Ciphergen Biosystems Inc. - ProteinChip™ technology

Publiceret Januar 2001

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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.

2001_1 ciph_slide1bg.gif
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.

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.

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.