Target Discovery and Validation Reviews and Protocols

Immunoproteomics 331
isoelectric focalization in reducing conditions. The second dimension corresponds
to a regular sodium dodecyl sulfate-PAGE in reducing conditions.
However, a gel gradient is indicated for optimal separations. For better reproducibility
of 2D patterns, it is recommended to migrate several 2D gels at a
time. After electrophoresis, proteins can be stained using Coomassie blue or
silver staining compatible with mass spectrometry analysis (13), or transferred
to polyvinylidene fluoride (PVDF) membrane. To monitor the transfer efficiency
and the amount of proteins before hybridization, a rapid staining can be
performed using red ponceau. This dye is water soluble and compatible with
following Western blotting. The hybridization step is realized with serum from
patient at a dilution 1/100–1/300. If background is generated, a lower dilution
can be used with subsequent extensive washes. For protein identification,
trypsin digestion is performed as described previously (14,15). After mass
spectrometry, spectra are used for database search by using programs MS-Fit,
Profound, or Mascott (http://prospector.ucsf.edu; www.hgmp.mrc.ac.uk/
GenomeWeb/prot-fragment.html, or www.matrixscience.com).
3.3. Relevant Examples
A study using the 2D gel-based Western blot approach has been used for
tumor antigen identification in breast cancer (15). This approach is described
in Fig. 1 as an example. In this study, sera from patients with breast cancer
and healthy individuals were used. The breast cancer cell line SUM-44 was
used as a source of tumor cell proteins for 2D-PAGE and for Western blot
analysis. A restricted reactivity against a set of three proteins with an estimated
molecular mass of 25 kDa was observed in 13% of the patients. These
protein spots were analyzed using mass spectrometry and were identified as
isoforms of the protein DJ-1.
Other interesting examples can be cited. Indeed, sera from patients with lung
adenocarcinomas exhibited a high frequency (60%) and specificity of autoantibodies
directed against annexins I and II (16). A high frequency of autoantibodies
also has been observed in sera from 70% patients with hepatocellular carcinoma
(HCC) (17). However, by contrast with lung adenocarcinoma, reactivity
against 13 protein spots has been observed. This observation may reflect the
Fig. 1. (Continued) then used for database search and protein identification. The
three protein spots were identified as the protein DJ-1. A validation of the identification
has been performed using Western blotting with specific monoclonal antibodies.
Finally, a relevance to cancer has definitely been established after immunochemistry
experiments that led to the observation of differential expression level and location of
the protein between breast cancer and the healthy tissue.