Biology Reference
In-Depth Information
10-15 orders of magnitude (
3, 4
). In order to visualize the
low-abundance proteins originating from damaged tissues and
tumors, it is necessary to remove the high-abundance classical pro-
teins and immunoglobulins. Numerous prefractionation methods
have been developed based on dye-based affi nity chromatography
(e.g., Cibacron Blue), peptide affi nity (e.g., refs.
5-7
), and immu-
noaffi nity depletion strategies. The immunoaffi nity systems have
shown the most promise due to their high selectivity, specifi city,
and reproducibility. Several devices using liquid chromatography
(LC) columns and spin fi lters incorporating antibodies from differ-
ent species are commercially available. One of these is the multiple
affi nity removal system (MARS) developed by Agilent Technologies
(Santa Clara, CA, USA), which has been shown to signifi cantly
improve coverage of the human serum proteome (
8
).
Here, the depletion of mouse serum using a MARS-M3 LC
column is outlined, and a subsequent processing strategy for label-
ing the resulting fractions with difference gel electrophoresis
(DIGE) fl uor minimal CyDyes (GE Healthcare, Uppsala, Sweden)
for comparative 2D DIGE is described. When separated by two-
dimensional electrophoresis (2DE) using large-format precast gels,
these samples produce highly reproducible, well-resolved spot
patterns.
2. Materials
The following protocol for high-abundance protein depletion and
subsequent 2DDIGE analysis is compatible with either serum or
plasma. Great care should be taken during sample collection and
storage as these factors can dramatically affect proteomic analysis
(
9
). Species specifi city is dependent on the MARS column used.
Presently, fi ve different MARS columns are available for humans
that remove albumin, albumin/immunoglobulin G and the top 6,
7, and 14 most abundant proteins, and a single column for mice
that depletes the top three most abundant proteins. The columns
come in three dimensions: 4.6 × 50, 4.6 × 100, and 10 × 100 mm.
Described here is the depletion of mouse serum using a
4.6×100 mm MARS-M3 column on a Hewlett Packard 1090
HPLC system. Volumes and instrument-specifi c parameters should
be modifi ed accordingly for alternative confi gurations. All solu-
tions should be prepared using high-purity water (ultrapure water:
resistivity ³ 18.2 M W cm, total organic content £ 1 ppb, at 25°C).
Reagents are analytical grade unless specifi ed otherwise. Products/
solutions should be maintained at room temperature unless other-
wise specifi ed. Prior to use, ensure all buffers are thawed completely
and mixed well by vortexing.
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