Target Discovery and Validation Reviews and Protocols

Immunoproteomics 329
by “shot gun” analysis by using tandem mass spectrometry (MS/MS). In both
methods, spectra are used for database searches, leading to protein identification.
It should be noted that MS/MS mass spectrometry allows the identification of a
protein from a single peptide. Indeed, after fragmenting that peptide, the second
spectrum (MS/MS or MS 2 ) is used for determining its sequence or a sequence
tag. Recent advances in proteomics have been realized by coupling liquid chromatography
with mass spectrometry (LC-MS/MS). LC-MS/MS is a powerful
tool for analyzing the composition of complex mixtures (12).
3. 2D Gel-Based Western Blot Approach
3.1. Strategy
A proteomic-based approach, using 2DE followed by Western blot experiments
with sera from cancer patients, has enabled the identification of tumor-associated
antigens that elicit a humoral response. The methodology to identify proteins
eliciting humoral response in cancer patients involves two essential steps: first,
the screening of autoantigens after serological analysis; and second, the identification
of the target molecule. The use of 2D-PAGE allows separating simultaneously
several thousand individual cellular proteins from tumor tissue or
tumor cell lines. Separated proteins are transferred onto membranes. Sera from
cancer patients obtained at time of diagnosis are used as primary antibody for
Western blot analysis and are screened individually for antibodies that react
against separated proteins. The comparison of series of Western blot obtained
with sera from cancer patients with those performed with sera from healthy
individuals led to determination of the occurrence of relevant autoantigens. An
important advantage of this methodology is that isoforms can be separately analyzed.
Notably, some modifications, such as glycosylation, can be immunogenic.
For identification, the recurrent protein spots that specifically react with sera
from cancer patients are located on silver-stained 2D gels after superimposition
with the blot. The proteins of interest are extracted from the gel and identified
by mass spectrometric analysis.
To confirm the identification of TAAs, a validation step is usually necessary.
This step can be performed using classical methods such as Western blot
experiments when antibodies are available. The relevance to cancer in terms
of expression and location may be investigated using ELISA, immunohistochemistry,
or by other techniques (Fig. 1).
3.2. Practical and Technical Aspects
2D gels are performed with 100–200 µg of proteins from whole cell extracts
of cell lines or biopsies (see Subheading 6.). The first dimension can be
realized using different pH gradients on different sizes of strips. A cocktail of
7 M urea, 2 M thiourea, and 2% CHAPS can be used as a regular buffer for