Biology Reference
In-Depth Information
Chapter 15
Comparative 2D DIGE Analysis of the Depleted Serum
Proteome for Biomarker Discovery
Megan Penno , Matthias Ernst , and Peter Hoffmann
Abstract
Serum is unarguably the most used diagnostic fl uid. As it circulates throughout the body, leakage peptides/
proteins from damaged and dying cells, host-response proteins including infl ammatory mediators, and
aberrant secretions from tumors and diseased tissues are released into serum, potentially providing a rich
source of disease biomarkers. Here, a method for extending access to the serum proteome by removing
highly abundant proteins prior to comparative two-dimensional difference gel electrophoresis (2D DIGE)
and subsequent protein digestion for identifi cation by mass spectrometry is described.
Key words:
Biomarkers, Serum, Depletion, Multiple affi nity removal system, Tryptic digestion
1. Introduction
The constituents of the serum proteome can be divided into eight
functional groups: (1) the classical serum proteins produced by
solid organs such as the liver; (2) the immunoglobulins; (3) long-
distance receptor ligands including the peptide and protein hor-
mones; (4) local receptor ligands such as cytokines and other
short-distance mediators of cellular functions; (5) temporary pas-
senger proteins that may navigate through the serum on their way
to the site of primary function; (6) foreign proteins derived from
bacterial, viral, parasitic, or proteinaceous agents within the circu-
lating blood volume; (7) tissue leakage products that normally
function within cells but are released into the serum as a result of
cell damage or apoptosis; and (8) aberrant secretions released from
tumors and other diseased tissues (
1
). Accordingly, serum is the
single most informative sample that can be collected from an indi-
vidual that describes their current state of health (
2
).
One of the most signifi cant challenges associated with analyzing
the serum proteome is the broad protein dynamic range spanning
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