Protein Chips and Bioinformatics: Essential Tools for Biomarker Discovery
Daniel W. Chan
Professor of Pathology, Oncology, Radiology and Urology; Director, Clinical Chemistry Division; Director, Biomarker Discovery Center; Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-7065
The discovery of serum biomarkers is important in the diagnosis of diseases such as cancer. High throughput proteomic techniques, such as protein chips, coupled with the advances in bioinformatics would allow the identification of biomarkers useful for clinical application. One example is the surface-enhanced laser desorption-ionization (SELDI) time of flight mass spectrometry (TOF-MS). Like many other types of high-throughput expression data, protein array data are often characterized by a large number of variables (the mass peaks) relative to a small sample size (the number of specimens). In analyzing such data to screen for disease- associated biomarkers, it is important to extract as much information as possible from a limited number of samples and to avoid selecting biomarkers whose performances are influenced mostly by non-disease related artifacts in the data. A software package was developed by our group to compute and rank the contribution of each individual peak towards the optimal separation of two diagnostic groups. Several cases of cancer biomarkers discovery from serum or plasma will be shown.
Proteomic Analyses Using a Fully Automated Chip-based Nanoelectrospray System
Colleen K. Van Pelt1, Sheng Zhang1, Xian Huang1, Gary Williams2, and Mark H. Allen2
1Advion BioSciences, Ithaca, New York; and 2Advion BioSciences, Ltd., Norwich, United Kingdom
A fully automated nanoelectrospray system used in combination with mass spectrometry has recently been developed. The NanoMate is a robotic infusion device that holds a 96-well plate, a rack of 96 disposable, conductive pipette tips, and an ESI Chip. The chip is a fully integrated monolithic electrospray device that consists of a 10 χ 10 array of nozzles etched from the planar surface of a silicon wafer using standard semiconductor processes. This automated nanoelectrospray system offers low sample consumption and enhanced ionization resulting in higher sensitivity, similar to conventional pulled-capillary nanoelectrospray. However, the automated system offers a simple, one-time spray optimization for a set of 96 samples, no carryover between samples, and enhanced spray stability.
To demonstrate the capability of the NanoMate for proteomic analyses the results from several studies will be discussed including the identification of proteins from 2D gels of E. coli crude cell extract, non-covalent interactions between the endoglucanase catalytic domain of the T. fusca bacterium and cellopentose, protein quantification of standard proteins using isotope-coded affinity tags (ICAT), and phosphorylated peptide analysis using an affinity chromatography column charged with ferric ions. Results from off-line collected liquid chromatography fractions from E. coli crude cell extracts will also be shown.
A Sample Preparation Platform Technology for Proteomic Applications
M. Walid Qoronfleh
Perbio Sciences, Milwaukee, Wisconsin 53202-1009
Proteomics is having a profound impact on biological research. In this post-genomics era, there is a need to develop reliable methods to quickly and conveniently analyze multiple protein samples. Equally important, pre-fractionation of complex protein mixtures and enrichment of desired protein fractions are critical to proteome analysis. To this end, we have developed SwellGel™ Technology, a versatile and novel affinity chroma- tography platform. This technology utilizes a selected chromatography media that is dehydrated to form uniform aggregates. The discs will instantly rehydrate upon addition of the protein sample where the researcher need only perform a few simple steps to recover their protein/ fraction of interest. Discs can be made in a variety of sizes (resin volume 25 μ^ ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and with different ligands attached to the matrix. SwellGel™ technology has been applied to affinity purification systems such as glutathione for GST-tagged proteins, metal chelates for His- tagged proteins, avidins for biotinylated molecule isolation, and proteins A/G for antibody purification. Data will be presented applying SwellGel discs to protein desalting/enrichment, the removal of abundant proteins from human serum- like albumin and immunoglobulin, and the isolation of phosphoproteins and phosphorylated peptides. The SwellGel™ format provides these chromatography systems with additional convenience, consistency, stability, and exceptional flexibility when compared to standard wet chromatography resins. These key features make SwellGel™ technology a significant advancement facilitating the sample handling needs for proteomic research.
Prioritising the Proteome: Identifying Pharmaceutical^ Relevant Targets by Linking Sequence to Function Through 3D Structure
VP Drug Discovery, Inpharmatica, 60 Charlotte Street, London W1T 2NU, United Kingdom
Inpharmatica is focussed on the application of protein 3-D structural data to the identification of distantly related homologs of pharmaceutically important gene families. We have developed a combined bioinformatics and chemoinformatics platform that significantly improves the yield and quality of functional annotation and correlates this annotation with an estimation of druggability. We will describe some specific examples of the application of this technology to the target discovery process.
From Gene Products to Disease Phenotype: Antibody Chips for Predictive Medicine in Cancer
Moncef Jendoubi, Christine Chavany, Jean-Francois Martini, Yue Xu, and Rosaura Valle Milagen Inc.
Characterizing the proteome has become essential to understanding phe- notype and thus disease, leading in turn to potentially finding new and better ways to cure disease. Milagen has developed a unique antibody- based approach termed ANTIBIOMIX™ to directly correlate gene products to disease. A major feature of ANTIBIOMIX™ is the high-throughput generation of polyclonal antibodies in a relatively short period of time and at a reasonable cost. Milagen will complete its 100,000 polyclonal antibody collection against human gene products by the end of 2002.
To directly identify proteins that underlie disease, Milagen antibody collections are applied in multiplex format to large collections of clinical samples, using our proprietary matrix protein array technology. Our technology can be applied to tissues and biological fluids. Based on differential profiles between matched normal and disease samples, we have thus identified several cancer-related targets, in major cancer types cancer, as well as secreted molecules in sera. We intend to apply this technology to other major human diseases.
The identification of cancer specific targets in body fluids leads to the development of a new generation of diagnostic products, namely antibody chips for predictive medicine, applicable to early detection, disease progression, metastatic indications, toxicity profile, and therapy response. Secreted biomarkers are candidates for the development of therapeutic products as well.
ANTIBIOMIX™ is bridging the gap between genotype (gene sequence) and phenotype (diseases state), leading to the rapid identification of a variety of disease specific proteins, and to the validation of novel targets for diagnostic and therapeutic applications.
From Cell to Gel to Well: A Modular Automated Platform for Proteomics Assays
Tecan-Munich, Feldkirchner Strasse 12a, 85551 Kirchheim, Germany
Current traditional proteomics techniques can be insufficient for many researcher’s needs in regards to: sensitivity (e.g., difficulties in detecting unknown, low abundant proteins), reproducibility (e.g., lack of robust, consistent methods for protein fractionation and 2D-PAGE), throughput (lack of automation systems and methods).
To help address these critical needs in Proteomics, Tecan has developed an automated modular platform. The complete platform consists of: a Cell Maintenance System (automation of cell culture procedures to insure cell preparation and treatment), a Free Flow Electrophoresis System (a novel semi-preparative, charged based, liquid separation technology for fractionation of cells, cell organelles and complex protein mixtures), a 2D-PAGE System (a fully automated system from IEF, Gel casting, SDS- PAGE, and Staining), and a Protein Processing System (consisting of a spot picking device followed by an in gel digestion and an interface to mass spectrometry). The instrumentations can be integrated in various combinations (e.g., Free Flow Electrophoresis combined directly with LC- MS) and allows researchers to construct a customized platform for different proteomics applications. The current study presents protein fraction- ation and identification data generated with the automated platform with samples from human serum, yeast and liver.
In the current study, special emphasis will be placed on the use of the combination of Free Flow Electrophoresis with classical 2D-PAGE. This technology is capable of overcoming one of the biggest challenges for proteome research: the reduction of the complexity of the protein samples by powerful early fractionation.
Published online: September 01, 2002
© 2002 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
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