Biology Reference
In-Depth Information
Chapter 20
Method for Protein Subfractionation of Cardiovascular
Tissues Before DIGE Analysis
Athanasios Didangelos , Xiaoke Yin , and Manuel Mayr
Abstract
Difference gel electrophoresis (DIGE) (Electrophoresis 18, 2071-2077, 1997,
1
)
is widely used in cardiovas-
cular research. However, the dynamic range limitations stemming from contaminating plasma pro-
teins and highly abundant extracellular matrix components can make cardiovascular tissues diffi cult to
analyze. Here we describe a novel methodology for biochemical subfractionation of cardiovascular tissues
before DIGE analysis.
Key words:
Cardiovascular, Heart, Proteomics, Vascular, Vessel
1. Introduction
Cardiovascular diseases are a major cause for morbidity and mortality
in the Western world. Cardiovascular studies using proteomics have
mainly focused on the analysis of whole tissue lysates or subcellular
organelle (
2-8
). Consequently, little is known about changes of
proteins in the extracellular space of cardiovascular tissues. The
extracellular space is composed of the extracellular matrix (ECM),
including, collagens, proteoglycans and glycoproteins and proteins
associated with the ECM such as growth factors, cytokines, and
proteinases. The characterization of these extracellular proteins is
diffi cult for two reasons: First, proteins that are bound to the ECM
are scarce and their identifi cation is hampered by the presence of
cellular proteins, and second, ECM proteins are usually cross-linked
in tight aggregates and are therefore diffi cult to extract and solubi-
lize. Moreover, they are subject to extensive posttranslational modi-
fi cations, in particular glycosylation, which alter their molecular
mass, charge, and electrophoretic properties. These characteristics not
only render the proteins diffi cult to identify by mass spectrometry
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