There is an increasing need among the translational medicine
community for automated, quantitative protein biomarker assays that
provide high predictive accuracy for disease diagnosis, prognosis and
treatment response. Ciphergen's ProteinChip® technology and Pattern
Track™ process were developed to facilitate the rapid translation of
biomarker discoveries into validated assays.
Introduction Traditional proteomic
methodologies, such as global digestion or gel-based techniques, involve
the protease digestion of numerous proteins and their subsequent
identification. Time often is wasted repeatedly developing antibody
assays before a marker is validated in a larger population. The Pattern
Track process takes a different approach — validation of biomarkers
before identification and assay development. This requires quantitative
and highly-reproducible methodology. To meet these needs, Ciphergen
developed the ProteinChip® System.
Study Design and Discovery The
Pattern Track process begins with careful study design and
implementation essential to the success of developing biomarker assays.
It is important that the technology used for the study not limit the
ability to ask the right clinical question or use the necessary number
and types of samples.
Figure 1. Serum samples from
control individuals and patients undergoing drug-treatment were
profiled on a cationic exchange ProteinChip Array. A specific
biomarker at 12.4 kDa is up-regulated in the treated serum
samples relative to the control samples. Mass (Da)/charge ratio
is shown on the x-axis and peak intensity is reported on the
y-axis.
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Initially, a small pilot study is conducted to scout for multiple
biomarker candidates with various profiling conditions. Ciphergen's
profiling technology is based on surface-enhanced laser desorption
ionization time-of-flight mass spectrometry (SELDI TOF-MS) utilizing the
ProteinChip System Series 4000, ProteinChip Arrays and software tools
for multi-marker analysis. Biological samples such as serum, tissue
extract, urine or culture supernatant may be applied directly to the
arrays and coated with chromatographic moieties, including anionic,
cationic, hydrophobic, hydrophilic or metal affinity. Proteins in the
complex sample are selectively retained on the surface, based on
chromatographic principles. The selectivity of the different
chromatographic surfaces provides increased resolution of proteins
expressed in complex samples. Interfering contaminants, such as salts
and detergents present in sample buffers, are washed away. Following
application of an energy-absorbing matrix, the captured proteins are
ionized and desorbed from the array in the Series 4000. The results are
displayed as mass spectra with mass-to-charge ratios versus
corresponding signal intensities (Figure 1).
Biomarker Validation, Identification
and Assay During the validation phase, optimal conditions,
determined in the pilot study, are applied to a larger sample set. The
best set of biomarkers — those that have the highest predictive value
— then are determined using multivariate analysis and biostatistical
algorithms. Multi-biomarker panels are superior to single markers in
that they are not affected by patient variability to the extent that
single markers are (1, 2). As a result, multiple markers provide higher
sensitivity, specificity and predictive accuracy.
The validated biomarkers then are purified for identification using
chromatographic and elution conditions determined during the discovery
phase. Purified biomarkers are proteolytically cleaved either on-spot or
in-gel, and resultant digests are analyzed by peptide mapping or, for
full sequence information, by tandem MS.
