila salivary glands
provide the opportunity to study the proteome in a synchronous
population of dying cells during development. Specifically,
the proteomes of salivary glands are analyzed from a
period beginning before steroid-triggered programmed
cell death and extending to its completion. By combining
the strength of our proteome technologies with the knowledge
in Drosophila genetics, the results gathered in this
study provide valuable information toward the difference
between autophagy and apoptosis and the novel protein
signaling pathways that mediate steroid-triggered cell
death. |
