Biomedical Engineering Reference
In-Depth Information
pH 4
pH 7
Cy2
mix std
Cy3
Cy5
0h a
8h a
24h a
40h a
0h a
24h a
40h a
8h a
40h c
0h a
Hydroxymethylglutaryl-CoA
+
1.22-fold
p
= 0.020
0.15
−
0.1
8h c
24h a
−
2.89-fold
p
= 0.00078
−
2.36
p
= 0.0021
−
0.4
0 h
8 h
24 h
40 h
Figure 13.1
Schematic of a typical DIGE experiment using a Cy2-labeled mixed-sample internal
standard to coordinate independent replicate samples from multiple conditions into a single
analysis. Tri-partite images from six gels are shown at left, with lines indicating Cy3 : Cy2 and
Cy5 : Cy2 ratios made within each gel followed by the normalization of ratios between gels as
described in the text. The enlarged gel indicates the typical resolution of intact protein forms
with isoelectric points between pH 4 and 7. Shown below are examples of protein expression
profiles. Adapted from Friedman
et al
. [20].
The strengths of 2D gel proteome analysis lie mainly in the resolution and quantification
of intact protein species, including charged post-translational modifications as well as pro-
teolytic forms, and facile protein identification directly from the resolved intact protein by
gel excision, in-gel digestion and MS. For quantitative studies, this platform provides the
highest statistical power when using the DIGE technology with the mixed-sample internal
standard method. Several studies have now been published that address the validation of the
DIGE technique with respect to quantitative assessment [27], as well as same/same exper-
iments that demonstrate low technical noise, obviating the need for technical replicates
and enabling a statistically powered experiment using a minimal number of independent
biological replicates (e.g. twofold changes with
N
4 independent samples [23]).
The limitations of 2D gel proteome analysis are almost exclusively tied to the isoelectric
focusing step of the 2D gel separation technology, which make it difficult to monitor proteins
of extreme pI (3
<
pI
<
11) or MW (10
< M
r
<
150 kDa), lower abundance proteins
(typically below 20 fmol), and hydrophobic integral membrane proteins. In addition, the
overall sensitivity of 2DE is limited by the amount of material accommodated by the 2DE
gel and how those proteins are distributed in abundance throughout the proteome. Total
=
