Biology Reference
In-Depth Information
Chapter 16
Differential Gel-Based Proteomic Approach
for Cancer Biomarker Discovery Using Human Plasma
Keun Na , Min-Jung Lee , Hye-Jin Jeong , Hoguen Kim ,
and Young-Ki Paik
Abstract
Two-dimensional fl uorescence difference gel electrophoresis (2D DIGE) has become a general platform
for analysis of various clinical samples such as biofl uids and tissues. In comparison to conventional 2-D
polyacrylamide gel electrophoresis (2D PAGE), 2D DIGE offers several advantages, such as accuracy and
reproducibility between experiments, which facilitate spot-to-spot comparisons. Although whole plasma
can be easily obtained, the complexity of plasma samples makes it challenging to analyze samples with
good reproducibility. Here, we describe a method for decreasing protein complexity in plasma samples
within a narrow pH range by depleting high-abundance plasma proteins. In combination with analysis of
differentially expressed spots, trypsin digestion, identifi cation of protein by mass spectrometry, and stan-
dard 2D PAGE and DIGE, this method has been optimized for comparison of plasma samples from
healthy donors and patients diagnosed with hepatocellular carcinoma.
Key words:
Two-dimensional fl uorescence difference gel electrophoresis, Narrow pH range, Plasma
proteomics, Hepatocellular carcinoma, Biomarker
1. Introduction
Human plasma is one of the most readily available clinical samples
for discovery of disease biomarkers because it is commonly col-
lected in the clinic and provides noninvasive, rapid analysis for any
type of disease (
1
). Most human plasma proteins are synthesized in
the liver, with the exception of
-globulin.
Separation of plasma proteins by electrophoresis offers a valu-
able diagnostic tool, as well as a way to monitor clinical progress
(
2
). However, plasma is known to contain a very complex pro-
teome with a dynamic range of more than ten orders and proteins
secreted by metabolic trauma from various organs in the human
body. For example, approximately 51-71% of plasma protein is
γ
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