Target Discovery and Validation Reviews and Protocols

Protein Arrays 339
fractions from chromatographic separations of complex protein mixtures (33).
The authors proposed arraying fractions from high-resolution multidimensional
chromatography separations, which serially combine different separation
phases such as reverse phase and ion exchange chromatography, producing as
many as 2000 fractions from a single protein pool. Twenty protein fractions
from the A549 lung cancer cell line were separated by isoelectric focusing and
arrayed. Solubilized proteins from the LoVo colon adenocarcinoma cell line
were separated into fractions, arrayed onto nitrocellulose-coated slides, and
hybridized with individual sera from patients with colon cancer, with lung
cancer, and healthy subjects (34). A distinct pattern of reactivity was observed
with sera from colon cancer relative to lung cancer. One fraction that exhibited
reactivity with 9 of 15 colon cancer sera was subjected to mass spectrometry,
leading to the identification of ubiquitin C-terminal hydrolase isoenzyme 3 as a
potential autoantigen. Notably, reverse phase microarray has the improvement
that novel cancer antigens may be identified.
1.2.3. Peptide-on-Plasmid Display
The advantage of combination of large cDNA expression libraries with
microarray technology is the direct connection of the DNA sequence information
from a particular clone to its recombinant, expressed protein. For the generation
of protein biochips, high-throughput subcloning of open reading frames from
the genome of humans, Saccharomyces cerevisiae (35,36), Arabidopsis
thaliana (37,38), or Caenorhabditis elegans (39) was performed. This cloning
approach is strongly dependent on the progress in genome sequencing projects
and the annotation of the human sequences (40). Thus, previously uncharacterized
proteins will be absent. Additionally, a clear determination of the expressed
sequence remains difficult because of differential splicing or posttranslational
modifications. For these reasons, this approach has proved most valuable in the
production of arrays containing proteins from well characterized organisms.
However, the use of cDNA expression libraries eliminates the need to construct
individual expression clones for every protein of interest and allows identification
of unknown sequences/proteins. In this respect, Büssow and colleagues
described a human fetal brain expression library (41). Recently, a mouse TH1
expression cDNA library was constructed (42).
1.2.4. Phage-Displayed Peptide/Recombinant Proteins
Peptide and cDNA phage display libraries also have been used to determine
the specificities of antibodies present in the whole sera of patients where
information about the parental antigens is unknown. This phage display-based
approach (termed “fingerprinting”) enables isolation of mimic peptides of
antigens eliciting a humeral response (19,20,43). Antibody fingerprinting is a