Method for Protein Subfractionation of Cardiovascular Tissues Before DIGE Analysis - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 288

(MS) but they are also responsible for many of the technical prob-
lems associated with two-dimensional gel electrophoresis (2-DE)
such as incomplete isoelectric focusing and poor resolution during
electrophoresis.
To analyze cellular as well as extracellular proteins, we have
recently applied a three-step methodology to cardiovascular tis-
sues, which allows a biochemical subfractionation of proteins based
on their different extractability properties ( 9 ): (1) Solubilization of
extracellular space proteins and plasma contaminants, which are
“loosely” bound to the ECM using nondenaturing, ionic condi-
tions; (2) Decellularization and retrieval of cellular proteins for
analysis by 2-DE; (3) Extraction and solubilization of the remain-
ing heavily cross-linked proteins which constitute the mature ECM.
The ECM and its associated proteins represent relatively simple
subproteomes and can be analyzed by gel-LC-MS/MS or shotgun
proteomics. The refi ned cellular proteome is largely devoid of
plasma proteins and extracellular matrix whereas the other frac-
tions contain less cellular proteins facilitating the identifi cation of
scant ECM components. This three-step protein subfractionation
procedure can be readily applied to clinical samples, including small
tissue biopsies, and allows the interrogation of changes in extracel-
lular as well as cellular proteins.

2. Materials

Prepare all solutions/buffers using double-distilled, sterile-fi ltered
deionized water (ddH 2 O) (>18.2 M resistance) and analytical

grade reagents (AnalR). Filter all solutions/buffers through 0.2-

Ω

m
fi lters. All washing and extraction buffers must be supplemented
with 25 mM ethylenediaminetetraacetic acid (EDTA) and include
broad-spectrum proteinase inhibitors: 2 mM of phenylmethyl-
sulfonyl fl uoride (PMSF) (serine proteinase inhibitor), aprotinin
(serine proteinase, including trypsin and related enzymes, inhibitor),
pepstatin (aspartic proteinase inhibitor), and leupeptin (cysteine,
serine, and threonine proteinase inhibitor). Alternatively, commer-
cially available proteinase inhibitor cocktails containing a range of
proteinase inhibitors can be used.

μ

1. 250 mM EDTA, pH 8.0, stock solution. Adjust pH using

12 M NaOH.

2.1. General Solutions

2. 100 mM Tris-HCl stock solution, pH 7.5. Adjust pH with

12 M HCl.

3. 1× phosphate buffered saline (PBS): 150 mM NaCl, 1.7 mM

KH 2 PO 4 , and 5 mM Na 2 HPO 4 . Adjust carefully to pH 7.4
using 6 M NaOH.