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Molecular & Cellular Proteomics 4:182-190, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

High Throughput Proteome Screening for Biomarker Detection^*

Sheng Pan, Hui Zhang, John Rush, Jimmy Eng^,¶, Ning Zhang, Dale Patterson^||, Michael J. Comb and Ruedi Aebersold^,**,

From the Institute for Systems Biology, Seattle, Washington 98103, Cell Signaling Technology, Inc., Beverly, Massachusetts 01915, ^¶ Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, ^|| Applied Biosystems, Framingham, Massachusetts 01701, and ^** Institute of Biotechnology, ETH Zurich and Faculty of Natural Sciences, University of Zurich, CH-8093 Zurich, Switzerland

Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Current methods, while highly developed and powerful, are falling short of their goal of routinely analyzing whole proteomes mainly because the wealth of proteomic information accumulated from prior studies is not used for the planning or interpretation of present experiments. The consequence of this situation is that in every proteomic experiment the proteome is rediscovered. In this report we describe an approach for quantitative proteomics that builds on the extensive prior knowledge of proteomes and a platform for the implementation of the method. The method is based on the selection and chemical synthesis of isotopically labeled reference peptides that uniquely identify a particular protein and the addition of a panel of such peptides to the sample mixture consisting of tryptic peptides from the proteome in question. The platform consists of a peptide separation module for the generation of ordered peptide arrays from the combined peptide sample on the sample plate of a MALDI mass spectrometer, a high throughput MALDI-TOF/TOF mass spectrometer, and a suite of software tools for the selective analysis of the targeted peptides and the interpretation of the results. Applying the method to the analysis of the human blood serum proteome we demonstrate the feasibility of using mass spectrometry-based proteomics as a high throughput screening technology for the detection and quantification of targeted proteins in a complex system.

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