July 1, 2007 (Vol. 27, No. 13)
David Hammond Ph.D.
Bead-based Combinatorial Chemistry for the Enrichment & Capture of Valuable Components
A major limitation to the discovery of new therapeutics and unique biomarkers of disease is the ability to identify, purify, and characterize valuable trace proteins in the presence of highly abundant components present in complex samples such as plasma. The abundant proteins such as albumin are present at millions of times greater concentration than trace components such as growth factors. Since state-of-the-art analytical techniques, including mass spectrometry and gel electrophoresis, are limited to detecting proteins within a range of about ten-thousand fold, trace proteins cannot be detected in the presence of more abundant components by these methods.
ProSpectrum Library™-based technology (Figure 1) addresses this fundamental limitation to discovering trace components by simultaneously decreasing the concentrations of the abundant proteins while enriching and capturing individual trace components such as proteins or protein complexes on affinity ligands (Figure 2). The roots of the technology and its applications lie in the choice of polymer, the construction of the peptide-based ProSpectrum Libraries, and their compatibility with processing diverse samples including whole blood in a broad range of formats and applications.
The ProSpectrum Libraries, available from Peptides International (www.pepnet. com), were designed and developed by American Red Cross scientists in 2001 to contain affinity ligands that would capture most, if not all, of the proteins present in any complex mixture. Since the foundation of the technology is fundamental affinity interactions between the proteins and the ligands, it is applicable to a vast number of important discovery and proteomic applications using a broad range of starting materials.
By decreasing the range of protein concentrations, ProSpectrum Libraries improve the detection of valuable trace entities in complex mixtures and increase the sensitivity of analytical assays. These characteristics make ProSpectrum Libraries a powerful tool for sample preparation, either for basic protein science research, or in the clinical laboratory as an adjunct to diagnostic tests. Moreover, the proteins may be analyzed en masse to obtain a precise protein profile for a given sample (e.g., a diseased patient). Since the libraries also preserve the concentration differential of specific proteins between samples ProSpectrum Libraries provide a particularly effective platform for novel biomarker discovery (Figure 3).
Current depletion-based sample-preparation methods improve the sensitivity of trace component detection from 10 to 100 fold, but are extremely expensive, highly specific for the target, (usually human plasma or serum), and further dilute trace components. Since the dynamic range of proteins is still orders of magnitude greater than that required for detecting all proteins within a sample, there remains a need to dig deeper into the proteomes of diverse samples using new approaches. ProSpectrum Libraries provide such an approach for enrichment of low-abundance proteins and are now being integrated with 2-D gel, mass spectrometry, ELISA, and functional assays to improve protein detection dramatically.
In addition to enrichment of trace proteins, ProSpectrum Libraries also provide technologies to support expression proteomics and characterization of differential expression of proteins across multiple diseases and complex clinical samples. One of these discovery technologies—the Bead blot—enables detection and identification of individual components from complex mixtures such as whole blood, identification of protein-protein and drug-protein interactions, and identification of a ligand to any target without the necessity of labeling or purifying the targets a priori, and without the interference of abundant proteins.
In essence, the Bead blot simultaneously evaluates millions of different affinity resins for their ability to capture and selectively release the molecular target of choice from test samples. The highly specific ligands found by Bead blot may be produced inexpensively at large scale for incorporation into standard diagnostic assays and/or for affinity purification of the target protein. Ligands that bind and remove pathogens such as prions at concentrations detectable only by infectivity assay were identified using the Bead blot technology. These ligands have been incorporated into a licensed filtration product that is being commercialized, following extensive safety, toxicity, stability, and hemocompatibility testing to reduce the amount of variant Creutzfeldt Jakob Disease from red blood cell concentrates for transfusion.
ProSpectrum Libraries can additionally be used as the basis of another technology called Functional Identification of Novel Activities (FIoNA) for discovery of novel protein activities. FIoNA allows discovery of the most diverse class of proteins in blood, the human antibody repertoire. FIoNA selects functional trace components from complex samples such as blood and tissue extracts based on the component’s biochemical or biological properties.
FIoNA starts at the endpoint of traditional drug discovery; it selects leads based on function and rapidly proceeds to the identification of the active proteins, thereby ensuring at the start of the selection process that the lead is relevant to the indication. Since only functional targets are analyzed, FIoNA identifies leads without generating overwhelming amounts of irrelevant data, thereby dramatically enhancing functionally relevant throughput. This technology is currently being utilized for the identification of specific antibodies active against melanoma.
ProSpectrum Libraries are also being applied in new formats such as ultra-high density arrays, placed into capillaries for direct interfacing with mass spectrometers, sorted by FACS, and combined with depletion strategies for deeper penetration of the proteome.
David Hammond, Ph.D., is the former executive director for plasma derivatives at the American Red Cross (ARC). He is now a consultant for the ARC, co-founder of Pathogen Removal and Diagnostic Technologies, and founder of Prolias. Web: www.proliastechnologies.com. E-mail: [email protected].