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The driving force behind the present era of genomics
and proteomics has been the emergence of new technologies that are
serving to improve our understanding of the complexity of living
organisms. Whilst the genome of an organism is static, the proteome
can alter with developmental changes, cellular trauma, or the onset
of disease. The information gained from DNA sequencing and mRNA
profiling does not take into account pre-translational events and
post-translational modifications of proteins. Because the set of
genes expressed at the mRNA level may not correlate with the protein
changes in a cell, protein biomarker discovery is a vital complement
to the work of genomics.
For studying the protein profile of a biological system, two-dimensional
gel electrophoresis (2-D PAGE) is the classical approach due to
its high resolving power. However, this technique requires large
quantities of sample, is time consuming and labour intensive, and
reproducibility between research groups is questionable.
Ciphergen® Biosystems was founded in 1996 with the goal of
bringing new technology to bear on the challenges of protein biology.
A bench-top technology that could enable rapid protein discovery
and assay development on a single platform was envisioned. Such
a technology would greatly complement existing technologies as mRNA
studies, 2-D gel electrophoresis, and antibody-based assays. The
ProteinChip system that emerged takes advantage of the combination
of affinity chromatography and time-of-flight mass-spectrometry,
a combination that is designated SELDI-TOF-MS (Surface-Enhanced
Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry) (1).
ProteinChip™ arrays and their applications
ProteinChip arrays carry 8 or 24 spots that are coated with
functional groups enabling the analysis of sub-groups of proteins
based on their affinity for a given surface. Classical principles
based on normal-phase, reverse-phase, IMAC (Immobilised Metal Affinity
Capture), and ion-exchange chromatography are employed to capture
proteins from complex biological samples. "Biochemical" arrays have
a pre-activated surface that is used to covalently tether a molecule
to the surface for subsequent capture of a specific target. In this
manner it is possible to customize an array for studying antibody-antigen,
protein-protein or DNA-protein interactions. Following capture,
arrays are washed to remove unbound proteins, buffers and contaminants,
reducing background "noise". The ProteinChip arrays are subsequently
introduced into the mass reader where the retained proteins are
ionised by a laser, and travel through a vacuum tube to be detected
and analyzed according to their mass over charge ratio.
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Figure 1: Fluid extracted from wound tissue
was applied to different ProteinChip Arrays: Reverse phase
in 10% acetonitrile; Anionic exchange, 100mM Tris pH 8.0;
Cationic exchange, 100mM Tris pH 8.0. Following 30 min incubation,
arrays were washed with binding buffer and analysed in the
ProteinChip Reader. Mass range shown 5-18kDa.
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Using SELDI-based ProteinChip technology, researchers can
produce differential maps of protein expression - "phenotypic fingerprints"
- directly from crude samples such as serum, urine or tissue extracts.
General applications include disease diagnosis, toxicological screening,
target-ligand interactions, peptide mapping, and immunoassay development.
Many research institutions in the United States and in Europe have
used ProteinChip systems to rapidly discover and analyze
protein biomarkers in disease areas as diverse as cancer (2), neurological
disorders, and drug-resistant pathogens (M. tuberculosis
and H. influenza).
A case study
In a recent study, wound fluids were examined using the ProteinChip
system. Figure 1 shows the same wound fluid exposed to three
different ProteinChip surfaces (reverse phase, anionic and
cationic exchange). This demonstrates that each chromatographic
surface has captured a different subset of proteins from the fluid,
depending on their biophysical characteristics. Increasing the number
of surfaces used, and examining different binding/washing conditions
can further extend the number of proteins captured and analysed
by the ProteinChip system. In this pilot study, wound fluids
extracted from different types of tissue trauma were compared. Figure
2 shows wound fluids from different patients examined on a cationic
exchange surface. The similarities within each group are striking,
and differences specific to each wound type were identified. These
differences will now be further investigated. Researchers are increasingly
using the ProteinChip system for the rapid, reproducible
generation of protein expression profiles. In the present study,
nine wound fluid samples were prepared and analysed on three different
surfaces in a few hours, illustrating the speed of protein analysis
with this technology.
Concluding remarks
The ProteinChip technology has proven a good complement
to other existing technologies in proteomics and will allow discovery
of proteins that are resilient to separation by other methods because
of characters such as small size or extreme pI values. The combination
of affinity chromatography and mass spectrometry is a powerful tool
for the rapid discovery of protein biomarkers. Samples do not require
complicated treatment prior to analysis and can be applied in minute
amounts. The ProteinChip arrays can be prepared on the bench
top in less than one hour, and subsequently, it is possible to rapidly
optimise experimental conditions.
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Figure 2: Comparison of wound fluids from
different patiens. Samples were applied (in microlitre quantities)
to a cationic exchange ProteinChip Array (in 100mM Tris pH
8.0). Following 30 min incubation, arrays were washed with
binding buffer and analysed in the ProteinChip Reader. Mass
range shown 5-18kDa.
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Applications for the ProteinChip technology can be found
in diverse fields of (protein) biology. An exciting application
could be found at the NASA Astrobiology Institute. They have recently
acquired a ProteinChip system for use in research on how
life arose in extreme and ancient environments on earth, and in
the ongoing effort to search for "signatures of life" in the solar
system (3). Only our imagination sets limits to the possible applications
of the ProteinChip technology in the field of biology.
References
1. Hutchens, T. W. and Yip, T-T. 1993. Rapid Commun. Mass Spectrom.
7:576-580.
2. Wright, G.L. Jr., Cazares, L.H., Leung, S.M., Nasim, S., Adam,
B., Yip, T., Schellhammer, P.F.,Gong, L. and Vlahou, A.2000. Prostate
Cancer and Prostatic Diseases, 2:264-276.
3. Carnegie Institution of Washington. 2000. News Release.
Ciphergen´s hovedkontor er beliggende i Fremont, USA. På verdensplan
er der solgt ca. 125 systemer (heraf 30 i Europa). Skandinavisk
kontor/laboratorier er beliggende på Symbion på Østerbro. Herfra
tilbydes en tæt kundekontakt, som bl.a. kan føre til afprøvning
af systemet på udvalgte prøver. Afprøvninger er ikke behæftet med
omkostninger og kan foregå på Symbion eller i ens eget laboratorium.
Kontakt os gerne på 39179741 eller læs mere om Ciphergen på www.ciphergen.com.
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