Proteomics - Biomedical Informatics in Translational Research - page 94

this time freezing of elution peaks on the target is that it is not subject to time con-
straints when analyzing the components, so detailed analyses can be performed on
the interesting peptides. The spotted plates can be archived for MALDI-MS analysis
and this decoupling of separation from high-performance MALDI-TOF/TOF MS
instruments enables several workflows to be interfaced to MALDI-MS for protein
identification.
The attributes of a MALDI-TOF instrument and its ability to interface with the
method-oriented front-end parallel sample preparation has led to the application of
this type of MS instrument for high-throughput profiling of blood sera from healthy
and diseased individuals to look for biomarker patterns. The seminal work of
Petricoin and colleagues [34] demonstrated that peaks obtained from the sur-
face-enhanced laser desorption and ionization (SELDI)-TOF analysis of clinical
samples can be used as discriminators to classify disease states and that prediction
models can be built based on the SELDI-TOF data for applications in the early
detection of cancer. Several front-end peptide fractionation techniques have been
developed that employ a wide variety of functionalized (SELDI) surfaces [35] or
functionalized magnetic beads [36] with hydrophobic, hydrophilic, anion exchange,
cation exchange, and immobilized metal or antibody affinity capture to selectively
enrich a subset of proteins/peptides from biological samples for SELDI-TOF and
MALDI-TOF profiling. These fractionation methodologies have been automated
for robotic handling of samples from parallel processes to spotting the samples
along with matrix onto high-density target plates, making this a robust platform for
the analysis of hundreds of samples per day in a high-throughput fashion for direct
marker analysis from body fluids. Currently, this SELDI/MALDI-TOF technique is
being explored to hunt for the peptide signatures from various biological fluids that
distinguish disease states using advanced pattern recognition tools.
Despite its usefulness in rapidly revealing pattern differences between sample
groups of interest, this method is limited to detecting patterns of peptides with a
mass range of 1 to 20 kDa due to sensitivity issues at high mass range and the size of
peptides that can be analyzed by TOF/TOF sequence analysis for the ultimate identi-
fication of discriminators. However, this technique has shown great promise just
using pattern differences as a biomarker for distinguishing among samples, although
there has been much debate about the reproducibility of the patterns across a differ-
ent set of samples and the association of these serum patterns to disease.
A more detailed study of serum samples from patients with three different solid
tumors and nonsolid tumor controls by Villanueva et al. [37] shed further light on
the origin of low molecular weight peptides. The study concluded that the formation
of most of the discriminatory peptides was due to the action of complement and ex
vivo exoproteases with a small number of cancer-specific discriminators. Although
this study showed evidence for ex vivo formation of low molecular weight peptides,
evidence has accumulated from other reports that protein fragments from the cells
and tissues that are produced in vivo due to tumor-related processes such as
apoptosis, tumor-stromal interaction, vascularization, and immune responses to
cancer cells are enriched on carrier proteins and circulate in the blood without being
filtered by the kidney. Selective enrichment methods for these carrier-bound factors
from Alzheimer's [38] and ovarian cancer [39] sera were demonstrated, and
MALDI-TOF analysis distinguished the diseased samples from controls in both